ACS Publications. Most Trusted. Most Cited. Most Read
Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Chem. Res. Toxicol. 2007, 20, 10, 1393-1408
  • Open Access
Article

Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat
Click to copy article linkArticle link copied!

View Author Information
Departments of Biochemistry, Chemistry, and Pharmacology, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
* To whom correspondence should be addressed. Tel: 615-343-7329 . Fax: 615-343-7534. E-mail: [email protected]
†Department of Biochemistry.
‡Department of Chemistry.
§Department of Pharmacology.
∥Mass Spectrometry Research Center.
Open PDFSupporting Information (1)

Chemical Research in Toxicology

Cite this: Chem. Res. Toxicol. 2007, 20, 10, 1393–1408
Click to copy citationCitation copied!
https://doi.org/10.1021/tx7001405
Published September 25, 2007

Copyright © 2007 American Chemical Society. This publication is licensed under these Terms of Use.

Request reuse permissions

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb’s hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes. Conjugates of the 1,4-enedial derivative of teucrin A, its presumed toxic metabolite, with lysine- and cysteine-containing peptides were synthesized and used to select ScFvs from a rodent phage-displayed library, which recognized the terpenoid moiety of the teucrin-derived adducts. Immunoaffinity isolation of adducted proteins from rat liver homogenates following administration of a toxic dose of teucrin A afforded a family of proteins that were identified by liquid chromatography/tandem mass spectrometry. Of the 46 proteins identified in this study, most were of mitochondrial and endoplasmic reticulum origin. Several cytosolic proteins were found, as well as four peroxisomal and two secreted proteins. Using Ingenuity Pathway Analysis software, two significant networks involving the target genes were identified that had major functions in gene expression, small molecule biochemistry, and cellular function and maintenance. These included proteins involved in lipid, amino acid, and drug metabolism. This study illustrates the utility of chemically synthesized biological conjugates of reactive intermediates and the potential of the phage display technology for the generation of affinity reagents for the isolation of adducted proteins.

Copyright © 2007 American Chemical Society

Subjects

what are subjects

Introduction

Click to copy section linkSection link copied!
Covalent adduction of proteins has been linked to the toxicity of many reactive metabolites that are generated upon the activation of xenobiotics by cytochrome P450 enzymes (1, 2). Likewise, protein damage by endogenous electrophiles, such as the lipid peroxidation product, 4-hydroxynonenal (4-HNE), is associated with a number of human diseases, including neurological and autoimmune disorders, as well as alcoholic liver disease (3-5). While analytical methods to identify reactive metabolites and their protein binding potencies in vitro are now routinely used in drug development to eliminate potentially toxic candidates, the ability to extend in vitro studies to in vivo toxicity is limited (6-8). Reactive intermediates modify a number of common abundant proteins, and there appears to be no single modified protein solely responsible for the toxic response. Thus, characterization of the entire spectrum of modified proteins is an important step in understanding mechanisms of toxicity.
A number of proteins adducted by reactive metabolites of drugs and xenobiotics, as well as endogenous reactive products, such as acetaminophen (APAP), bromobenzene (BB), and 4-HNE, have been identified from intact animals (3, 4, 9-22). Detection of adducted proteins was accomplished by the use of radioactively labeled toxicants or by employing adduct-specific antibodies for immunoblotting. These approaches are limited by sensitivity in the case of radiochemical detection or by specificity in the case of adduct-specific antibodies. These limitations preclude detection of the complete profile of adducts.
Our laboratory is interested in the chemistry and biology of teucrin A, a major constituent of the neo-clerodane diterpenoid fraction of the hydroalcoholic extract of germander (Teucrium chamaedrys). Germander has been used to treat obesity and is a flavoring agent in alcoholic beverages (Scheme 1) (23, 24). Oral administration of teucrin A or germander extracts causes depletion of intracellular glutathione and damage to cellular protein thiols, resulting in elevated plasma ALT activity and significant centrilobular liver injury in mice (25, 26). Isolated diterpenoid fractions from germander cause apoptosis in isolated rat hepatocytes (27). The toxicity of teucrin A in mice has been attributed to metabolic activation of the 3-substituted furan ring to an electrophilic metabolite (25). This hypothesis is supported by the observation that a tetrahydrofuran analogue is not toxic (25). By analogy to other furan-containing compounds, such as methylfuran, furosemide, 4-ipomeanol, menthofuran, and aflatoxin (28-35), 1,4-enedials or possible epoxide precursors are the likely reactive metabolites (Scheme 2) (34-37).

Scheme 1

Scheme 1. Proposed Mechanism for the Bioactivation of 3-Substituted Furans and the Formation of Reactive Electrophilic Enedial Metabolites
High Resolution Image
Download MS PowerPoint Slide

Scheme 2

Scheme 2. Synthesis of the Enedial Derivative of Teucrin A and N-Terminal Biotinylated Peptide Conjugatesa
High Resolution Image
Download MS PowerPoint Slide

Scheme a(a) Dimethyldioxirane (DMDO), acetone. (b) Btn-AKDVY, 0.1 M phosphate buffer, pH 7.4. (c) Btn-ACDVY, 0.2 M phosphate buffer, pH 7.4.

Previously, we reported the synthesis of the enedial derivative of teucrin A (1) and the structural characterization of its conjugates with N-acetyl lysine methyl ester (NAL) and N-acetyl cysteine (NAC). In addition, we prepared conjugates of 1 to the lysine-containing peptides, RKDVY and N-terminal biotinylated RKVDY (38). The ability to efficiently synthesize stable peptide adducts of 1 provided a tool for the development of selective antibodies against the teucrin epitope (39). Adducted peptides were used to screen a phage-displayed single-chain antibody library for single-chain fragment variables (ScFvs) that recognize the teucrin-adducted peptides independently of the sequence of the peptide. Candidates ScFvs were further screened to identify ScFvs that detect adducted proteins by Western blotting and that immunoprecipitate them. These antibodies were used to validate the production of teucrin–protein adducts in vivo and to enrich them for mass spectrometric analysis. We report here a comprehensive set of protein targets of activated teucrin A. We also describe the possible consequences of modification based on network analysis of the modified proteins.

Materials and Methods

Click to copy section linkSection link copied!

Reagents and Solvents

HPLC grade solvents for column chromatography and HPLC were obtained from Fisher (Pittsburg, PA) and were used as received. Reagent grade chemicals were obtained from Aldrich (Milwaukee, WI). Biocytin [Ser25] protein kinase C fragment 19–31 and KKRAARATS amide were purchased from Sigma-Aldrich (St. Louis, MO). 5-mer peptides were purchased from Sigma-Genosys (The Woodlands, TX). Thin-layer chromatography was performed on silica gel GF glass plates from Analtech (Newark, DE). The chromatograms were visualized under UV (254 nm), fluorescence, or by staining with sulfuric acid solution, followed by heating. Column chromatography was performed using silica gel 60–100 mesh from Fischer. Teucrin A was a generous gift from Dr. Corrado Galli, University of Milan (Italy). Purification and characterization of teucrin A were performed as described previously (38). DMDO was prepared as previously reported (38, 40).

Instrumental Analysis

UV spectra were recorded using a Hewlett-Packard UV/vis model 89500 spectrometer. Mass spectra were recorded on a Finnigan TSQ 7000 triple-quadrupole spectrometer under positive or negative ion mode. 1H NMR spectra were recorded on a Bruker 300, 400, and 500 MHz NMR spectrometers using acetone-d6 and DMSO-d6 as solvent and internal standard. HPLC analysis was performed on Waters 1525 Binary HPLC Pump with Waters 2996 Photodiode Array Detector on the reversed phase Jupiter C18 5 μm 250 mm × 4.6 mm or 150 mm × 4.6 mm column at 1 mL/min (Phenomenex, CA). Semipreparative HPLC was performed using Jupiter C18 5 μm 250 mm × 10 mm column at 4 mL/min.
Enedial derivative of teucrin A (1) was prepared and characterized as previously reported (38). Teucrin A (10 mg, 0.029 mmol) was dissolved in 0.5 mL of anhydrous acetone-d6, and the solution was cooled in the acetone/dry ice bath. Cold DMDO-d6 (1.2 mL, 0.034 mmol) was added under argon atmosphere. The reaction mixture was allowed to warm up to room temperature, and the progress of the reaction was monitored by 1H NMR. The reaction was complete in 2 h. Upon disappearance of the starting material, nitrogen was blown through the solution to eliminate unreacted DMDO-d6, and the product was stored as acetone-d6 solution at −20 °C.

Synthesis of Teucrin A-Adducted Biotinylated Peptides

Btn-AKDVY (2 mg, 2.14 μmol) and Btn-ACDVY (2 mg, 2.20 μmol) were dissolved in 0.2 mL of 0.2 M sodium phosphate buffer, pH 7.4. The acetone solution of 1 (1 equiv, 26 mM) was added, and mixtures were incubated at 37 °C. The reactions were monitored by HPLC/UV looking at the disappearance of the starting peptides. Adducted products were purified by HPLC and analyzed by LC-MS/MS. Liquid chromatography was performed using a gradient at 1 mL/min as follows: from 0 to 1 min, hold 100% A; from 1 to 20 min, linear gradient to 50% A; and from 20 to 25 min, linear gradient to 100% A, where A = water + 0.1% acetic acid and B = acetonitrile + 0.1% acetic acid.

Phage Antibody Selection on Teucrin A Dial-Btn-AKDVY

A rodent phage displayed recombinant antibody library (∼2.9 × 109 members) was generated according to a previously described protocol (41) with minor modifications. The library was used to obtain teucrin A-specific ScFv antibodies. ScFv antibodies stemming from the library bear an E-tag peptide sequence. E-tagged ScFv antibodies bound to antigen can be detected using the an anti-E tag monoclonal antibody conjugated to peroxidase (Anti-E/HRP, G. E. Healthcare catalog no. 27941301) and purified from Escherichia coli using an anti-E tag monoclonal antibody column (G. E. Healthcare catalog no. 17-1362-01). For phage antibody selections, Nunc Maxisorb Immunotubes were coated with 1 mL of streptavidin diluted to 5 µg/mL in phosphate-buffered saline (PBS) for 1 h at room temperature (RT) and then blocked with PBS containing 0.1% Tween 20 (PBS-T) for 1 h at RT.
One milliliter of teucrin A-adducted Btn-AKDVY diluted to 1 µg/mL PBS-T was added to streptavidin-coated tubes. The tubes were incubated for 1 h at RT, then washed six times (10 seconds per wash) with PBS-T to remove unbound teucrin A-adducted Btn-AKDVY. The phage-displayed antibody library was diluted to 1012 phage per mL in PBS-T and incubated for 2 h at RT to block nonspecific phage-antibody binding activity. One milliliter of phage antibody library in PBS-T was added to teucrin A-adducted Btn-AKDVY on streptavidin-coated tubes. After a 2 h incubation at RT, the tubes were washed six times (10 seconds per wash) with PBS-T to remove unbound phage antibodies. After the first round of selection, phage-displayed antibodies, bound to teucrin A-adducted Btn-AKDVY on streptavidin-coated tubes, were eluted with 1 mL of 100 mM triethylamine for 10 min at RT. The pH of the eluted phage antibodies, in triethylamine, was adjusted to a pH of 7–8 with 0.5 mL of 1 M Tris, pH 7.4–8.
The phage antibodies were then used to infect log phase E. coli TG1 cells, incubated at 37 °C for 1 h. Infected E. coli TG1 cells were plated onto 2xYT AG agar plates (17 g of Bacto-tryptone, 10 g of Bacto-yeast extract, 5 g of NaCl, 20 g of glucose, 100 mg of ampicillin, and 15 g agar per liter, final concentration), incubated overnight at 30 °C, and helper phages were rescued with M13KO7 to produce phage-displayed antibodies, in bacterial culture supernatant, for use in a second round of phage antibody selection according to methods described previously (42). One microliter of Tween 20 was added to 1 mL of phage-displayed antibodies in bacterial culture supernatant to block nonspecific phage-antibody binding activity. After a 2 h incubation at RT, the blocked phage-displayed antibodies were used for a second round of selection on teucrin A-adducted Btn-AKDVY on streptavidin-coated tubes. The protocols used to carry out the first round of phage antibody selection were also used to perform the second round of selection.
The protocols used to identify antigen-specific ScFv by an enzyme-linked immunsorbant assay (ELISA) and to grow and purify ScFv have been described (42) and modified as follows to obtain teucrin A-specific ScFv. Briefly, bacterial colonies stemming from the second round of phage antibody selection were picked from 2xYT AG agar plates into 384 well microtiter plates containing 2xYT AI (17 g of Bacto-tryptone, 10 g of Bacto-yeast extract, 5 g of NaCl, 100 mg of ampicillin, and 1 mM IPTG, final concentration) and induced to express soluble ScFv recombinant antibodies. For ELISA, Nunc 384 well microtiter plates were coated with streptavidin (5 µg/mL PBS, 25 µL/well) for 1 h at RT and then blocked with PBS-T. Teucrin A-adducted Btn-AKDVY or ACDVY (positive control) or Btn-AKDVY or ACDVY (negative control) diluted to 1 µg/mL PBS-T, 25 µL/well was then added to microtiter wells. After a 1 h at RT incubation, plates were washed six times with PBS-T (10 seconds per wash), emptied, and then used for assay as described (42). Bacterial colonies expressing ScFv reactive by ELISA with the positive but not negative control were subsequently identified and used to produce teucrin A-specific ScFv.

Teucrin A Treatment of Rats

Animal protocols were performed under the approval of Vanderbilt University and in accordance with the Institutional Animal Care and Use Committee policies. Male Sprague Dawley rats (225–250 g) with vascular catheters surgically implanted in the femoral and jugular veins were obtained from Charles River Laboratories (Wilmington, MA). The rats were allowed to feed a standard diet for 48 h ad libitum and were starved overnight prior to the experiment. Animals, in triplicate, were dosed orally by gavage a single dose of teucrin A at 25 mg/animal (100 mg/kg bw) in minimum DMSO in corn oil or DMSO in corn oil alone (total volume, 1 mL). The plasma levels of alanine amino transferase (ALT) were monitored over time (0.4 mL blood samples were taken at 0, 1, 3, 6, 10 and 24 h) using ALT Colorimetric Assay Kit (Teco Diagnostics, Anaheim, CA). The animals were sacrificed 24 h after the dose, and the livers were excised. Liver homogenate was separated into cytosolic and particulate protein and analyzed by Western blotting for teucrin A adducts using teucrin A-specific ScFv monoclonal antibodies as described below.

Rat Liver Homogenization and Fractionation

Thawed livers were weighed and finely scissor-minced with a scalpel. The tissue was transferred into a 50 mL Potter–Elvehjem glass container, 20 mL of ice cold homogenization buffer (20 mM Tris-HCl, pH 8.0, and 250 mM sucrose, containing 1 tablet of Roche protease inhibitor mix) was added, and the livers were homogenized. The mixtures were centrifuged at 250g for 15 min at 4 °C, and the supernatant was collected. The pellet was rehomogenized in an equal volume of homogenization buffer and centrifuged. Combined supernatants (homogenates) were centrifuged at 184000g for 50 min at 4 °C. The supernatant was collected as a cytosolic fraction. The pellet was resuspended in homogenization buffer and centrifuged at 184000g for 50 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 20 mM Tris-HCl, pH 8.0, as a particulate. The particulate was then incubated for 20 min on ice in the buffer containg 0.1% Triton X-100 and centrifuged at 184000g for 50 min at 4 °C, and the resulting supernatant was collected as solubilized protein from particulate (membrane proteins). Cytosolic fractions were desalted on a Sephadex G25 resin with 20 mM Tris-Hcl, pH 8.0, at 4 °C. Protein concentrations were determined by BCA assay (Pierce).

Western Analysis Using 2I2 ScFv Antibody

Protein samples were separated on 10% SDS-PA gels and transferred to nitrocellulose membranes (Biorad). The membranes were blocked in 5% nonfat dry milk in PBS for 1 h at RT on a rotary shaker. Blocked membranes were transferred into a solution of primary ScFv antibody and anti-E/HRP secondary antibody diluted 1:20000 in 5% nonfat dry milk/PBS and incubated for 1 h at RT, subsequently washed with DI water, and incubated in 5% nonfat dry milk/PBS/0.1% Tween 20 for 1 h (the solution was changed at least two times during this period). Membranes were washed again in DI water, the ECL substrate solution (Pierce no. 34077) was applied for 5 min at RT, and the excess solution was dried with filter paper. The membranes were placed in the cassette, and the film was exposed for 3–60 s and developed.

Anti-E Sepharose Bead Preparation

NHS-activated Sepharose beads (G. E. Healthcare catalog no. 17-0906-01) were washed with 10 volumes of cold 1 mM HCl, suspended in 2 mL of PBS, and incubated with 1 mg of anti-E tag monoclonal antibody (G. E. Healthcare catalog no. 27-9412-01) at 18 °C for 4 h, followed by the addition of 4 mL of 110 mM Tris-HCl, 10 mM ethanolamine, pH 7.4, and was allowed to react at 18 °C for 3 h. The beads were washed in three cycles alternating 3× column volume of 100 mM Tris-HCl, pH 8.0, and 3× column volume of 100 mM ammonium formate, pH 4.0. Finally, the beads were washed 3× column volume of water and stored in 1.5 mL of 25% ethanol at 4 °C.

2I2 ScFv Immunoprecipitation of Teucrin A Enedial-Adducted BSA

The 2I2 ScFv antibody was affinity-purified from the periplasmic extracts of E. coli using an anti-E tag monoclonal antibody column (G. E. Healthcare catalog no. 17-1362-01). Anti-E tag Sepharose beads (20 µL) were transferred on a mini-spin column (Biorad) and washed 3 × 200 µL of PBS-T. Then, 60 µL of PBS-T was added, followed by 15 µL of 2I2 ScFv (0.23 mg/mL) and 200 µL of teucrin A enedial-adducted BSA or control BSA (2 mg protein/mL). The mixtures were incubated for 1 h at RT. To wash off unbound and nonspecifically bound proteins, the beads were washed 5 × 50 µL of PBS-T, followed by 3 × 50 µL of PBS and and 3 × 50 µL of water. Proteins retained on the beads were sequentially eluted with 1 M sodium chloride (50 µL) and increasing concentrations of acetonitrile (5, 20, and 50%, 50 µL each) and, finally, 50 µL of 50% ACN/1% formic acid. Sodium chloride eluates were desalted using a protein desalting columns (Biorad). Fractions containing formic acid were neutralized with 5 M NaOH. All fractions were then supplemented with 5× Laemmli buffer and heated for 10 min at 95 °C, and 10 µL of each sample was loaded on 12% Tris-Gly gel (Invitrogen). Proteins were separated using SDS Tris-Gly running buffer (Invitrogen) at 140 V for 85 min and visualized by Coomassie Blue staining (Colloidal Blue from Invitrogen). Protein-containing bands were excised and in-gel digested with trypsin. Resulting peptide mixtures were analyzed by LC-MS/MS.

2I2 ScFv Immunoprecipitation of Rat Liver Membrane Fractions (RLM)

Anti-E tag Sepharose beads (30 µL) were transferred on a mini-spin column (Biorad) and washed 3 × 200 µL of PBS/0.1% Tween 20. Then, 80 µL of PBS-T was added, followed by 20 µL of 2I2 ScFv (0.23 mg/mL) and 200 µL of solubilized rat liver membrane fractions (3 mg protein/mL). The mixtures were incubated on a shaker for 2 h at 18 °C, followed by washing with PBS/Tween 20, PBS, and water. The proteins were eluted from the beads using 2× Laemmli buffer and heated at 95 °C for 10 min. The samples were cleaned up on 12% Tris-Gly gel (Invitrogen) using Tris-Gly SDS running buffer before enzymatic digestion and mass spectral analyses.

2I2 ScFv Immunoprecipitation of Rat Liver Cytosolic Fractions (RLC)

Anti-E tag Sepharose beads (30 µL) were transferred on a mini-spin column (Biorad), washed 3 × 200 µL of PBS-T, and resuspended in 30 µL of PBS-T. A random nonspecific ScFv was added (30 µL, 0.33 mg/mL), followed by 200 µL of RLC (3 mg protein/mL). The mixtures were incubated for 1 h at ambient temperature. The supernatant was collected (15 µL was collected for Western blot analysis) and transferred into a vial containing a fresh mix of washed anti-E beads (100 µL) and anti-teucrin A-specific 2I2 antibody (90 µL, 0.23 mg/mL). The mixtures were incubated at ambient temperature for 1 h (or 4 °C overnight), followed by a washing procedure after collecting the supernatant as follows: (i) 100 µL of PBS-T, 15 min; (ii) 100 µL of PBS, 10 min; (iii) 100 µL of water; (iv) 2 × 100 µL of 1 M NaCl, 15 min; (v) 5–50% aqueous acetonitrile gradient, 100 µL fraction/15 min each; and (vi) 100 µL of 50% aqueous acetonitrile/0.1%formic acid. Sodium chloride eluates were desalted using protein desalting columns (Biorad). Fractions containing formic acid were neutralized with 5 M NaOH. All fractions were then supplemented with 5× Laemmli buffer and heated for 10 min at 95 °C, and 10 µL of each sample was loaded on 12% Tris-Gly gel (Invitrogen). Proteins were separated using SDS Tris-Gly running buffer (Invitrogen) at 140 V for 85 min and visualized by Coomassie Blue staining (Colloidal Blue from Invitrogen) or by silver staining (kit from Amersham Biosciences) or transferred onto a nitrocellulose membrane (Biorad) for Western blotting.

In-Gel Digestion and LC-MS/MS Analysis

Protein samples were run either just 1.5 cm into a 10% SDS-PAGE gel (for rat samples) to clean up the samples from residual detergents and salts as previously described (43) or fully into the gel, and specific protein bands were excised. Protein bands were cut out from gels and in-gel digested with trypsin or chymotrypsin as previously described (44). LC-MS analysis of the resulting peptides was performed using a Thermo LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Thermo Surveyor HPLC pump, nanospray source, and Xcalibur 1.4 instrument control. The peptides were separated on a packed capillary tip, 100 µm × 11 cm, with C18 resin (Jupiter C18, 5 μm, 300 Å; Phenomonex, Torrance, CA) using an inline solid phase extraction column that was 100 µm × 6 cm packed with the same C18 resin [using a frit generated from liquid silicate Kasil 1 (45) similar to that previously described (46), except the flow from the HPLC pump, which was split prior to the injection valve]. The flow rate during the solid-phase extraction phase of the gradient was 1 µL/min, and during the separation phase, it was 700 nL/min. Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. A 95 min gradient was performed with a 15 min washing period (100% A for the first 10 min followed by a gradient to 98% A at 15 min) to allow for solid-phase extraction and removal on any residual salts. After the initial washing period, a 60 min gradient was performed where the first 35 min was a slow, linear gradient from 98 to 75% A, followed by a faster gradient to 10% A at 65 min, and an isocratic phase at 10% A to 75 min. MS/MS scans were acquired using an isolation width of 2 m/z, an activation time of 30 ms, an activation Q of 0.250, and 30% normalized collision energy using one microscan and a maximum injection time of 100 for each scan. The mass spectrometer was tuned prior to analysis using the synthetic peptide TpepK (AVAGKAGAR). Typical tune parameters were as follows: spray voltage of between 1.8 kV, a capillary temperature of 150 °C, a capillary voltage of 50 V, and tube lens of 100 V. The MS/MS spectra of the peptides were acquired using data-dependent scanning, in which one full MS spectrum was followed by three MS/MS spectra.

Database Searching and Data Analysis

The “ScanDenser” algorithm read tandem mass spectra stored as centroided peak lists from Thermo RAW files and transcoded them to DTA files. Spectra that contained fewer than 26 peaks or that had less than 2e1 measured TIC did not result in DTAs. DTAs from singly charged spectra were created if 90% of the TIC occurred below the precursor ion; all other spectra were processed as both doubly and triply charged DTAs. Proteins were identified using the TurboSEQUEST version 27 (revision 12) algorithm (Thermo Electron, San Jose, CA) (47) on a high-speed, multiprocessor Linux cluster in the Advanced Computing Center for Research & Education at Vanderbilt University, using the Sequest algorithm using either the rat or the bovine subset of the Uniref100 database. The databases were reversed so that false discovery rates could be determined, and the reversed version of each protein sequence was appended to the forward database for a total of 108994 and 112404 sequences in the rat and bovine databases, respectively. The false discovery rates were calculated as previously described (48). Differential modifications for carbamidomethylation of cysteines and oxidation of methionines as well as teucrin A adducts with a mass shift of 342 on lysines and 342 and 376 on cysteines were allowed with peptide and fragment ion tolerances of 2.5 and 1.0 Da, respectively. Up to 10 missed cleavages and only fully tryptic peptides were allowed. Protein matches were preliminarily filtered using the following criteria: if the charge state of the peptide is 1, the Xcorr is greater than or equal to 1, the RSp is less than or equal to 5, and the Sp is greater than or equal to 350; if the charge state is 2, the Xcorr is greater than or equal to 1.8, the RSp is less than or equal to 5, and the Sp is greater than or equal to 350; and if the charge state is 3, the Xcorr is greater than or equal to 2.5, the RSp is less than or equal to 5, and the Sp is greater than or equal to 350. Once filtered based on these scores, all protein matches that had less than two peptide matches were eliminated from the search.

Network Generation and Functional Analysis Using Ingenuity Pathways Analysis

The networks and functional analyses were generated through the use of Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). A data set containing gene identifiers and corresponding expression values was uploaded into the application. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. These genes, called Focus Genes, were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of these Focus Genes were then algorithmically generated based on their connectivity. The Functional Analysis of a network identified the biological functions and/or diseases that were most significant to the genes in the network. The network genes associated with biological functions and/or diseases in the Ingenuity Pathways Knowledge Base were considered for the analysis. Fischer’s exact test was used to calculate a p value determining the probability that each biological function and/or disease assigned to that network is due to chance alone. Canonical pathways analysis identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the data set. The significance of the association between the data set and the canonical pathway was measured in two ways: (i) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed, and (ii) Fischer’s exact test was used to calculate a p value determining the probability that the association between the genes in the data set and the canonical pathway is explained by chance alone. A network is a graphical representation of the molecular relationships between genes/gene products. Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base. Human, mouse, and rat orthologues of a gene are stored as separate objects in the Ingenuity Pathways Knowledge Base but are represented as a single node in the network. Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes (e.g., P for phosphorylation and T for transcription).

Results

Click to copy section linkSection link copied!

Synthesis of Biotinylated Peptides Containing a Teucrin A Epitope

To generate an anti-teucrin A adduct-specific antibody, a hapten containing an appropriate epitope was designed, synthesized, and structurally characterized. First, teucrin A was oxidized with DMDO affording 1 (Scheme 2), as previously described (38). N-terminal biotinylated peptide AKDVY was reacted with 1 in phosphate buffer, pH 7.4, at 37 °C overnight. The reaction products were isolated by HPLC and characterized by mass spectrometry. The major HPLC peak eluting at 21.8 min exhibited a base peak at m/z 639.0, corresponding to the [M + 2H]2+, and a molecular ion at m/z 1277.0, representing the [M + H]+. The observed masses matched the predicted formation of the anticipated pyrroline-2-one product. Because there are two possible regioisomers of the pyrroline-2-one ring that can be formed in this reaction, specifically three- or four-substituted pyrrolinones, 1H NMR and COSY spectra of the HPLC purified conjugate were measured and examined for the presence of signals characteristic of the two regioisomers (38, 49). A signal observed at 7.3 ppm corresponded to the H4 proton of the three-substituted pyrroline-2-one 2, as described for the conjugate of 1 with NAL (Figure 1) (38). A cross-peak in the COSY spectrum between the signal at 7.3 ppm and a signal at 4.2 ppm, which is consistent with the reported value for the methylene group protons H5 of the three-substituted pyrrolinone ring, confirmed the structural assignment. Two triplet signals at 5.34 and 5.49 ppm were assigned to H12 of the teucrin A moiety. Previously, we reported evidence for an equilibrium between the enedial and the hydroxyenal tautomers in aqueous environments, resulting in epimerization of the H12 proton of 1 and formation of diastereomeric products in the reactions of 1 with NAL or NAC. This is consistent with the observation of two triplets for the H12 proton (Scheme 3) (38).

Figure 1

Figure 1. 1H NMR and COSY spectra (500 MHz, DMSO-d6) of the conjugate of 1 with Btn-AKDVY peptide (2) with the labeled structure showing the positions of the relevant protons.

High Resolution Image
Download MS PowerPoint Slide

Scheme 3

Scheme 3. Proposed Mechanism of Pyrroline-2-one Adduct Formation and Epimerization of C12
High Resolution Image
Download MS PowerPoint Slide
To obtain an ScFv antibody useful for the investigation of proteins adducted with activated teucrin A in vivo, the selection was designed so that the antibody would recognize not only lysine but potential cysteine conjugates as well; thus, its recognition would be independent of adduct structure or peptide sequence. For this purpose, N-terminal biotinylated peptide, ACDVY, was reacted with 1 under the same conditions as described above for the Lys-containing protein. The reaction afforded one major product, which was isolated by HPLC and characterized by mass spectrometry. The m/z of the base peak of the isolated material was 1307.65, corresponding to the [M + Na]+ of the hydrated cyclic Michael adduct 3 and the m/z of the molecular ion [M + H]+ was 1285.56. We previously showed that hydrated adducts of NAC with 1 are unstable to extensive purification (38) so the Btn-ACDVY peptide conjugates were not characterized by NMR.

Selection of a Teucrin A-Specific ScFv Antibody

To obtain a tool to study teucrin A-adducted proteins in vitro and in vivo, a monoclonal ScFv antibody was isolated and characterized from a rodent phage-displayed library (Figure 2). The purified teucrin A enedial-peptide conjugate with biotinylated AKDVY peptide 2 was used as an antigen for two rounds of phage antibody selection. An ELISA was used to detect teucrin A-specific ScFv. Thirty-two ScFv antibodies interacted strongly with teucrin A-adducted Btn-AKDVY peptide conjugate and weakly with unmodified Btn-AKDVY peptide. These ScFvs were further screened against the purified teucrin A-peptide conjugate with the biotinylated cysteine-containing ACDVY peptide, 3, and positive clones were selected based on their ability to recognize both lysine and cysteine conjugates equally well (Figures S1 and S2, Supporting Information). Four ScFv clones (2E18, 2F7, 2I2, and 2G7) were further characterized by screening against synthesized teucrin A enedial-peptide conjugates that contained variable amino acid sequences (RKVDY, Btn-RKDVY, biocytin [Ser25] protein kinase C fragment 19–31, and KKRAARATS amide) to ensure that the ScFv antibodies were teucrin A-specific and not teucrin A-amino acid sequence-specific.

Figure 2

Figure 2. Process of the anti-teucrin A ScFv antibody selection from the rodent phage-displayed library.

High Resolution Image
Download MS PowerPoint Slide

Western Blotting with an Anti-Teucrin A ScFv Antibody Using Teucrin A Enedial-Adducted BSA

Western blotting and immunoprecipitation procedures with the ScFv antibody (designated 2I2) were developed to detect and enrich teucrin A-adducted proteins from complex protein mixtures. Four other teucrin-specific ScFv were also characterized for use in Western blot. For Western blot protocol optimization purposes, bovine serum albumin (BSA) was chosen as a representative protein and reacted with two equivalents of 1 at 37°C overnight in phosphate buffer pH 7.4 to generate a teucrin A-adducted BSA standard. As a control, BSA was incubated under the same conditions in the absence of 1. For Western blot analysis, 50 µg of teucrin A-adducted or control BSA were resolved by SDS polyacrylamide gel electrophoresis using a single-well pre-cast prep gel. Proteins were transferred to nitrocellulose and Western blot analysis was performed using ScFv in E. coli periplasmic cell extracts. E-tagged ScFv bound to teucrin A-adducted BSA on nitrocellulose was visualized using an Anti-E/HRP conjugate, an HRP-chemiluminescent substrate film. E. coli periplasmic extract containing unpurified 2I2 could be diluted 1:20:000 for use in Western blot and exhibited the highest affinity for the adducted BSA and the lowest background of the four clones tested (Figure S3, Supporting Information). The 2I2 ScFv specifically detected protein bands in lanes where adducted BSA was loaded, whereas there was no detectable signal in lanes loaded with control BSA. The 2I2 ScFv detected 2.5 ng of the adducted BSA in Western blots using a film exposure time of ten seconds or less (Figure 3). The immunoreactive high molecular weight bands (>200 kDa) in the Western blot analysis resulted from adduction of protein contaminants in the purchased BSA as shown by Colloidal Blue staining and confirmed by LC-MS/MS (Figure S3 and Table S1, Supporting Information).

Figure 3

Figure 3. Western blot of BSA adducted in vitro with teucrin A enedial using the periplasmic extracts of the selected 2I2 ScFv clone.

High Resolution Image
Download MS PowerPoint Slide
To confirm the modifications caused by 1 on the BSA protein, the adducted sample was subjected to LC-MS/MS analysis. First, the protein sample was purified by SDS polyacrylamide gel electrophoresis and stained with Colloidal Blue. The protein band corresponding to the adducted BSA was excised and in-gel digested with trypsin. LC-MS/MS analysis of the trypsin digest revealed numerous lysines modified by 1. The increases in molecular weight of the modified peptides that were observed corresponded to the formation of the pyrroline-2-one between the lysine residues and 1 as described above for the peptide conjugates. A single cysteine residue (C56) was covalently modified, resulting in the formation of the conjugate 3. The residues that were found adducted in BSA are listed in Table 1. Several lysine-modified peptides were detected numerous times in repeated analyses, possibly due to preferential adduction by the electrophile. The residues detected five or more times were mapped onto the crystal structure of human serum albumin (HSA) (Figure 4) (50). The residues copiously modified were distributed throughout the HSA structure and most of the adducted residues were in solvent accessible regions. The detailed kinetics and reactivity studies were not performed.
Table 1. Residues Adducted in BSA by 1 in Vitroa
residue no.peptides detected*
K374 (351), K568 (545)≥10
K36 (12), K138 (R114), K160 (136), K204 (181), K304 (281)5–7
K28, K100, K412, K420, K455, K463, 4894
K140, K235, K248, K251, K256, K266, K346, K5483
K75, K88, K117, K183, K245, K263, K285, K297, K299, K399, K401, K437, K489, K547, K561<2
C582
a

The asterisk shows the number of times that the peptides with adducted residues were detected in three independent analyses. Numbers in parentheses are the corresponding residues in the crystal stucture of HSA. The complete MS data are available in the Supporting Information (Table S1b and MS/MS figures).

Figure 4

Figure 4. Crystal structure of human serum albumin (PDB: 1AO6 ) with the highlighted residues corresponding to the ones significantly adducted in bovine serum albumin (BSA) by teucrin A enedial 1 in vitro (lysines in red, cysteine in yellow, and L233 in white). The right frame is rotated 90° with respect to the left frame.

High Resolution Image
Download MS PowerPoint Slide

Immunoprecipitation of Teucrin A Enedial-Adducted BSA Using 2I2 ScFv

To ensure that the 2I2 ScFv antibody was capable of enriching the teucrin A-adducted proteins, immunoprecipitation was performed with BSA adducted with 1 and unadducted BSA using the 2I2 ScFv antibody. Sepharose beads covalently cross-linked with anti-E tag IgG antibody, the affinity-purified 2I2 ScFv antibody and adducted or control BSA were incubated as described in Materials and Methods. An optimized procedure for the selective elution of the bound proteins is illustrated in Figure 5. 1M Sodium chloride was found to selectively elute adducted BSA bound to the beads without disrupting the ScFv-anti-E tag bindings. Further washes with increasing concentrations of acetonitrile (from 5 to 50%) and the addition of formic acid (1%) eluted the residual adducted BSA. The final acidic wash also released the ScFv from the beads. Minimal non-specific protein binding was observed in the control BSA immunoprecipitation by Colloidal blue as well as silver staining. In LC-MS/MS analysis, the enrichment of the BSA peptides in the samples treated with 1 over the control in the sodium chloride elution fraction was consistently over fourfold. In addition, nine modified peptides were identified in the sodium chloride elution fraction, all of which were abundantly present in the tryptic digest of the adducted BSA without immunoprecipitation, including the peptide shown in Figure 6. Thus, the enrichment procedure consisting of the selective elution of adducted protein with sodium chloride and gradient elution of bound protein with increasing concentrations of acetonitrile appeared to give satisfactory results and was further used to immunoprecipitate proteins adducted with activated teucrin A in vivo in rat liver. False discovery rates in these experiments were 1% or less.

Figure 5

Figure 5. Flow chart of the immunoprecipitation procedure with the 2I2 ScFv antibody.

High Resolution Image
Download MS PowerPoint Slide

Figure 6

Figure 6. Representative MS/MS spectrum of a BSA peptide modified on the lysine residue by teucrin A enedial.

High Resolution Image
Download MS PowerPoint Slide

Identification of Teucrin A Protein Adducts in Vivo by Western Blot Analysis

In order to generate a toxic response in vivo, we needed to determine an optimal dose of teucrin A in rodents. Previous studies found that the hydroalcoholic extract of T. chamaedrys was generally well-tolerated by male Sprague-Dawley rats up to 13 weeks at 56 mg/kg/day administered orally (teucrin A equivalent of 0.4 mg/kg/day) (23). Chronic exposure of rats to approximately 2 mg/kg/day of teucrin A over 13 weeks lead to significant changes in the liver, mainly hepatocellular hypertrophy and steatosis (23). A higher chronic dose of 10 mg/kg/day of teucrin A was poorly tolerated (23). Subchronic four week dosing of 10 mg/kg/day caused liver modifications of degenerative origin and necrosis of isolated rat hepatocytes (23). A single intragastric administration of a germander tea lyophilisate (1250 mg/kg) or the furan neoclerodane diterpenoid fraction (125 mg/kg) produced similar mid-zonal liver cell necrosis at 24 hours in mice (51). In another in vivo study, a single oral administration of an ethanolic extract of germander (2000–4000 mg/kg) to mice caused increases in plasma alanine aminotransferase (ALT) activity and mid-zonal hepatic necrosis 24 hours after dosing (25). A single administration of an acetone extract (500 mg/kg) of the herb caused similar effects that were attenuated by pre-treatment with the cytochrome P450 inhibitor piperonyl butoxide (PBO) (26). Teucrin A caused mid-zonal hepatic necrosis at 150 mg/kg, comparable to that of the 500 mg/kg dose of the acetone extract (26).
For the purpose of our study, male Sprague Dawley rats were orally administered by gavage a single dose of teucrin A (100 mg/kg) and sacrificed after 24 hours. ALT activity in plasma was monitored over 24 hours as a marker of liver toxicity. The dose induced a threefold increase in plasma ALT within 6 hours and by 24 hours the plasma ALT activity was elevated fivefold over control, indicating significant ALT leakage from the injured liver (Figure 7A). The livers were excised and liver homogenates were separated into cytosolic (RLC) and membrane (RLM) fractions by differential centrifugation, such that the membrane fraction contained endoplasmic reticulum, peroxisomal and mitochondrial membranes. RLC and RLM proteins were then separated by SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by Western blotting. To visualize the putative covalent adducts that should form in vivo upon metabolism of teucrin A in the liver, we used the periplasmic extracts of the 2I2 ScFv antibody as described above. The 2I2 ScFv was able to selectively detect very low concentrations of teucrin-adducted material in RLC and RLM samples. Immunoreactivity was not observed with untreated RLC and RLM samples. In the treated samples, both cytosolic and membrane proteins were extensively modified by in vivo activated teucrin A (Figure 7B).

Figure 7

Figure 7. (A) Time course of the plasma alanine aminotrasferase (ALT) activity in Spague–Dawley rats (n = 3) after oral administration of 100 mg/kg of teucrin A. (B) Western blot of the rat liver proteins adducted in vivo by teucrin A metabolite(s) detected with the periplasmic extracts of the selected 2I2 ScFv clone.

High Resolution Image
Download MS PowerPoint Slide

Immunoprecipitation and LC-MS/MS Analysis of Teucrin A-Adducted Proteins from in Vivo Samples

Using the 2I2 ScFv, immunoprecipitation of the teucrin A-adducted proteins from the rat liver homogenates was performed as described for BSA. In-gel enzymatic digestion of the resulting adduct-enriched protein mixtures was followed by LC-MS/MS analysis. Table 2 lists 46 proteins that were consistently present in the treated samples from three different animals in duplicate immunoprecipitations and mass spectral analysis. There were 39 unique proteins, which were not detected in the immunoprecipitated samples from the control homogenates. The additional seven proteins (designated with a in Table 2) were detected in the control samples, but the enrichment in the treated samples was consistently over fourfold higher, as determined from the increase in the number of unique and total peptides identified per protein, along with an increase in the sequence coverage. Thus, the respective proteins were considered selectively enriched due to modification by the teucrin A metabolite(s). False discovery rates in these experiments were less than 0.5%.
Table 2. Proteins Covalently Modified by Teucrin A in Vivo in the Rat
locationproteinUniprot/Swiss-ProtgeneMW% sequence coverage (min–max)b no. of unique peptides (min–max)
mitochondriaATP synthase α-chain, mit.p.P15999ATP5A159753.66.1–12.73–7
ATP synthase β-chain, mit.p.P10719ATP5B56353.616.4–216–7
acyl-CoA dehydrogenase, long-chain specific, mit.p.P15650ACADL47872.913.3–17.94–6
acyl-CoA dehydrogenase, very long-chain specific, mit.p.P45953ACADVL70749.38.5–17.35–8
electron transfer flavoprotein α-subunit, mit.p.P13803ETFA34976.421.9–28.85–7
electron transfer flavoprotein, β-polypeptideQ68FU3ETFB27687.423.1–27.15–5
hydroxymethylglutaryl-CoA synthase, mit.p.P22791HMGCS256911.913.6–20.76–11
methylmalonate-semialdehyde dehydrogenase, mit.p.Q02253ALDH6A157807.615.5–26.27–12
trifunctional enzyme α subunit, mit.p.Q64428HADHA82512.912.6–16.17–10
60 kDa heat shock protein, mit.p. (Hsp60)P63039HSPD160970.55.8–83–4
glutamate dehydrogenase 1, mit.p.P10860GLUD161427.99.7–12.94–5
prohibitin-2 (B-cell receptor-associated protein)Q5XIH7PHB233312.410–13.43–4
3-ketoacyl-CoA thiolase,a mit.p.P13437ACAA241870.927.5–39.38–10
aldehyde dehydrogenase,a mit.p.P11884ALDH256488.416.4–21.48–10
carbamoyl-phosphate synthase (ammonia),a mit.p.P07756CPS1164579.925.9–30.131–36
ER78 kDa glucose-regulated protein precursor (GRP78, Bip)P06761HSPA57234714.2–23.27–11
NADH-cytochrome b5 reductase (Diaphorase)P20070CYB5R334255.717.5–23.54–5
protein disulfide-isomerase A6 precursor cusQ63081PDIA648760.217.5–25.65–7
retinol dehydrogenase 2P50170RDHS35597.124–31.97–11
retinol dehydrogenase 3P50169N/A35662.46.3–15.12–4
cytochrome P450 2A1P11711CYP2A1256009.17.1–143–6
cytochrome P450 2A2P15149CYP2A256345.56.3–12.62–5
cytochrome P450 2C7P05179CYP2C3856187.17.1–12.93–4
cytochrome P450 2D3P12938CYP2D1356641.811.6–14.65–8
dimethylaniline monooxygenase (N-oxide-forming) 3Q9EQ76FMO359960.310.2–12.25–6
transitional endoplasmic reticulum ATPase (TER ATPase)P46462VCP895344.2–7.52–4
UDP-glucuronosyltransferase 1-1 precursor, microsomalQ64550UGT1A159662.85.4–11.43–5
cytochrome P450 2D1aP10633CYP2D957175.424–33.512–19
epoxide hydrolase 1aP07687EPHX152581.622.6–27.310–13
fatty aldehyde dehydrogenaseaP30839ALDH3A254081.614.5–20.95–9
peroxisomeacyl-coenzyme A dehydrogenase, short chainQ6IMX3N/A44967.66–8.52–3
bile acid CoA:amino acid N-acyltransferaseQ63276BAAT46464.76.9–14.83–5
catalaseP04762CAT596266.3–12.73–5
long-chain fatty acid-CoA ligase 1aP18163ACSL178178.730.9–33.919–20
cytoplasm4-trimethylaminobutyraldehyde dehydrogenaseQ9JLJ3ALDH9A153652.711.3–19.64–8
fructose-bisphosphate aldolase BP00884ALDOB3952516.8–195–6
argininosuccinate synthaseP09034ASS46496.313.3–28.26–8
cystathionine γ-lyaseP18757CTH43605.38.8–15.83–5
glyceraldehyde-3-phosphate dehydrogenaseP04797GAPDH35704.917.5–24.44–5
heat shock 90 kDa protein 1-β (HSP84)P34058HSP90AB183341.36.8–15.54–9
10-formyltetrahydrofolate dehydrogenaseP28037ALDH1L199126.53.7–6.82–4
alcohol dehydrogenase 1P06757ADH1C39514.17.2–9.93–4
betaine-homocysteine S-methyltransferaseO09171BHMT44976.47.1–12.53–5
glutathione S-transferase μ1 (GSTM1-1)P04905GSTM525894.96.42
excretedβ2 globinP11517HBB15847.234.9–35.64–5
serum paraoxonase/lactonase 3Q68FP2PON339458.210.73
a

Tryptic peptides detected in samples from control animals. Proteins from teucrin A-treated samples were enriched over 4-fold based on peptide count and sequence coverage.

b

Represents the range of minimal to maximal coverage/unique peptide count between three samples obtained from three different animals. Complete MS data are available in the Supporting Information (Table S3a,b).

Among the broad range of targets affected most by teucrin A treatment in rat liver were mitochondrial and endoplasmic reticulum (ER) proteins. Damaged mitochondrial proteins included enzymes involved in lipid metabolism, cell respiration and energy production, as well as the essential mitochondrial chaperone Hsp60. Several peroxisomal proteins involved in fatty acid metabolism were identified, as well as catalase, the peroxisomal enzyme responsible for the degradation of hydrogen peroxide. ER-resident proteins adducted by teucrin A represented a number of proteins involved in drug metabolism and clearance, including several cytochrome P450 enzymes, UDP-glucuronosyl transferase and epoxide hydrolase. Additionally, ER proteins with chaperone and maintenance functions were present. Among the cytosolic proteins identified were several proteins involved in amino acid and glucose metabolism, along with glutathione S-transferase M1 and the cytosolic chaperone, Hsp90.

Functional Analysis of the Adducted Proteins Using Ingenuity Pathway Analysis

Two cellular networks were generated through the use of Ingenuity Pathways Analysis (Figure 8A and 8B). The SwissProt gene identifiers of the modified proteins in table 1, called focus genes, were uploaded into the application and overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. The networks were then generated algorithmically based on the connectivity of the focused genes (Table 3). Furthermore, Functional Analysis identified the biological functions and diseases that were most significant to the data set (Table 4).

Figure 8

Figure 8. Networks and canonical pathways generated by Ingenuity Pathways Analysis for the targets of teucrin A, called focused genes. (A) The direct interactions between the focused genes (gray) and the transcriptional regulators in network 1. (B) The direct interactions between the focused genes (gray) and the transcriptional regulators in network 2. (C) The cellular pathways, functions, and diseases associated with the focused genes. Abbreviations: A, activation; E, expression; T, transcription; PD, protein–DNA interaction; PP, protein–protein interaction. Network 1: BRCA1, breast cancer 1, early onset; DGKA, diacylglycerol kinase α; PPARA, peroxisome proliferative activated receptor α; PPARG, peroxisome proliferative activated receptor γ; SP1, Sp1 transcription factor; SRC, v-src sarcoma (Schmidt–Ruppin A-2) viral oncogene homologue; SREBF2, sterol regulatory element binding transcription factor 2; TP53, tumor protein 53. Network 2: ABCB11, ATP-binding cassette, subfamily B (MDR/TAP), member 11; AKR1C4, aldo-keto reductase family 1, member C4; CYP2C9, cytochrome P450, family 2, subfamily C, polypeptide 9; ESR1, estrogen receptor 1; FOS, v-fos FBJ murine osteosarcoma viral oncogene homologue; GATA4, GATA binding protein 4; HNF4A, hepatocyte nuclear factor 4α; RXRA, retinoid X receptor α; TCF1, transcription factor 1, hepatic (hepatic nuclear factor HNF1); UGT2B1, UDP glucuronosyltransferase 2 family, polypepetide B1; UGT2B7, UDP glucuronosyltransferase 2 family, polypeptode B7; XBP1, X-box binding protein 1.

High Resolution Image
Download MS PowerPoint Slide
Table 3. Genes Directly Associated with the Protein Targets of Teucrin A Metabolite(s) (Bold) Linked to the Affected Cellular Functions by Ingenuity Pathway Analysis
IDgenesscorefocus genestop functions
1ACAA2, ACADL, ACADVL, ACSL1, ALDH3A2, ALDH9A1, ANXA8, ASS, BRCA1, CAT, CTSF (includes EG:8722), CYB5R3, CYP2D9, CYP4A14, DDIT4, DGKA, FXYD3, GSTM5, HBB (includes EG:3043), HSP90AB1, LHX1, LPIN1, LY6D, MGLL, PERP (includes EG:64065), PEX11A, PHLDA1, PPARA, PPARG, PTPRV, SP1, SRC, SREBF2, TP53, VCP2614gene expression, lipid metabolism, small molecule biochemistry
2ABCB11, ADH1C (includes EG:126), AKR1C4, ALDH2, ALDOB, BAAT, CTH, CTSL2, CYP2C9, EPHX1, ESR1, FOS, GATA4, HADHA, HMGCS2, HNF4A, HSPA5, MPG, NCOA4 (includes EG:8031), NRIP2, PCBD1, PDIA6, PHB2, PKLR, RBP1, RXRA, SLC10A1, SLC10A2, SNCG, TCF1,UGT1A1 UGT1A9 (includes EG:54600), UGT2B1, UGT2B7, XBP12112lipid metabolism, small molecule biochemistry, drug metabolism
3AR, ATP5A1, ATP5A2, ATP5B, ATP5C1, ATP5C2, ATP5D, ATP5E, ATP5J, BAX, CA2, CEBPA, CPS1, DHFR, FASN, GAPDH, GP9, HDAC1 (includes EG:3065), HIST2H2BE, HSPD1, MAPKAPK2, MYCN, NFATC1, NFYB, NR3C1, PPARG, PTPN3 (includes EG:5774), RAF1, RIN1, SNCA, TBP, TUBB2A, XRCC5, YWHAG, YWHAZ75cancer, gene expression, cell cycle
Table 4. Cellular Functions Associated with the Protein Targets of Teucrin A Metabolite(s) Identified by Ingenuity Pathway Analysis
functionsignificanceagenes
amino acid metabolism2.83 × 10–4–1.76 × 10–2ASS, GLUD1, BAAT, BHMT, CAT
carbohydrate metabolism8.82 × 10–3–8.82 × 10–3UGT1A1, GAPDH
cell death3.16 × 10–3–4.76 × 10–2ALDH2, HSPD1, CYB5R3, CAT, HSP90AB1, HADHA, HSPA5, GAPDH
cell signaling6.36 × 10–3–8.82 × 10–3HSPD1, ASS, CAT
cellular assembly and organization4.42 × 10–3–4.33 × 10–2ALDOB, VCP, CYB5R3, HSPA5, GAPDH
cellular function and maintenance4.75 × 10–4–4.75 × 10–4VCP, HSP90AB1, HSPA5
drug metabolism3.11 × 10–3–4.76 × 10–2CYP2C38, FMO3, CYB5R3, CAT, ADH1C (includes EG:126), RDHS, HSP90AB1, CYP2A12, UGT1A1
endocrine system development and function3.11 × 10–3–4.33 × 10–2CYB5R3, HSP90AB1, UGT1A1
energy production8.82 × 10–3–3.05 × 10–2HSPD1, ACADVL, ATP5B
hepatic system disease4.42 × 10–3–8.82 × 10–3BAAT, CAT, HADHA, UGT1A1
lipid metabolism2.06 × 10–4–4.76 × 10–2BAAT, ACADVL, HMGCS2, CAT, CYB5R3, ADH1C (includes EG:126), RDHS, HSP90AB1, ACADL, HADHA, ACSL1, UGT1A1
metabolic disease4.38 × 10–3–4.42 × 10–3ASS, GLUD1, ACADVL, CAT, HADHA, CTH, UGT1A1
molecular transport4.42 × 10–3–2.62 × 10–2HBB (includes EG:3043), ADH1C (includes EG:126), ACSL1, HADHA, ACADL, UGT1A1, GAPDH
protein folding3.05 × 10–2–3.05 × 10–2HSP90AB1, PDIA6
small molecule biochemistry2.06 × 10–4–4.76 × 10–2HSPD1, ALDH2, HBB (includes EG:3043), HMGCS2, BHMT, FMO3, CYB5R3, ADH1C (includes EG:126), HSP90AB1, UGT1A1, ASS, ALDH9A1, GLUD1, BAAT, ACADVL, CAT, RDHS, ACSL1, ACADL, HADHA, ATP5B, GAPDH
a

Fischer’s exact test was used to calculate a p value, determining the probability that the association between the genes in the data set and the canonical pathway is explained by the chance alone.

In Network 1, 14 focused genes were associated with 21 additional genes by direct interactions, which were primarily linked with gene expression, lipid metabolism, and small molecule biochemistry. Figure 8A shows the relations between these focused genes and some important transcriptional regulators involved in the functions associated with Network 1, including peroxisome proliferator-activated receptors (PPAR-α and PPAR-γ), p53 oncogene, transcription factor SP1, sterol regulatory element-binding factor 2 (SREBF2), and tumor suppressor BRCA1. Twelve focused genes were directly linked with 23 genes in the knowledge database, together composing Network 2, primarily associated with lipid metabolism, small molecule biochemistry and drug metabolism. Figure 8B depicts the interactions of these focused genes with transcriptional regulators within this network, such as retinoid X receptor α (RXRA), estrogen receptor (ESR1), hepatocyte nuclear factor 4 α (HNF4α), and transcription factors TCF1, GATA4 and FOS. Network 3 was associated with only five focused genes, which did not achieve statistical significance (< 10). The focused genes involved in Network 3 are mitochondrial proteins Hsp60, carbamoylphosphate synthase, ATP-binding proteins α and β, and cytosolic glyceraldehyde-3-phosphate dehydrogenase. Due to the low score of Network 3, the assignment of focused genes into this network may be coincidental. Table 4 groups the focus genes based on the cellular function or a disease that they are involved in, which is illustrated in figure 8C. The most significant cellular functions are lipid metabolism, small molecule biochemistry, amino acid metabolism, and cellular function and maintenance, followed by drug metabolism and cell death. Among diseases linked to the analyzed focused genes are metabolic, hepatic system and inflammatory disease. Finally, the targets of teucrin A are associated with cellular assembly and organization, molecular transport, cell signaling, and energy production.

Discussion

Click to copy section linkSection link copied!
The identification of protein targets of reactive metabolites of xenobiotics, as well as endogenous reactive products, is becoming the focus of chemical toxicologists in an effort to elucidate the importance of protein adduction in the development of human disease and injury (52). Covalent protein adduction correlates with the toxicity of many drugs and xenobiotics, including furan-containing compounds (53-55). Furans are activated by cytochrome P450 enzymes into reactive enedial derivatives that are powerful electrophiles, capable of reacting with cellular nucleophiles, including DNA and proteins (29, 36, 56-62). Reaction products of several activated furans (such as furan itself and ipomeanol) with model amino acids have been characterized, but the proteins adducted by their toxic metabolites generated in vivo and potentially involved in their toxicity remain unidentified (32, 49). We previously reported the characterization of the reaction products of 1, the presumed metabolite of the furano-diterpenoid teucrin A, with NAC and NAL methyl ester, as well as with lysine-containing peptides (38). Here, we present identification of the proteins adducted by activated teucrin A in vivo in rat liver using a novel approach for the enrichment of teucrin A-adducted proteins that employs an ScFv antibody selected from a rodent phage display library.
Phage display libraries are used for a variety of purposes, ranging from mapping protein-protein interactions, identification of specific binding reagents for receptors and enzymes, determination of enzyme-substrate specificity, and the creation of vaccines (38, 63-65). Antibody phage display is perhaps the most common application for the identification of high-affinity specific binding reagents used in the development of human therapeutics and diagnostics, and cell- or tissue-specific markers (65-68). The advantage of the phage-displayed antibody library is that the displayed protein is physically linked to the genetic information inside the carrier phage and thus the initial antibody gene inventory can be diversified by recombination, random mutation or site-directed mutagenesis to optimize binding affinity. Consequently, specific antibodies against virtually any target epitope can be isolated very rapidly in vitro through phage antibody selection on antigen. This intrinsic quality of phage display technology offers an enormous potential for its use in the identification of protein targets of reactive electrophiles. The ability to synthesize and purify peptide conjugates of activated chemicals provides a means for the generation of structurally characterized epitopes for ScFv antibody selection. Despite the significant potential and versatility of this methodology and its existence for over 20 years, a rather small number of reports describe its application for the detection of specific protein adducts and literature on the use of ScFv antibodies for immunoprecipitation of adducted proteins is unavailable. An ScFv antibody against synthetic peptide conjugates of isoketals, the reactive rearrangement products of F2-isoprostanes, was generated and used in tissue samples to visualize the localization of isoketal adducts produced in vivo (39). In the present study, we synthesized conjugates of enedial derivative of teucrin A with lysine- and cysteine-containing biotinylated peptides, that served as epitopes for the selection of an ScFv antibody that was essentially independent of the peptide sequence and capable of specifically recognizing teucrin A-modified peptides.
To further optimize the ScFv antibody selected in the ELISA for the detection of teucrin A-adducted proteins, we prepared teucrin A enedial 1-adducted BSA. The adducted BSA was analyzed by LC-MS/MS to determine the modifications. Two equivalents of 1 resulted in the adduction of numerous lysines and one cysteine residue, as listed in Table 1. On the basis of the mass shifts, the conjugates corresponded to the structures 2 and 3 in Scheme 2. The residues from the significantly modified peptides are highlighted in the structure of human serum albumin (HSA) in Figure 4 (50). It appears that 1 reacts with solvent accessible lysine residues without a considerable selectivity, although a rigorous kinetic analysis was not performed. It is interesting to note that K233 (highlighted in white in Figure 4) is the lysine residue in HSA that is adducted by HNE; this residue does not appear to be a preferential site of modification by 1 (69). K233 resides at the end of the fatty acid-binding groove of HSA, which has been proposed to orient the lipid-derived nonpolar HNE molecule and facilitate attack by K233. The diterpenoid structure of teucrin A is polar and is not expected to bind in this hydrophobic region. In addition, 1 appears to be more reactive than HNE (A.D., unpublished observation), and it is likely scavenged by the available nucleophiles before any noncovalent interactions occur. The modified C34, the only free cysteine residue in both HSA and BSA, is a target of many drugs and endogenous cysteine-reactive molecules, as well as oxidative and nitrosative modifications (70-75).
The selected ScFv antibody recognized BSA adducted with 1 in the Western blot analysis as shown in Figure 3. The anti-teucrin A ScFv antibody was then used to enrich teucrin A-adducted proteins generated in vivo in rat liver. The list of detected proteins along with their cellular localization is provided in Table 2. Numerous ER-associated proteins were detected in the analysis, among them enzymes involved in xenobiotic metabolism. In particular, the adduction of microsomal epoxide hydrolase (mEH) can be correlated to the interesting observation that autoantibodies against mEH were present in humans who developed hepatitis after exposure to germander preparations (76, 77). The formation of autoantibodies supports the idea of covalent adducts playing a role in the development of an immune response against the adducted protein. The sensitivity of mEH may also indicate the involvement of a putative epoxide intermediate as the modifying agent (Scheme 1), although there may be other reasons for the generation of autoantibodies unrelated to modification by germander-derived compounds (78, 79). Moreover, among ER-associated proteins detected in our analysis were several cytochrome P450 enzymes (CYP 2A1, 2A2, 2C7, 2D1, and 2D3) and UDP-glucuronosyl transferase 1-1. Interestingly, CYPs as well as UDP-glucuronosyl transferases are targets of autoantibodies in drug-induced hepatitis, further corroborating the possible involvement of the drug-modified proteins in the development of an autoimmune response (80-82). Prolonged exposure of the immune system to the peptides generated from degraded modified proteins can lead to the development of immune defense. Besides drug-induced hepatitis, alcohol-induced liver disease also has a strong immune component due to the liver protein damage caused by reactive products of lipid peroxidation arising from the alcohol-initiated oxidative stress (3, 83, 84).
Even though teucrin A is activated by CYP 3A in rats, enzymes from this family were not consistently detected in our analysis. This is potentially due to low amounts of these enzymes expressed in rat liver (85-88). Unlike in humans, CYP3As are not the major P450 enzymes in rat liver, and to obtain significant expression of these enzymes, induction by phenobarbital or dexamethasone is typically used. However, this was not performed in our experiment. It is also possible that other CYPs are capable of activating teucrin A into electrophilic species, although preliminary in vitro experiments in our laboratory showed complete inhibition of teucrin A metabolism by troleandomycin, a specific CYP3A4 inhibitor, in the presence of other major human CYPs (1A2, 2C8, 2C9, 2C19, 2D6, and 2E1). Interestingly, CYP3A4 was not covalently modified by activated teucrin A in vitro, as determined by Western blot with anti-teucrin A ScFv antibody. Instead, a band with a higher molecular weight showed a signal in the blot (Figure S5, Supporting Information).
Among other ER-associated proteins that were found to be modified by activated teucrin A were proteins with chaperone and maintenance functions, such as GRP78 (BiP), PDI A6, and transitional ER ATPase. GRP78 functions primarily as a chaperone and is an important signaling molecule during reaction to stress (89-91). It plays a major role in the initiation of the ER stress response upon accumulation of damaged and unfolded proteins, and it also activates the nutrient deprivation response, in both cases promoting cell survival (5, 92). Recently, GRP78 has been identified as a specific proteolytic target of AB5 subtilase cytotoxin, a member of the AB5 family of bacterial toxins such as Schiga, cholera, and pertusis (93). The A subunit of AB5 subtilase is a serine protease that cleaves GRP78 at a single site, resulting in its selective inactivation, thereby triggering death in the target cells. PDIs participate in the correct protein folding of newly synthesized proteins through the maintenance of proper disulfide bridges (94-97). GRP78 is among the genes that are significantly up-regulated in response to subtoxic doses of 4-HNE, a lipid peroxidation-derived α,β-unsaturated aldehyde that covalently modifies proteins during oxidative stress (98). Additionally, 4-HNE has been shown to covalently modify PDI A6 in vivo and inhibit the enzyme in vitro by modification of a catalytic cysteine residue (99). GRP78 and several protein disulfide isomerases, including PDI A6, are also targets of activated BB in vivo (12, 13). Transitional ER ATPase (or valosin-containing protein) is involved in the ER, Golgi apparatus, and nuclear envelope membrane fusion, assembly and maintenance, protein transport, and cell cycle regulation (100-104). The down-regulation of this protein results in ER membrane swelling. It has numerous chaperone activities, and it is required in ubiquitin-proteasome protein degradation (105, 106).
Teucrin A activation resulted in extensive damage of mitochondrial proteins. Several proteins involved in respiration were adducted, including α- and β-chains of ATP synthase, α- and β-subunits of electron-transfer flavoprotein, and trifunctional enzyme, α-subunit. In addition, an essential mitochondrial chaperone, Hsp60, was modified by the teucrin A metabolite(s). A number of enzymes involved in the biosynthesis of fatty acids and small molecule precursors, such as long-chain and very long-chain Acyl-CoA dehydrogenases and carbamoyl-phosphate synthase, respectively, were also adducted. Together, this widespread protein damage may lead to mitochondrial dysfunction. Disruption of mitochondrial function can lead to pore formation and release of cytochrome c, resulting in apoptosis. The loss of membrane potential can cause the release of the contents into the cytosol, leading to necrosis. Analogous mechanisms operate in the toxicity of APAP, whose reactive electrophilic metabolite N-acetyl-para-benzoquinone imine (NAPQI) adducts preferentially mitochondrial proteins involved in respiration (107). Among other NAPQI mitochondrial targets found in this study were aldehyde dehydrogenase and glutamate dehydrogenase. Mitochondrial aldehyde dehydrogenase is also covalently modified by 4-HNE and 4-oxononenal (4-ONE), as well as its oxidation product, 4-oxononenoic acid (4-ONEA) (108). 4-HNE is a reversible inhibitor of the enzyme that does not modify the active site cysteine residue (Cys302) up to 500 μM concentrations. On the other hand, the more potent irreversible inhibitors 4-ONE and 4-ONEA inhibit the enzyme and covalently bind the active site cysteine at a concentration of 50 µM.
The most abundant protein targets of activated teucrin A in cytosol were enzymes involved in amino acid metabolism, such as argininosuccinate synthase, cystathionine γ-lyase, and betaine-homocysteine S-methyltransferase. Two important enzymes involved in the biotransformations of sugars, fructose-biphosphate aldolase B and glyceraldehyde-3-phosphate dehydrogenase, were adducted in our study. Glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase is also the target of 4-HNE and NAPQI (109, 110). 4-HNE inactivates the enzyme in vitro via covalent modification of a surface cysteine and/or histidine residues, whereas NAPQI inactivates it presumably by arylation of Cys-149 in the active site of the enzyme. An important enzyme in purine biosynthesis, 10-formyltetrahydrofolate dehydrogenase (10-FDH), is adducted by teucrin A metabolites, as well as NAPQI (111). Covalent modification of 10-FDH can lead to its faster degradation and an up-regulation of new protein synthesis. Ectopic expression of 10-FDH leads to G1 cell cycle arrest and triggers apoptosis through activation of p53 in cancer cells (112).
Among cytosolic chaperones, heat shock protein 90 (Hsp90) was the target of activated teucrin A. Heat shock proteins, such as Hsp90 and Hsp70, bind pro-apoptotic signaling kinases, such as JNK1, and prevent their activation (5). Additionally, Hsp90 and Hsp70 bind heat shock factor 1 (HSF1). Covalent damage to these chaperones by electrophiles generated in cells leads to dissociation with their respective binding partners, thus activating their downstream cascades. The release of HSF1 activates the expression of cytoprotective genes, such as chaperones Hsp70 and Hsp40, to boost cellular protection against stress. Hsp90 is also the target of 4-HNE in alcoholic liver disease (4). Another cytosolic enzyme essential in the response to electrophilic stress that was adducted by the teucrin A-derived reactive metabolite(s) was glutathione S-transferase M1. Aside from its glutathione-conjugating activity, GST M1 regulates certain pro-apoptotic kinases, such ASK1 and MEKK1, via their sequestration into protein complexes (113, 114). Modification of critical nucleophilic residues on GST M1 resulting in conformational change is thought to be the mechanism for release of its binding partners, leading to activation of apoptosis (115). GSTs are targets for other electrophiles as well. Cysteine 86 of rat GST M1 and to a lesser extent GST M2 is covalently modified in vivo by acrylonitrile, an industrial vinyl monomer that is an acute toxicant and animal carcinogen (116). Rat GSTs of class M contain three cysteine residues, C86, C114, and C173, none of which is essential for its catalytic activity as shown by site-directed mutagenesis (117, 118). C86 of GST M1 is a preferential site of covalent modification by acrylonitrile because of its lowered pKa that results from interaction with His 84; this is unique for rat GST M1. NAPQI covalently modifies GST P1 (119). The site of modification was not determined. The most reactive cysteine residue in GST P1 is C47, which has an extremely low pKa due to its interaction with K54 (120). Metabolites of BB covalently modify GST A1 and GST A2 at Cys 111 (121). 4-HNE covalently binds to and inhibits GSTs in vitro with the order of reactivity and inhibition potency in the presence of GSH being P > A > M, which is changed upon dialysis of GSTs against S-hexyl-GSH to A > M > P; this order parallels the number of cysteine residues within the sequence (122). The primary GST responsible for GSH conjugation of 4-HNE is GSTA4 (123, 124).
Several peroxisomal enzymes also were targets of teucrin A in our study. Among them was catalase, which protects cells from peroxisome-derived hydrogen peroxide (125, 126). Covalent modification of this enzyme could lead to its inhibition and an increase in the production of reactive oxygen species, promoting cellular damage. Other peroxisomal targets of activated teucrin A were short-chain acyl-CoA dehydrogenase, bile acid CoA:amino acid N-acyltransferase, and long-chain fatty-acid-CoA ligase, all of which are involved in fatty acid biosynthesis. The accumulation of fatty acid due to adduction of fatty acid-metabolizing peroxisomal and mitochondrial proteins can lead to activation of peroxisome proliferator-activating receptor α (PPARα) and peroxisome proliferation, promoting oxidative stress (127, 128).
Our results reveal that activation of teucrin A in vivo results in the covalent modification of numerous proteins in the liver. However, despite enrichment of modified proteins using a specific ScFv antibody, we were not able to demonstrate the presence of teucrin A-modified peptides in the mass spectral experiments. Despite the progress in mass spectrometry methods, identification of modified peptides––the ultimate proof of the modification––remains a challenge, especially in complex in vivo samples. This can be attributed to the dilution effect of the complex peptide mixtures and multiple resultant peptide conjugates, the low level of total covalent binding, and the potential changes in the chromatographic behavior of the modified peptides. Previously reported sites of covalent modification, such as Cys 111 in GST A1 and GST A2 by the benzoquinone metabolite of BB, was possible only after glutathione affinity purification of the GTSs and their chromatographic separation, followed by HPLC linear ion-trap quadrupole Fourier transform mass spectrometry to search for predicted modified tryptic peptides (121). Similarly, Cys 86 modified by acrylonitrile in GST M1 was identified after rigorous GST affinity purification and HPLC separation with the help of radiolabeling (116).
Together with the results of the current study, it is obvious that diverse reactive intermediates covalently bind similar cellular targets, especially abundant proteins such as chaperones, PDIs, and GSTs. Additionally, the amount of the adduction by agents such as BB or APAP typically does not exceed one modification per 10 protein molecules (121). Therefore, the in vivo covalent modification of a protein does not necessarily mean a complete loss of its critical function. However, some reactive metabolites exhibit preferential binding to defined subsets of proteins, which can result in the disruption of the particular cellular function that can have overall detrimental downstream consequences. APAP, in a mouse model of drug-induced liver disease, disrupts mitochondrial function and causes the loss of mitochondrial potential, resulting in the release of cytochrome c and the initiation of apoptosis in hepatocytes. This correlates well with the ability of NAPQI to preferentially bind to mitochondrial proteins involved in respiration. The structural analogue of APAP, 3′-hydroxyacetanilid (AMAP), is also metabolized by P450s into the electrophilic species, 2-acetamido-para-benzoquinone. While the covalent binding of NAPQI to proteins correlates with the liver toxicity observed after APAP overdose, AMAP is not toxic despite the covalent binding of its metabolite to proteins (129). The metabolite of AMAP preferentially modifies cytosolic proteins in hepatocytes. Although the functional significance of these observations is uncertain, the differences in the toxicity profiles of the two compounds and the subcellular targets of the two quinones are evident.
The importance of characterizing the complete spectrum of modified proteins by different reactive intermediates is critical to determining the mechanisms of toxicity induced by protein damage. Additionally, it will be important to assess the enzymatic activity of the protein targets to determine the relevance of the covalent binding in toxicity. A more complete understanding of these processes could be achieved with the knowledge of the proteome changes following the exposure to the reactive intermediates. We are currently investigating the effects of the toxic dose of teucrin A on individual protein levels in rat liver. Knowledge databases, such as Ingenuity Pathway Analysis used in this study, will be an essential tool for finding the linkages between the protein targets and the proteome and gene expression changes induced by reactive intermediates and for understanding the cellular pathways responsible for their action.
Our current results illustrate that antibody phage display technology is a potentially powerful approach for studying protein adduction by miscellaneous compounds. Antibody selection is accomplished by rapid panning methodologies that employ rigorously identified peptide adduct antigens. No in vivo immunizations are required, and the stability and thereby identity of the antigen are unequivocally established. Multiple strategies are available to optimize the affinity of the selected antibody for the antigen. Using an anti-teucrin A-specific ScFv, we showed that teucrin A covalently modifies proteins following activation in the rat liver in vivo. Immunoenrichment of the adducted proteins with the ScFv antibody enabled us to identify a broad range of protein targets of the teucrin A metabolite(s) in vivo in rat liver. We were able to show that mitochondrial and ER-associated proteins are the major targets of teucrin A, along with enzymes involved in cell maintenance and small molecule metabolism. This study represents the first complete proteomic analysis of the in vivo protein targets of an activated furan-containing xenobiotic.

Supporting Information

Click to copy section linkSection link copied!

Detailed mass spectrometric analysis of the peptide fragments of target proteins. This information is available free of charge via the Internet at http://pubs.acs.org.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
    • Lawrence J. Marnett - Departments of Biochemistry, Chemistry, and Pharmacology, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146;  Email: [email protected]
  • Authors
    • Alexandra Druckova - Departments of Biochemistry, Chemistry, and Pharmacology, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
    • Raymond L. Mernaugh - Departments of Biochemistry, Chemistry, and Pharmacology, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
    • Amy-Joan L. Ham - Departments of Biochemistry, Chemistry, and Pharmacology, A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Acknowledgment

Click to copy section linkSection link copied!

This work was supported by research grants from the National Foundation for Cancer Research and the National Institute of Health (ES-013125).

References

Click to copy section linkSection link copied!

This article references 129 other publications.

  1. 1
    Guengerich, F. P., and Liebler, D. C.1985 Enzymatic activation of chemicals to toxic metabolites Crit. Rev. Toxicol. 14 259 307
    Google Scholar
    1
    Enzymic activation of chemicals to toxic metabolites
    Guengerich, F. Peter; Liebler, Daniel C.
    Critical Reviews in Toxicology (1985), 14 (3), 259-307CODEN: CRTXB2; ISSN:0045-6446.
    A review with 333 refs. on the enzymol. related to the bioactivation of xenobiotics, the key chem. principles which govern the prodn. and fate of reactive compds., and some biol. and biochem. factors which are important in considering potential toxicity.
  2. 2
    Pumford, N. R.1997 Protein targets of xenobiotic reactive intermediates Annu. Rev. Pharmacol. Toxicol. 37 91 117
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Petersen, D. R., and Doorn, J. A.2004 Reactions of 4-hydroxynonenal with proteins and cellular targets Free Radical Biol. Med. 37 937 945
    Google Scholar
    3
    Reactions of 4-hydroxynonenal with proteins and cellular targets
    Petersen, Dennis R.; Doorn, Jonathan A.
    Free Radical Biology & Medicine (2004), 37 (7), 937-945CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
    A review. Peroxidative degrdn. of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidn., redn., and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidn. and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo prodn. of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alc.-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biol. signaling.
  4. 4
    Carbone, D. L., Doorn, J. A., Kiebler, Z., Ickes, B. R., and Petersen, D. R.2005 Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease J. Pharmacol. Exp. Ther. 315 8 15
    Google Scholar
    4
    Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease
    Carbone, David L.; Doorn, Jonathan A.; Kiebler, Zachary; Ickes, Brian R.; Petersen, Dennis R.
    Journal of Pharmacology and Experimental Therapeutics (2005), 315 (1), 8-15CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
    Lipid peroxidn. during oxidative stress leads to increased concns. of thiol-reactive α,β-unsatd. aldehyde, including 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepd. from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alc.-treated animals. In vitro chaperoning expts. using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concns. of this aldehyde ranging from 23 to 450 μM. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concns. as low as 6 μM (1:1 M ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by liq. chromatog./tandem mass spectrometry peptide anal. identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alc.-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.
  5. 5
    West, J. D., and Marnett, L. J.2006 Endogenous reactive intermediates as modulators of cell signaling and cell death Chem. Res. Toxicol. 19 173 194
    Google Scholar
    5
    Endogenous Reactive Intermediates as Modulators of Cell Signaling and Cell Death
    West, James D.; Marnett, Lawrence J.
    Chemical Research in Toxicology (2006), 19 (2), 173-194CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    A review. The prodn. of endogenous reactive intermediates, the mechanistic features of cell death, and the signaling pathways that these intermediates ultimately alter to influence cell death processes are discussed.
  6. 6
    Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A.2004 Drug-protein adducts: An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development Chem. Res. Toxicol. 17 3 16
    Google Scholar
    6
    Drug-Protein Adducts: An Industry Perspective on Minimizing the Potential for Drug Bioactivation in Drug Discovery and Development
    Evans, David C.; Watt, Alan P.; Nicoll-Griffith, Deborah A.; Baillie, Thomas A.
    Chemical Research in Toxicology (2004), 17 (1), 3-16CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    A review. It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug-protein adducts and organ toxicity. Because the potential does exist, however, for these processes to be causally related, the general strategy at Merck Research Labs. has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metab. vectors and typically involves the initial use of small mol. trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) prepns. from preclin. species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Labs. The availability of a dedicated Labeled Compd. Synthesis group, coupled to a close working relationship between Drug Metab. and Medicinal Chem., represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.
  7. 7
    Baillie, T. A., Cayen, M. N., Fouda, H., Gerson, R. J., Green, J. D., Grossman, S. J., Klunk, L. J., LeBlanc, B., Perkins, D. G., and Shipley, L. A.2002 Drug metabolites in safety testing Toxicol. Appl. Pharmacol. 182 188 196
    Google Scholar
    7
    Drug metabolites in safety testing
    Baillie, Thomas A.; Cayen, Mitchell N.; Fouda, Hassan; Gerson, Ronald J.; Green, James D.; Grossman, Scott J.; Klunk, Lewis J.; LeBlanc, Bernard; Perkins, Darcy G.; Shipley, Lisa A.
    Toxicology and Applied Pharmacology (2002), 182 (3), 188-196CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Science)
    A review, summarizing the deliberations of a multidisciplinary committee, sponsored by the Pharmaceutical Research and Manufacturers of America, on current "best practices" within the U.S. pharmaceutical industry in assessing the role of drug metabolites as potential mediators of the toxicity of new drug products. Input to the document was obtained from numerous sources, including members of the pharmaceutical industry, academic investigators, and representatives of regulatory agencies who attended a workshop on the subject in Nov. 2000. The overall goal of the paper is to define practical and scientifically based approaches to the use of metabolite data that address contemporary issues in the safety evaluation of drug candidates. Although there remains a lack of consensus on how best to deal with several aspects of this complex subject, this paper raises a no. of points to consider, which emphasize the need to treat drug metabolite issues on a case-by-case basis. It is hoped that the discussion will promote continued dialog among industrial scientists and regulators charged with ensuring the clin. safety of new therapeutic agents.
  8. 8
    Baillie, T. A., and Kassahun, K.2001 Biological reactive intermediates in drug discovery and development: A perspective from the pharmaceutical industry Adv. Exp. Med. Biol. 500 45 51
    Google Scholar
    8
    Biological reactive intermediates in drug discovery and development: A perspective from the pharmaceutical industry
    Baillie, Thomas A.; Kassahun, Kelem
    Advances in Experimental Medicine and Biology (2001), 500 (Biological Reactive Intermediates VI), 45-51CODEN: AEMBAP; ISSN:0065-2598. (Kluwer Academic/Plenum Publishers)
    A review. The detection of chem.-reactive, electrophilic metabolites poses a particular problem in the discovery and development of drug candidates in pharmaceutical research, inasmuch as it is not possible to accurately predict the likely toxicol. consequences of these intermediates in animal safety studies or in clin. trials. Advances in anal. instrumentation (notably liq. chromatog.-tandem mass spectrometry [LC-MS/MS]) have facilitated the detection of reactive intermediates through the identification of the glutathione (GSH) adducts to which they normally give rise, while the increased use of radiolabeled tracers in drug development permits an early assessment to be made of the propensity of a drug candidate to undergo covalent binding to cellular macromols. Unfortunately, these advances in anal. methods for the detection and characterization of reactive drug metabolites have far outstripped our understanding of the mechanisms of foreign compd.-mediated toxicities at the mol. level, and of the role of both covalent binding and oxidative stress in the cascade of events that lead ultimately to cellular injury or immune-mediated toxicities. In light of these uncertainties, it seems reasonable to argue that one should attempt to minimize, through structural modification, the extent to which a drug candidate undergoes metab. to reactive intermediates. Therefore, it becomes imperative to have close collaboration between Drug Metab. scientists and their counterparts in Medicinal Chem. during both the discovery and early development phases. Bioactivation of troglitazone and the hepatotoxicity of troglitazone metabolites is discussed as an example.
  9. 9
    Carbone, D. L., Doorn, J. A., and Petersen, D. R.2004 4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase Free Radical Biol. Med. 37 1430 1439
    Google Scholar
    9
    4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase
    Carbone, David L.; Doorn, Jonathan A.; Petersen, Dennis R.
    Free Radical Biology & Medicine (2004), 37 (9), 1430-1439CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
    The lipid peroxidn. product 4-hydroxynonenal (4-HNE) was shown to interfere with protein function. The goal of this study was to det. the effects of substrate modification by 4-HNE on protein degrdn. Equine liver alc. dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approx. 1.5-fold faster than control, while treatment with concns. up to 100-fold molar excess aldehyde were inhibitory to degrdn. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS anal. of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through mol. modeling expts. a conformational shift in both zinc-contg. regions was predicted, with an approx. doubling of the distance between the structural zinc and its resp. chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degrdn. of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.
  10. 10
    Thome-Kromer, B., Bonk, I., Klatt, M., Nebrich, G., Taufmann, M., Bryant, S., Wacker, U., and Kopke, A.2003 Toward the identification of liver toxicity markers: A proteome study in human cell culture and rats Proteomics 3 1835 62
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Sampey, B. P., Korourian, S., Ronis, M. J., Badger, T. M., and Petersen, D. R.2003 Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury Alcohol Clin. Exp. Res. 27 1015 1022
    Google Scholar
    11
    Immunohistochemical Characterization of Hepatic Malondialdehyde and 4-Hydroxynonenal Modified Proteins During Early Stages of Ethanol-Induced Liver Injury
    Sampey, Brante P.; Korourian, Soheila; Ronis, Martin J.; Badger, Thomas M.; Petersen, Dennis R.
    Alcoholism: Clinical and Experimental Research (2003), 27 (6), 1015-1022CODEN: ACRSDM; ISSN:0145-6008. (Lippincott Williams & Wilkins)
    BACKGROUND: Chronic ethanol consumption is assocd. with hepatic lipid peroxidn. and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alc.-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alc.-induced liver injury. METHODS: Female Sprague Dawley rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet contg. ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathol. anal., and induction of cytochrome P 4502E1 (CYP2E1). Immunohistochem. methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quant. image anal. RESULTS: After 36 days of treatment, rats receiving the alc.-contg. diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis assocd. with only minor inflammation and necrosis. Alc. administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quant. immunohistochem. anal. revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepd. from rats treated with alc. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphol. and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells assocd. with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alc.-induced lipid peroxidn. is an early event during alc.-mediated liver injury and may be a sensitizing event resulting in the prodn. of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.
  12. 12
    Koen, Y. M., and Hanzlik, R. P.2002 Identification of seven proteins in the endoplasmic reticulum as targets for reactive metabolites of bromobenzene Chem. Res. Toxicol. 15 699 706
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Koen, Y. M., Williams, T. D., and Hanzlik, R. P.2000 Identification of three protein targets for reactive metabolites of bromobenzene in rat liver cytosol Chem. Res. Toxicol. 13 1326 1335
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Rombach, E. M., and Hanzlik, R. P.1999 Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal protein sulfhydryl groups using specific antibodies Chem. Res. Toxicol. 12 159 163
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Hartley, D. P., Kolaja, K. L., Reichard, J., and Petersen, D. R.1999 4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: Immunochemical detection and lobular localization Toxicol. Appl. Pharmacol. 161 23 33
    Google Scholar
    15
    4-Hydroxynonenal and Malondialdehyde Hepatic Protein Adducts in Rats Treated with Carbon Tetrachloride: Immunochemical Detection and Lobular Localization
    Hartley, Dylan P.; Kolaja, Kyle L.; Reichard, John; Petersen, Dennis R.
    Toxicology and Applied Pharmacology (1999), 161 (1), 23-33CODEN: TXAPA9; ISSN:0041-008X. (Academic Press)
    The metab. of CCl4 initiates the peroxidn. of polyunsatd. fatty acids producing α,β-unsatd. aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compds. like CCl4. To det. the rate at which CCl4-initiated lipid peroxidn. results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl4 (1.0 mL/kg) and euthanized 0-72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immunohistochem. and immunopptn./Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immunohistochem. in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidn. and hepatocellular necrosis increased. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was obsd. 24 h after CCl4 administration and immunopos. hepatocytes were obsd. in zone 2 as well as zone 3. Immunopos. cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administration, at least four adducted proteins were immunopptd. from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to mol. wts. of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be assocd. with hepatocellular injury following CCl4 exposure. (c) 1999 Academic Press.
  16. 16
    Rombach, E. M., and Hanzlik, R. P.1998 Identification of a rat liver microsomal esterase as a target protein for bromobenzene metabolites Chem. Res. Toxicol. 11 178 184
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Hartley, D. P., and Petersen, D. R.1997 Profiles of hepatic cellular protein adduction by malondialdehyde and 4-hydroxynonenal. Studies with isolated hepatocytes Adv. Exp. Med. Biol. 414 123 131
    Google Scholar
    17
    Profiles of hepatic cellular protein adduction by malondialdehyde and 4-hydroxynonenal: studies with isolated hepatocytes
    Hartley, Dylan P.; Petersen, Dennis R.
    Advances in Experimental Medicine and Biology (1997), 414 (Enzymology and Molecular Biology of Carbonyl Metabolism 6), 123-131CODEN: AEMBAP; ISSN:0065-2598. (Plenum)
    The prodn. of aldehydic products during a time course of iron-initiated lipid peroxidn. was quantitated in freshly isolated hepatocytes. Aldehyde-adducted proteins were detected by use of polyclonal antibodies produced against malondialdehyde- or 4-hydroxynonenal-protein adducts.
  18. 18
    Hartley, D. P., Kroll, D. J., and Petersen, D. R.1997 Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: Detection of 4-hydroxynonenal- and malondialdehyde-protein adducts Chem. Res. Toxicol. 10 895 905
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Hartley, D. P., Lindahl, R., and Petersen, D. R.1995 Covalent modification of class 2 and class 3 aldehyde dehydrogenase by 4-hydroxynonenal Adv. Exp. Med. Biol. 372 93 101
    Google Scholar
    19
    Covalent modification of class 2 and class 3 aldehyde dehydrogenase by 4-hydroxynonenal
    Hartley, Dylan P.; Lindahl, Ronald; Petersen, Dennis R.
    Advances in Experimental Medicine and Biology (1995), 372 (Enzymology and Molecular Biology of Carbonyl Metabolism 5), 93-101CODEN: AEMBAP; ISSN:0065-2598. (Plenum)
    The authors used two forms of aldehyde dehydrogenase active site models to assess the role of 4-hydroxynonenal covalent interactions in differential enzymic inactivation of these specific isoforms of aldehyde dehydrogenase.
  20. 20
    Bambal, R. B., and Hanzlik, R. P.1995 Bromobenzene 3,4-oxide alkylates histidine and lysine side chains of rat liver proteins in vivo Chem. Res. Toxicol. 8 729 735
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Slaughter, D. E., and Hanzlik, R. P.1991 Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles Chem. Res. Toxicol. 4 349 359
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Narasimhan, N., Weller, P. E., Buben, J. A., Wiley, R. A., and Hanzlik, R. P.1988 Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites Xenobiotica 18 491 499
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    De Vincenzi, M., Maialetti, F., and Silano, M.2003 Constituents of aromatic plants: Teucrin A Fitoterapia 74 746 749
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Bedir, E., Manyam, R., and Khan, I. A.2003 Neo-clerodane diterpenoids and phenylethanoid glycosides from Teucrium chamaedrys L Phytochemistry 63 977 983
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Kouzi, S. A., McMurtry, R. J., and Nelson, S. D.1994 Hepatotoxicity of germander (Teucrium chamaedrys L.) and one of its constituent neoclerodane diterpenes teucrin A in the mouse Chem. Res. Toxicol. 7 850 856
    Google Scholar
    25
    Hepatotoxicity of Germander (Teucrium chamaedrys L.) and One of Its Constituent Neoclerodane Diterpenes Teucrin A in the Mouse
    Kouzi, Samir A.; McMurtry, Randolph J.; Nelson, Sidney D.
    Chemical Research in Toxicology (1994), 7 (6), 850-6CODEN: CRTOEC; ISSN:0893-228X.
    The hepatotoxicity of the herbal plant germander and that of one of its major furanoneoclerodane diterpenes, teucrin A, were investigated in mice. Teucrin A was found to cause the same midzonal hepatic necrosis as obsd. with exts. of the powd. plant material. Evidence that bioactivation of teucrin A by cytochromes P 450 (P 450) to a reactive metabolite(s) is required for initiation of the hepatocellular damage is provided by results of expts. on the induction and inhibition of P 450 and from studies on the effects of glutathione depletion. Pretreatment of mice with the P 450 inducer phenobarbital enhanced the hepatotoxic response, as indicated by an increase in plasma alanine aminotransferase (ALT) levels and hepatic necrosis, while pretreatment with the P 450 inhibitor piperonyl butoxide markedly attenuated the toxic response. Hepatotoxicity of teucrin A also was increased following pretreatment with the inhibitor of glutathione synthesis buthionine sulfoximine. Most importantly, the THF analog of teucrin A, obtained by selective chem. redn. of the furan ring, was not hepatotoxic, a result that provides strong evidence that oxidn. of the furan ring moiety of the neoclerodane diterpenes is involved in the initiation of hepatocellular injury caused by germander.
  26. 26
    Lekehal, M., Pessayre, D., Lereau, J. M., Moulis, C., Fouraste, I., and Fau, D.1996 Hepatotoxicity of the herbal medicine germander: Metabolic activation of its furano diterpenoids by cytochrome P450 3A Depletes cytoskeleton-associated protein thiols and forms plasma membrane blebs in rat hepatocytes Hepatology 24 212 218
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Fau, D., Lekehal, M., Farrell, G., Moreau, A., Moulis, C., Feldmann, G., Haouzi, D., and Pessayre, D.1997 Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes Gastroenterology 113 1334 1346
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Boyd, M. R., Grygiel, J. J., and Minchin, R. F.1983 Metabolic activation as a basis for organ-selective toxicity Clin. Exp. Pharmacol. Physiol. 10 87 99
    Google Scholar
    28
    Metabolic activation as a basis for organ-selective toxicity
    Boyd, Michael R.; Grygiel, John J.; Minchin, Rodney F.
    Clinical and Experimental Pharmacology and Physiology (1983), 10 (1), 87-99CODEN: CEXPB9; ISSN:0305-1870.
    A discussion with many refs.
  29. 29
    Falzon, M., McMahon, J. B., Schuller, H. M., and Boyd, M. R.1986 Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines Cancer Res. 46 3484 3489
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M.1988 Preparation of the 8,9-epoxide of the mycotoxin aflatoxin b1: The ultimate carcinogenic species J. Am. Chem. Soc. 110 7929 7931
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Smela, M. E., Currier, S. S., Bailey, E. A., and Essigmann, J. M.2001 The chemistry and biology of aflatoxin B (1): From mutational spectrometry to carcinogenesis Carcinogenesis 22 535 545
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Baer, B. R., Rettie, A. E., and Henne, K. R.2005 Bioactivation of 4-ipomeanol by CYP4B1: Adduct characterization and evidence for an enedial intermediate Chem. Res. Toxicol. 18 855 864
    Google Scholar
    32
    Bioactivation of 4-Ipomeanol by CYP4B1: Adduct Characterization and Evidence for an Enedial Intermediate
    Baer, Brian R.; Rettie, Allan E.; Henne, Kirk R.
    Chemical Research in Toxicology (2005), 18 (5), 855-864CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromols. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liq. chromatog./electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping mol. Reaction mixts. contg. equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resoln. mass spectrometry and two-dimensional NMR anal. following large-scale isolation of the biol. derived material provided evidence for an N-substituted cysteinyl pyrrole deriv. of IPO, analogous to that characterized previously in model chem. studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P 450/30 min. A panel of 14 com. available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were obsd. with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s.
  33. 33
    Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P.2002 Biotransformation reactions of five-membered aromatic heterocyclic rings Chem. Res. Toxicol. 15 269 299
    Google Scholar
    33
    Biotransformation Reactions of Five-Membered Aromatic Heterocyclic Rings
    Dalvie, Deepak K.; Kalgutkar, Amit S.; Khojasteh-Bakht, S. Cyrus; Obach, R. Scott; O'Donnell, John P.
    Chemical Research in Toxicology (2002), 15 (3), 269-299CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    A review with 278 refs. is given on the biotransformation pathways of most commonly used 5-membered arom. heterocyclic rings. The effect of physicochem. properties such as the electronic effects of heteroatoms, aromaticity, and acidity/basicity (pKa) of the rings are correlated on their biotransformation. The generation of reactive metabolites following oxidn. or redn. of these heterocycles and the subsequent toxicol. consequences are discussed.
  34. 34
    Thomassen, D., Knebel, N., Slattery, J. T., McClanahan, R. H., and Nelson, S. D.1992 Reactive intermediates in the oxidation of menthofuran by cytochromes P-450 Chem. Res. Toxicol. 5 123 130
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    McClanahan, R. H., Thomassen, D., Slattery, J. T., and Nelson, S. D.1989 Metabolic activation of (R)-(+)-pulegone to a reactive enonal that covalently binds to mouse liver proteins Chem. Res. Toxicol. 2 349 355
    Google Scholar
    There is no corresponding record for this reference.
  36. 36
    Ravindranath, V., Burka, L. T., and Boyd, M. R.1984 Reactive metabolites from the bioactivation of toxic methylfurans Science 224 884 886
    Google Scholar
    36
    Reactive metabolites from the bioactivation of toxic methylfurans
    Ravindranath, Vijayalakshmi; Burka, Leo T.; Boyd, Michael R.
    Science (Washington, DC, United States) (1984), 224 (4651), 884-6CODEN: SCIEAS; ISSN:0036-8075.
    Acetylacrolein [5729-47-5] and methylbutenedial [4360-53-6] were identified as the principal reactive intermediates of 2-methylfuran (I) [534-22-5] and 3-methylfuran [930-27-8] that were produced. They bound covalently to tissue macromols. in hepatic and pulmonary microsomal systems in vitro. Therefore, the reactive compds. may be the ultimate toxic metabolites responsible for target tissue alkylation and toxicity produced by the parent furans in vivo. Epoxides, even if formed transiently, appear not to play a major role in the covalent binding nor in the toxicity of the furans.
  37. 37
    Chen, L. J., Hecht, S. S., and Peterson, L. A.1995 Identification of cis-2-butene-1,4-dial as a microsomal metabolite of furan Chem. Res. Toxicol. 8 903 906
    Google Scholar
    37
    Identification of cis-2-Butene-1,4-dial as a Microsomal Metabolite of Furan
    Chen, Ling-Jen; Hecht, Stephen S.; Peterson, Lisa A.
    Chemical Research in Toxicology (1995), 8 (7), 903-6CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    The rat liver microsomal metab. of furan was examd. in the presence of NADPH and semicarbazide. HPLC anal. of incubation mixts. revealed the formation of a metabolite that coeluted with stds. for the bis-semicarbazone adduct of cis-2-butene-1,4-dial. The formation of this compd. required the presence of NADPH, semicarbazide, and microsomes. Preparative isolation and chem. characterization of this metabolite confirmed the structural assignment. These data provide evidence that the reactive aldehyde, cis-2-butene-1,4-dial, is a major metabolic product of furan.
  38. 38
    Druckova, A., and Marnett, L. J.2006 Characterization of the amino acid adducts of the enedial derivative of teucrin A Chem. Res. Toxicol. 19 1330 1340
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Davies, S. S., Talati, M., Wang, X., Mernaugh, R. L., Amarnath, V., Fessel, J., Meyrick, B. O., Sheller, J., and Roberts, L. J.2004 Localization of isoketal adducts in vivo using a single-chain antibody Free Radical Biol. Med. 36 1163 1174
    Google Scholar
    39
    Localization of isoketal adducts in vivo using a single-chain antibody
    Davies, Sean S.; Talati, Megha; Wang, Xiahong; Mernaugh, Raymond L.; Amarnath, Venkataraman; Fessel, Joshua; Meyrick, Barbara O.; Sheller, James; Roberts, L. Jackson
    Free Radical Biology & Medicine (2004), 36 (9), 1163-1174CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
    Isoketals are highly reactive γ-ketoaldehydes formed by the oxidn. of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2α). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the no. of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.
  40. 40
    Murray, R. W., and Jeyaraman, R.1985 Dioxiranes––Synthesis and reactions of methyldioxiranes J. Org. Chem. 50 2847 2853
    Google Scholar
    There is no corresponding record for this reference.
  41. 41
    Pope, T., Embelton, J., and Mernaugh, R. L. (1996) Constructionand use of antibody gene repertories. In Antibody Engineering:A Practical Approach (McCafferty, J., Chiswell, D., and Hoogenboom, H., Eds.) IRL Press, Oxford, England.
    Google Scholar
    There is no corresponding record for this reference.
  42. 42
    Vinion-Dubiel, A. D., McClain, M. S., Cao, P., Mernaugh, R. L., and Cover, T. L.2001 Antigenic diversity among Helicobacter pylori vacuolating toxins Infect. Immun. 69 4329 4336
    Google Scholar
    42
    Antigenic diversity among Helicobacter pylori vacuolating toxins
    Vinion-Dubiel, Arlene D.; McClain, Mark S.; Cao, Ping; Mernaugh, Raymond L.; Cover, Timothy L.
    Infection and Immunity (2001), 69 (7), 4329-4336CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
    Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 IgG1κ anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.
  43. 43
    Lapierre, L. A., Avant, K. M., Caldwell, C. M., Ham, A. J., Hill, S., Williams, J. A., Smolka, A. J., and Goldenring, J. R.2007 Characterization of immunoisolated human gastric parietal cells tubulovesicles: Identification of regulators of apical recycling Am. J. Physiol. Gastrointest. Liver Physiol. 292 (5) G1249 62
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Ham, A.-J. (2005) Proteolytic digestion protocols. In The Encyclopediaof Mass Spectrometry, Volume 2 Biological Applications Part A: Proteinsand Peptides (Caprioli, R. M., and Gross, M. L., Eds.) Vol. 2, pp 10– 17, Elsevier Ltd., Kidlington, Oxford, United Kingdom.
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Cortes, H. J., Pfeiffer, C. D., Richter, B. E., and Stevens, T. S.1987 Porous ceramic bed supports for fused-silica packed capillary columns used in liquid-chromatography J. High Resolut. Chromatogr. Chromatogr. Commun. 10 446 448
    Google Scholar
    45
    Porous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography
    Cortes, H. J.; Pfeiffer, C. D.; Richter, B. E.; Stevens, T. S.
    HRC & CC, Journal of High Resolution Chromatography and Chromatography Communications (1987), 10 (8), 446-8CODEN: HCJCDB; ISSN:0344-7138.
    Porous ceramic bed supports for fused silica packed capillary columns utilized in liq. chromatog. were prepd. by polymg. solns. contg. potassium silicate in-situ within a column to create a mech. stable, rugged, and easily constructed termination. The effect of the bed support length on efficiency, and comparisons to glass wool bed supports, were considered in terms of column efficiencies and hydrodynamic variables. Results obtained indicate better performance for the ceramic bed support.
  46. 46
    Licklider, L .J., Thoreen, C. C., Peng, J. M., and Gygi, S. P.2002 Automation of nanoscale microcapillary liquid chromatography-tandem mass spectromentry with a vented column Anal. Chem. 74 3076 3083
    Google Scholar
    There is no corresponding record for this reference.
  47. 47
    Yates, J. R., Eng, J. K., Mccormack, A. L., and Schieltz, D.1995 Method to correlate tandem mass-spectra of modified peptides to amino-acid-sequences in the protein database Anal. Chem. 67 1426 1436
    Google Scholar
    There is no corresponding record for this reference.
  48. 48
    Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P.2005 Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations Nat. Methods 2 667 675
    Google Scholar
    48
    Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations
    Elias, Joshua E.; Haas, Wilhelm; Faherty, Brendan K.; Gygi, Steven P.
    Nature Methods (2005), 2 (9), 667-675CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Researchers have several options when designing proteomics expts. Primary among these are choices of exptl. method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, the authors compared triplicate measurements of the yeast proteome by liq. chromatog. tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, the authors selected scoring criteria yielding 1% estd. false pos. identifications at max. sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.
  49. 49
    Chen, L. J., Hecht, S. S., and Peterson, L. A.1997 Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan Chem. Res. Toxicol. 10 866 874
    Google Scholar
    There is no corresponding record for this reference.
  50. 50
    He, X. M., and Carter, D. C.1992 Atomic structure and chemistry of human serum albumin Nature 358 209 215
    Google Scholar
    50
    Atomic structure and chemistry of human serum albumin
    He, Xiao Min; Carter, Daniel C.
    Nature (London, United Kingdom) (1992), 358 (6383), 209-15CODEN: NATUAS; ISSN:0028-0836.
    The three-dimensional structure of human serum albumin has been detd. crystallog. to a resoln. of 2.8 Å. It comprises three homologous domains that assemble to form a heart-shaped mol. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chem. The structure explains numerous phys. phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.
  51. 51
    Loeper, J., Descatoire, V., Letteron, P., Moulis, C., Degott, C., Dansette, P., Fau, D., and Pessayre, D.1994 Hepatotoxicity of germander in mice Gastroenterology 106 464 472
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Baillie, T. A.2006 Future of toxicology––Metabolic activation and drug design: Challenges and apportunities in chemical toxicology Chem. Res. Toxicol. 19 889 893
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Pumford, N. R., Halmes, N. C., and Hinson, J. A.1997 Covalent binding of xenobiotics to specific proteins in the liver Drug Metab. Rev. 29 39 57
    Google Scholar
    53
    Covalent binding of xenobiotics to specific proteins in the liver
    Pumford, Neil R.; Halmes, N. Christine; Hinson, Jack A.
    Drug Metabolism Reviews (1997), 29 (1 & 2), 39-57CODEN: DMTRAR; ISSN:0360-2532. (Dekker)
    A review with 67 refs.
  54. 54
    Guengerich, F. P.2005 Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation Arch. Biochem. Biophys. 433 369 378
    Google Scholar
    There is no corresponding record for this reference.
  55. 55
    Boyd, M. R.1981 Toxicity mediated by reactive metabolites of furans Adv. Exp. Med. Biol. 136Part B 865 879
    Google Scholar
    There is no corresponding record for this reference.
  56. 56
    Gordon, W. P., Huitric, A. C., Seth, C. L., McClanahan, R. H., and Nelson, S. D.1987 The metabolism of the abortifacient terpene, (R)-(+)-pulegone, to a proximate toxin, menthofuran Drug Metab. Dispos. 15 589 594
    Google Scholar
    56
    The metabolism of the abortifacient terpene, (R)-(+)-pulegone, to a proximate toxin, menthofuran
    Gordon, W. Perry; Huitric, Alain C.; Seth, Cynthia L.; McClanahan, Robert H.; Nelson, Sidney D.
    Drug Metabolism and Disposition (1987), 15 (5), 589-94CODEN: DMDSAI; ISSN:0090-9556.
    (R)-(+)-Pulegone (I) is metabolized by hepatic microsomal monooxygenses of the mouse to a hepatotoxin. The formation of a toxic metabolite is apparently mediated by cytochromes P 450 of the phenobarbital class inasmuch as phenobarbital pretreatment of mice increases, whereas β-naphthoflavone pretreatment decreases, the extent of hepatic necrosis caused by pulegone. Furthermore, 2 inhibitors of cytochromes P 450, cobaltous chloride and piperonyl butoxide, block toxicity. An analog of I that was labeled with deuterium in the allylic Me groups was significantly less hepatotoxic than the parent compd. Apparently, oxidn. of an allylic Me group is required for generation of a hepatotoxic metabolite. Menthofuran was identified as a proximate toxic metabolite of I, and investigations with I-d6 and 18O2 strongly indicate that menthofuran is formed by a sequence of reactions that involve: (1) oxidn. of an allylic Me group, (2) intramol. cyclization to form a hemiketal, and (3) dehydration to form the furan.
  57. 57
    Kobayashi, T., Sugihara, J., and Harigaya, S.1987 Mechanism of metabolic cleavage of a furan ring Drug Metab. Dispos. 15 877 881
    Google Scholar
    There is no corresponding record for this reference.
  58. 58
    Parmar, D., and Burka, L. T.1993 Studies on the interaction of furan with hepatic cytochrome P-450 J. Biochem. Toxicol. 8 1 9
    Google Scholar
    58
    Studies on the interaction of furan with hepatic cytochrome P-450
    Parmar, Devendra; Burka, Leo T.
    Journal of Biochemical Toxicology (1993), 8 (1), 1-9CODEN: JBTOEB; ISSN:0887-2082.
    In vitro incubation of rat liver microsomes with [14C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was obsd. with microsomes from phenobarbital-treated rats and a four- to five-fold increase was obsd. with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chems. contg. an amine group (semi carbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [14C]-furan to microsomal protein. A decrease in cytochrome P 450 (P 450) content and decreases in the activities of P 450-dependent aniline hydroxylase, 7-ethoxycoumarin O-deethylase (ECD), and 7-ethoxyresorufin O-deethylase (ERD) was obsd. 24 h after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P 450 catalyzed metab. could be binding with nucleophilic groups within the P 450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and ECD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P 450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after in vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P 450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P 450.
  59. 59
    Sahali-Sahly, Y., Balani, S. K., Lin, J. H., and Baillie, T. A.1996 In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4 Chem. Res. Toxicol. 9 1007 1012
    Google Scholar
    There is no corresponding record for this reference.
  60. 60
    Racha, J. K., Rettie, A. E., and Kunze, K. L.1998 Mechanism-based inactivation of human cytochrome P450 1A2 by furafylline: Detection of a 1:1 adduct to protein and evidence for the formation of a novel imidazomethide intermediate Biochemistry 37 7407 7419
    Google Scholar
    There is no corresponding record for this reference.
  61. 61
    Zhang, K. E., Naue, J. A., Arison, B., and Vyas, K. P.1996 Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: Evidence for furan bioactivation Chem. Res. Toxicol. 9 547 554
    Google Scholar
    There is no corresponding record for this reference.
  62. 62
    Boyd, M. R., and Dutcher, J. S.1981 Renal toxicity due to reactive metabolites formed in situ in the kidney: Investigations with 4-ipomeanol in the mouse J. Pharmacol. Exp. Ther. 216 640 646
    Google Scholar
    62
    Renal toxicity due to reactive metabolites formed in situ in the kidney: investigations with 4-ipomeanol in the mouse
    Boyd, Micheal R.; Dutcher, John S.
    Journal of Pharmacology and Experimental Therapeutics (1981), 216 (3), 640-6CODEN: JPETAB; ISSN:0022-3565.
    The in vitro metab. and covalent binding of 4-ipomeanol (I) [32954-58-8] was mediated by O-requiring, NADPH-dependent, CO-inhibitable microsomal enzymes present in the livers, lungs and kidneys of adult male mice. These activities were inhibitable by piperonyl butoxide and they were markedly enhanced in hepatic microsomes from C57/6J mice, but not DBA/2J mice, pretreated with 3-methylcholanthrene. The i.p. administration of I to adult male mice resulted in the covalent binding of large amts. of its metabolite(s) in the lungs and kidneys. The material bound in the kidneys was located predominantly in the proximal renal cortical tubules. The covalent binding and toxicity of I to the renal tubules could be prevented by pretreatment of the animals with piperonyl butoxide. The hepatic covalent binding and toxicity of I were enhanced and the pulmonary and renal covalent binding and toxicity were decreased in C57BL/6J mice pretreated with 3-methylcholanthrene; however, this pretreatment did not significantly alter the tissue covalent binding or toxicity of I in noninducible DBA/2J mice. These results support the view that renal damage by I in the mouse is caused by reactive I metabolite(s) formed in situ in the kidney.
  63. 63
    Smothers, J. F., Henikoff, S., and Carter, P.2002 Tech.Sight. Phage display. Affinity selection from biological libraries Science 298 621 622
    Google Scholar
    There is no corresponding record for this reference.
  64. 64
    Smothers, J. F., and Henikoff, S.2001 Predicting in vivo protein peptide interactions with random phage display Comb. Chem. High Throughput Screening 4 585 591
    Google Scholar
    64
    Predicting in vivo protein-peptide interactions with random phage display
    Smothers, James F.; Henikoff, Steven
    Combinatorial Chemistry and High Throughput Screening (2001), 4 (7), 585-591CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers)
    A review. Binding sites in protein complexes occasionally map to small peptides within one or more proteins. Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest. The natural substrates for the protein are typically known in advance. However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two-hybrid screening. Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo. Here, we review an approach that addresses this problem. First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display. Next, searching programs detect similar blocks of conserved sequences within naturally-occurring proteins to predict partner proteins. Finally, the significance of an interaction is tested using site-specific mutagenesis, binding competition or co-immunopptn. expts. This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biol.
  65. 65
    Yip, Y. L., and Ward, R. L.1999 Epitope discovery using monoclonal antibodies and phage peptide libraries Comb. Chem. High Throughput Screening 2 125 138
    Google Scholar
    65
    Epitope discovery using monoclonal antibodies and phage peptide libraries
    Yip, Yum L.; Ward, Robyn L.
    Combinatorial Chemistry and High Throughput Screening (1999), 2 (3), 125-138CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers)
    A review with 148 refs. Phage display is a biol. system which facilitates the cloning and rapid selection of peptides from large combinatorial libraries. In comparison to the chem. combinatorial approach, the advantages of phage display lie in its simplicity and replicability. While phage display has many diverse applications, this review focuses on the use of phage peptide libraries to discover epitopes recognized by monoclonal antibodies. As monoclonal antibodies are useful tools for the detection of proteins and for the investigation of mol. interactions, the identification of their epitopes will serve to elucidate the structure and function of proteins, as well as aid in the discovery of new drugs and the development of vaccines.
  66. 66
    Wang, L. F., and Yu, M.2004 Epitope identification and discovery using phage display libraries: Applications in vaccine development and diagnostics Curr. Drug Targets 5 1 15
    Google Scholar
    66
    Epitope identification and discovery using phage display libraries: Applications in vaccine development and diagnostics
    Wang, Lin-fa; Yu, Meng
    Current Drug Targets (2004), 5 (1), 1-15CODEN: CDTUAU; ISSN:1389-4501. (Bentham Science Publishers Ltd.)
    A review. Antigenic epitopes are the part (contact points) of an antigen involved in specific interaction with the antigen-binding site (the paratope) of an antibody or a T-cell receptor. Detailed anal. of epitopes is important both for the understanding of immunol. events and for the development of more effective vaccine and diagnostic tools for various diseases. Identification and characterization of epitopes is a complex process. Although various methods have been developed in this area, there still lacks a simple common approach which can be applied to all epitopes. Since its first introduction more than a decade ago, phage display technol. has made a major impact in this area of research. With the exponential growth in this area, it is impractical to review the entire literature detailing all possible applications. Instead, this review aims to focus on specific applications related to the discovery and identification of epitopes which have potential as vaccine candidates or can be used in disease diagnosis.
  67. 67
    Mancini, N., Carletti, S., Perotti, M., Canducci, F., Mammarella, M., Sampaolo, M., and Burioni, R.2004 Phage display for the production of human monoclonal antibodies against human pathogens New Microbiol. 27 315 328
    Google Scholar
    67
    Phage display for the production of human monoclonal antibodies against human pathogens
    Mancini, N.; Carletti, S.; Perotti, M.; Canducci, F.; Mammarella, M.; Sampaolo, M.; Burioni, R.
    New Microbiologica (2004), 27 (4), 315-328CODEN: NMEIB2; ISSN:1121-7138. (Edizioni Internazionali srl, Div. EDIMES)
    A review. In the last decade an increasing no. of antibodies have made their way from the research benchtops into the clinics and many more are currently under clin. trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the mol. dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.
  68. 68
    Azzazy, H. M., and Highsmith, W. E., Jr2002 Phage display technology: Cclinical applications and recent innovations Clin. Biochem. 35 425 445
    Google Scholar
    68
    Phage display technology: clinical applications and recent innovations
    Azzazy, Hassan M. E.; Highsmith, W. Edward, Jr.
    Clinical Biochemistry (2002), 35 (6), 425-445CODEN: CLBIAS; ISSN:0009-9120. (Elsevier Science Inc.)
    A review. Phage display is a mol. diversity technol. that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technol. is the direct link that exists between the exptl. phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized mols. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technol.
  69. 69
    Szapacs, M. E., Riggins, J. N., Zimmerman, L. J., and Liebler, D. C.2006 Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions Biochemistry 45 10521 10528
    Google Scholar
    There is no corresponding record for this reference.
  70. 70
    Ivanov, A. I., Christodoulou, J., Parkinson, J. A., Barnham, K. J., Tucker, A., Woodrow, J., and Sadler, P. J.1998 Cisplatin binding sites on human albumin J. Biol. Chem. 273 14721 14730
    Google Scholar
    70
    Cisplatin binding sites on human albumin
    Ivanov, Andrei I.; Christodoulou, John; Parkinson, John A.; Barnham, Kevin J.; Tucker, Alan; Woodrow, John; Sadler, Peter J.
    Journal of Biological Chemistry (1998), 273 (24), 14721-14730CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Reactions of cisplatin with albumin are thought to play an important role in the metab. of this anticancer drug. These reactions were investigated via (1) labeling of cisplatin with 15N and use of 2-dimensional [1H,15N]NMR spectroscopy, (2) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (3) chem. modification of Cys, Met, and His residues, (4) reactions of bound Pt with thiourea, and (5) gel filtration chromatog. In contrast to previous reports, it was shown that the major S-contg. binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Addnl. monofunctional adducts involving other Met residues and Cys-34 were also obsd. During the later stages of reactions of cisplatin with albumin, release of NH3 occurred due to the strong trans influence of Met S, which weakens the Pt-NH3 bonds, and protein crosslinking was obsd. The consequences of these findings for the biol. activity of cisplatin-albumin complexes are discussed.
  71. 71
    Fabisiak, J. P., Sedlov, A., and Kagan, V. E.2002 Quantification of oxidative/nitrosative modification of CYS(34) in human serum albumin using a fluorescence-based SDS-PAGE assay Antioxid. Redox Signaling 4 855 865
    Google Scholar
    71
    Quantification of Oxidative/Nitrosative Modification of CYS34 in Human Serum Albumin Using a Fluorescence-Based SDS-PAGE Assay
    Fabisiak, James P.; Sedlov, Andrey; Kagan, Valerian E.
    Antioxidants & Redox Signaling (2002), 4 (5), 855-865CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)
    The SH group represented by cysteine in proteins is fundamental to the redox regulation of protein structure and function. Albumin is the most abundant serum protein whose redox modification modulates its physiol. function, as well as serves as a biomarker of oxidative stress. Measurement of selective Cys modification (S-oxidn./nitrosation, electrophilic substitution) on specific proteins, however, is problematic within complex biol. mixts. such as plasma. We have utilized a maleimide fluorogenic SH reagent, ThioGlo-1, to develop a fluorescence-based quant. assay of SH modification of human serum albumin (hSA) using SDS-PAGE. Fully reduced native albumin contg. one free SH (Cys34) per mol. was utilized as a model protein to characterize the kinetics of ThioGlo-1 reaction using a soln.-based spectrofluorometric assay. Optimum labeling of hSA Cys34 was achieved within 10 min at 60°C using a threefold molar excess of ThioGlo-1 relative to hSA and required SDS. Comparison of the soln. spectrofluorometric assay to fluorescent image anal. of hSA bands localized by SDS-PAGE revealed that SH groups in hSA could be quantified after gel electrophoresis. The soln.- and gel-based methods were in excellent concordance in their ability to quantify SH modification of hSA following exposure to phenoxyl radicals and nitric oxide. The application of ThioGlo-1 staining and SDS-PAGE quantified the degree of hSA modification in complex human plasma exposed to oxidative or nitrosative stress and revealed that hSA is more sensitive to S modification than other SH-contg. plasma proteins.
  72. 72
    Shen, B., and English, A. M.2005 Mass spectrometric analysis of nitroxyl-mediated protein modification: Comparison of products formed with free and protein-based cysteines Biochemistry 44 14030 14044
    Google Scholar
    72
    Mass Spectrometric Analysis of Nitroxyl-Mediated Protein Modification: Comparison of Products Formed with Free and Protein-Based Cysteines
    Shen, Biao; English, Ann M.
    Biochemistry (2005), 44 (42), 14030-14044CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Although biol. active, nitroxyl (HNO) remains one of the most poorly studied NOx. Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addn. product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 μM), human calbindin D28k (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivs. in incubates contg. 0.9-2.5 mM AS. Ions at M + n × 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramol. sulfinamide crosslinking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were obsd. for HCalB, which addnl. formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramol. disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3- → HNO + NO2-), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.
  73. 73
    Talib, J., Beck, J. L., and Ralph, S. F.2006 A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]- to human serum albumin J. Biol. Inorg. Chem. 11 559 570
    Google Scholar
    73
    A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]- to human serum albumin
    Talib, Jihan; Beck, Jennifer L.; Ralph, Stephen F.
    JBIC, Journal of Biological Inorganic Chemistry (2006), 11 (5), 559-570CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)
    Electrospray ionization (ESI) mass spectrometry was used to examine the reactions of the clin. used antiarthritic agent [Au(S2O3)2]3-, and AuPEt3Cl, a deriv. of another clin. used agent auranofin, with human serum albumin (HSA) obtained from a human volunteer. Both compds. reacted readily with HSA to form complexes contg. one or more covalently attached gold fragments. In the case of AuPEt3Cl, binding was accompanied by the loss of the chloride ligand, while for [Au(S2O3)2]3- the mass spectral data indicated binding of Au(S2O3) groups. Expts. performed using HSA with Cys34 blocked by reaction with iodoacetamide were consistent with reaction of both gold compds. with this amino acid. Sep. blocking expts. using diethylpyrocarbonate and AuPEt3Cl also provided evidence for histidine residues acting as lower-affinity binding sites for this gold compd. ESI mass spectra of solns. contg. [Au(S2O3)2]3- or [Au(CN)2]-, and HSA, provided evidence for the formation of protein complexes in which intact gold mols. were noncovalently bound. In the case of [Au(S2O3)2]3-, these noncovalent complexes proved to be transitory in nature. However, for [Au(CN)2]- a noncovalent complex contg. a single gold mol. bound to HSA was found to be stable, and constituted the main adduct formed in solns. contg. low-to-medium Au-to-HSA ratios. Evidence was also obtained for the formation of a covalent adduct in which a single Au(CN) moiety was bonded to Cys34 of the protein. AuPEt3Cl reacted to a much lower extent with HSA that had Cys34 modified by formation of a disulfide bond to added cysteine, than with unmodified HSA. This suggests that the extent of modification of the protein in vivo may have an important influence on the transport and bioavailability of gold antiarthritic drugs.
  74. 74
    Ascoli, G. A., Domenici, E., and Bertucci, C.2006 Drug binding to human serum albumin: Abridged review of results obtained with high-performance liquid chromatography and circular dichroism Chirality 18 667 679
    Google Scholar
    There is no corresponding record for this reference.
  75. 75
    Bertucci, C., and Domenici, E.2002 Reversible and covalent binding of drugs to human serum albumin: Methodological approaches and physiological relevance Curr. Med. Chem. 9 1463 1481
    Google Scholar
    75
    Reversible and covalent binding of drugs to human serum albumin: methodological approaches and physiological relevance
    Bertucci, Carlo; Domenici, Enrico
    Current Medicinal Chemistry (2002), 9 (15), 1463-1481CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers)
    A review. Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concn. of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metab., distribution and excretion. The free concn. of a drug can also be affected by interaction with coadministered drugs or by pathol. conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clin. impacts for drugs that have a relatively narrow therapeutic index. This article reviews the physiol. role of HSA in in the human body and the pharmacol. consequences of drug-HSA binding; it then focuses on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatog. on immobilized HSA has been shown to be a rapid and effective tool for the characterization of binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. Also discussed is the potential offered by the combined use of CD on the same protein/drug system in soln., not only for the detn. of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs is addressed. The effect of reversible or covalent binding of drugs is also discussed and examples of physiol. relevance reported.
  76. 76
    De Berardinis, V., Moulis, C., Maurice, M., Beaune, P., Pessayre, D., Pompon, D., and Loeper, J.2000 Human microsomal epoxide hydrolase is mthe target of germander-induced autoantibodies on the surface of human hepatocytes Mol. Pharmacol. 58 542 551
    Google Scholar
    There is no corresponding record for this reference.
  77. 77
    Loeper, J., De Berardinis, V., Moulis, C., Beaune, P., Pessayre, D., and Pompon, D.2001 Human epoxide hydrolase is the target of germander autoantibodies on the surface of human hepatocytes: Enzymatic implications Adv. Exp. Med. Biol. 500 121 124
    Google Scholar
    77
    Human epoxide hydrolase is the target of germander autoantibodies on the surface of human hepatocytes: Enzymatic implications
    Loeper, Jacqueline; De Berardinis, Veronique; Moulis, Claude; Beaune, Philippe; Pessayre, Dominique; Pompon, Denis
    Advances in Experimental Medicine and Biology (2001), 500 (Biological Reactive Intermediates VI), 121-124CODEN: AEMBAP; ISSN:0065-2598. (Kluwer Academic/Plenum Publishers)
    Wild germander (Teuchrium chamaedrys L.) was traditionally used as a folk medicine tor its choleretic and antiseptic properties. In 1991, germander consumed to treat obesity, caused an epidemic of cytolytic hepatitis. Thirty cases of hepatotoxicity were first reported including cases with pos. rechallenge. For these patients, an early recurrence was obsd. despite lack of other features of hypersensitivity (Castot and Larrey, 1992). In mice, germander toxicity required CYP3A-dependent metab. (Loeper, and al., 1994), more specifically, the metabolic activation of the furan ring of the diterpenoid teucrin A (TA) (Kouzi, and al., 1994). TA-toxicity was via CYP3A-generated electrophilic metabolites that were detoxified by glutathione conjugation, depleted cellular thiols and caused apoptosis in isolated rat hepatocytes (Lekehal, and al., 1996; Fau and al., 1997). To explain this immune process, patient's sera consuming germander tea in great quantity were tested by Western blot anal. They contained autoantibodies directed against human microsomal epoxide hydrolase (hmEH), that was located both, in the endoplasmic reticulum and the plasma membrane (PM) of human hepatocyte and hmEH-expressing yeast. Germander-induced autoantibodles (GIAA) were shown to recognize hmEH on the cell surface. To implicate hmEH in the metabolic activation of TA we used a humanized yeast strain expressing human P 450-reductase and cytochrome b5 transformed with CYP3A4 and/or hmEH cDNAs. TA was metabolized by CYP3A4 into a metabolite which concn. diminished in presence of hmEH. Incubations of CYP3A4 and hmEH with TA inactivated hmEH in a time-dependent-manner, in agreement with formation of reactive teuchrin A-metabolite that could covalently alter and inhibit hmEH. This modified enzyme may bypass the immunol. tolerance that normally exists for hmEH. In conclusion for the first time, we have shown that anti-hmEH autoantibodies can develop in plant-induced hepatitis. The hmEH is present and functional in the human hepatocyte PM and hmEH epitopes are exposed on the outer surface of the PM, allowing the autoantibodies to possibly participate in the immune destruction of hepatocytes. CYP3A4-mediated metabolic activation of TA into a reactive epoxide could modify and inactivate hmEH. This altered protein may bypass immune tolerance and trigger the immune response against hmEH.
  78. 78
    Boitier, E., and Beaune, P.2000 Xenobiotic-metabolizing enzymes as autoantigens in human autoimmune disorders. An update Clin. Rev. Allergy Immunol. 18 215 239
    Google Scholar
    78
    Xenobiotic-metabolizing enzymes as autoantigens in human autoimmune disorders; An update
    Boitier, Eric; Beaune, Philippe
    Clinical Reviews in Allergy & Immunology (2000), 18 (2), 215-239CODEN: CRAIF2; ISSN:1080-0549. (Humana Press Inc.)
    A review with 146 refs. Topics discussed include drug-induced autoimmune diseases in which autoantibodies against cytochromes P 450 or xenobiotic-metabolizing enzymes; tienilic acid-induced hepatitis; dihydralazine-induced hepatitis; halothane-induced hepatitis; mol. targets of autoimmune disease nonassocd. with a toxic chem.; uridine diphosphate-glucouronosyltransferase as autoantibody target; glutathione-S-transferase as autoantibody target; pathogenesis of autoimmune disease; and mol. mimicry.
  79. 79
    Strassburg, C. P., Obermayer-Straub, P., and Manns, M. P.2000 Autoimmunity in liver diseases Clin. Rev. Allergy Immunol. 18 127 139
    Google Scholar
    79
    Autoimmunity in liver diseases
    Strassburg, Christian P.; Obermayer-Straub, Petra; Manns, Michael P.
    Clinical Reviews in Allergy & Immunology (2000), 18 (2), 127-139CODEN: CRAIF2; ISSN:1080-0549. (Humana Press Inc.)
    A review with 31 refs. Topics discussed include the role of hepatotropic virus infection in genuine autoimmune hepatitis, the characterization and evaluation of novel autoantigens in hepatic disease as part of autoimmune polyendocrine syndrome type 1, and the investigation of autoimmune features in hepatitis D infection.
  80. 80
    Boitier, E., and Beaune, P.1999 Cytochromes P450 as targets to autoantibodies in immune mediated diseases Mol. Aspects Med. 20 84 137
    Google Scholar
    80
    Cytochromes P450 as targets to autoantibodies in immune mediated diseases
    Boitier E; Beaune P
    Molecular aspects of medicine (1999), 20 (1-2), 84-137 ISSN:0098-2997.
    There is no expanded citation for this reference.
  81. 81
    Mizutani, T., Shinoda, M., Tanaka, Y., Kuno, T., Hattori, A., Usui, T., Kuno, N., and Osaka, T.2005 Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2 Drug Metab. Rev. 37 235 252
    Google Scholar
    81
    Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2
    Mizutani, Takaharu; Shinoda, Masakazu; Tanaka, Yuta; Kuno, Takuya; Hattori, Asuka; Usui, Toru; Kuno, Nayumi; Osaka, Takashi
    Drug Metabolism Reviews (2005), 37 (1), 235-252CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis, Inc.)
    A review. Autoimmune hepatitis (AIH) is a disease of unknown etiol., characterized by liver-related autoantibodies. Autoimmune hepatitis is subdivided into two major types: AIH type 1 is characterized by the detection of ANA, SMA, ANCA, anti-ASGP-R, and anti-SLA/LP. Autoimmune hepatitis type 2 is characterized to be mainly related with drug-metabolizing enzymes as autoantigens, such as anti-LKM (liver-kidney microsomal antigen)-1 against CYP2D6, anti-LKM-2 against CYP2C9-tienilic acid, anti-LKM-3 against UGT1A, and anti-LC1 (liver cytosol antigen)-1 and anti-APS (autoimmune polyglandular syndrome type-1) against CYP1A2, CYP2A6, and others. Anti-LKM-1 sera inhibited CYP2D6 activity in vitro but did not inhibit cellular drug metab. in vivo. CYP2D6 is the major target autoantigen of LKM-1 and expressed on plasma membrane (PM) of hepatocytes, suggesting a pathogenic role for anti-LKM-1 in liver injury as a trigger. Anti-CYP1A2 was obsd. in dihydralazine-induced hepatitis, and radiolabeled CYP1A2 disappeared from the PM with a half-life of less than 30 min, whereas microsomal CYP1A2 was stably radiolabeled for several hours. Main antigenic epitopes on CYP2D6 are as 193-212, as 257-269, and as 321-351; and D263 is essential. The third epitope is located on the surface of the protein CYP2D6 and displays a hydrophobic patch that is situated between an arom. residue (W316) and histidine (H326). Some drugs such as anticonvulsants (phenobarbital, phenytoin, and carbamazepine) and halothane are suggested to induce hepatitis with anti-CYP3A and anti-CYP2E1, resp. Autoantibodies against CYP11A1, CYP17, and/or CYP21 involved in the synthesis of steroid hormones are also detected in patients with adrenal failure, gonadal failure, and/or Addison disease.
  82. 82
    Obermayer-Straub, P., Strassburg, C. P., and Manns, M. P.2000 Target proteins in human autoimmunity: cytochromes P450 and UDP- glucuronosyltransferases Can. J. Gastroenterol. 14 429 439
    Google Scholar
    82
    Target proteins in human autoimmunity: cytochromes P450 and UDP- glucuronosyltransferases
    Obermayer-Straub P; Strassburg C P; Manns M P
    Canadian journal of gastroenterology = Journal canadien de gastroenterologie (2000), 14 (5), 429-39 ISSN:0835-7900.
    Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are targets of autoantibodies in several hepatic and extrahepatic autoimmune diseases. Autoantibodies directed against hepatic CYPs and UGTs were first detected by indirect immunofluorescence as antiliver and/or kidney microsomal antibodies. In autoimmune hepatitis (AIH) type 2, liver and/or kidney microsomal (LKM) type 1 autoantibodies are detected and are directed against CYP2D6. About 10% of AIH-2 sera further contain LKM-3 autoantibodies directed against family 1 UGTs. Chronic infections by hepatitis C virus and hepatitis delta virus may induce several autoimmune phenomena, and multiple autoantibodies are detected. Anti-CYP2D6 autoantibodies are detected in up to 4% of patients with chronic hepatitis C, and anti-CYP2A6 autoantibodies are detected in about 2% of these patients. In contrast, 14% of patients with chronic hepatitis delta virus infections generate anti-UGT autoantibodies. In a small minority of patients, certain drugs are known to induce immune-mediated, idiosyncratic drug reactions, also known as 'druginduced hepatitis'. Drug-induced hepatitis is often associated with autoantibodies directed against hepatic CYPs or other hepatic proteins. Typical examples are tienilic acid-induced hepatitis with anti-CYP2C9, dihydralazine hepatitis with anti-CYP1A2, halothane hepatitis with anti-CYP2E1 and anticonvulsant hepatitis with anti-CYP3A. Recent data suggest that alcoholic liver disease may be induced by mechanisms similar to those that are active in drug-induced hepatitis. Autoantibodies directed against several CYPs are further detected in sera from patients with the autoimmune polyglandular syndrome type 1. Patients with autoimmune polyglandular syndrome type 1 with hepatitis often develop anti-CYP1A2; patients with adrenal failure develop anti-CYP21, anti- CYP11A1 or CYP17; and patients with gonadal failure develop anti-CYP11A1 or CYP17. In idiopathic Addison disease, CYP21 is the major autoantigen.
  83. 83
    Mottaran, E., Stewart, S. F., Rolla, R., Vay, D., Cipriani, V., Moretti, M., Vidali, M., Sartori, M., Rigamonti, C., Day, C. P., and Albano, E.2002 Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease Free Radical Biol. Med. 32 38 45
    Google Scholar
    83
    Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease
    Mottaran, Elisa; Stewart, Stephen F.; Rolla, Roberta; Vay, Daria; Cipriani, Valentina; Moretti, MariaGrazia; Vidali, Matteo; Sartori, Massimo; Rigamonti, Cristina; Day, Christopher P.; Albano, Emanuele
    Free Radical Biology & Medicine (2001), 32 (1), 38-45CODEN: FRBMEH; ISSN:0891-5849. (Elsevier Science Inc.)
    Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alc. liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hydroxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alc. cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alc. as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead obsd. in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition expts. showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidn. contribute to the development of immune responses assocd. with alc. liver disease.
  84. 84
    Stewart, S. F., Vidali, M., Day, C. P., Albano, E., and Jones, D. E.2004 Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease Hepatology 39 197 203
    Google Scholar
    There is no corresponding record for this reference.
  85. 85
    Corcos, L., and Lagadic-Gossmann, D.2001 Gene induction by phenobarbital: An update on an old question that receives key novel answers Pharmacol. Toxicol. 89 113 122
    Google Scholar
    85
    Gene induction by phenobarbital: an update on an old question that receives key novel answers
    Corcos, Laurent; Lagadic-Gossmann, Dominique
    Pharmacology & Toxicology (Copenhagen, Denmark) (2001), 89 (3), 113-122CODEN: PHTOEH; ISSN:0901-9928. (Munksgaard International Publishers Ltd.)
    A review is given. Phenobarbital has long been used as a sedative and antiepileptic drug. The drug is the representative of a myriad of lipophilic mols. able to evoke a pleiotropic response in the liver and also in prokaryotes and flies. A great deal of novel information was obtained in recent years regarding the mechanism of cytochrome P 450 (CYP) gene induction by phenobarbital. Most importantly, a nuclear orphan receptor, the constitutive androstane receptor was identified as a primary determinant of the transcriptional activation of CYP genes in response to phenobarbital-like inducers in mammals. Another nuclear receptor, the pregnane X receptor can also mediate some of the phenobarbital response, but the functional overlap of the 2 inductive pathways is only partial. The response of mammalian CYP2B genes to phenobarbital was abolished in the liver of mice carrying a null allele of the constitutive androstane receptor gene, whereas that of CYP3A genes was lost in pregnane X receptor knock-out mice.
  86. 86
    Czekaj, P.2000 Phenobarbital-induced expression of cytochrome P450 genes Acta Biochim. Pol. 47 1093 1105
    Google Scholar
    There is no corresponding record for this reference.
  87. 87
    Gibson, G. G., Plant, N. J., Swales, K. E., Ayrton, A., and El-Sankary, W.2002 Receptor-dependent transcriptional activation of cytochrome P4503A genes: Induction mechanisms, species differences and interindividual variation in man Xenobiotica 32 165 206
    Google Scholar
    There is no corresponding record for this reference.
  88. 88
    Joannard, F., Galisteo, M., Corcos, L., Guillouzo, A., and Lagadic-Gossmann, D.2000 Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: Involvement of several serine/threonine protein kinases and phosphatases Cell. Biol. Toxicol. 16 325 337
    Google Scholar
    88
    Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases
    Joannard, F.; Galisteo, M.; Corcos, L.; Guillouzo, A.; Lagadic-Gossmann, D.
    Cell Biology and Toxicology (2000), 16 (5), 325-337CODEN: CBTOE2; ISSN:0742-2091. (Kluwer Academic Publishers)
    We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P 450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compds. one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot anal. of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the neg. role of the cAMP/protein kinase A pathway and the pos. role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca2+/calmodulin-dependent protein kinases II (independently of Ca2+) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function resp. as pos. and neg. regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A.
  89. 89
    Haas, I. G.1994 BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum Experientia 50 1012 1020
    Google Scholar
    There is no corresponding record for this reference.
  90. 90
    Kleizen, B., and Braakman, I.2004 Protein folding and quality control in the endoplasmic reticulum Curr. Opin. Cell Biol. 16 343 349
    Google Scholar
    90
    Protein folding and quality control in the endoplasmic reticulum
    Kleizen, Bertrand; Braakman, Ineke
    Current Opinion in Cell Biology (2004), 16 (4), 343-349CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)
    A review and discussion. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least 2 main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasizes more than ever that chaperones act in concert with co-factors and with each other.
  91. 91
    Sommer, T., and Jarosch, E.2002 BiP binding keeps ATF6 at bay Dev. Cell 3 1 2
    Google Scholar
    91
    BiP binding keeps ATF6 at bay
    Sommer, Thomas; Jarosch, Ernst
    Developmental Cell (2002), 3 (1), 1-2CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
    A study by Shen et al. in this issue of Developmental Cell shows that transport to the Golgi complex and subsequent proteolytic activation of the stress-regulated transcription factor ATF6 is initiated by the dissocn. of the ER chaperone BiP from ATF6. This demonstrates that BiP is a key element in sensing the folding capacity within the ER and provides mechanistic insights on how the activation of membrane-bound transcription factors can be regulated.
  92. 92
    Zhang, K., and Kaufman, R. J.2006 Protein folding in the endoplasmic reticulum and the unfolded protein response Handb. Exp. Pharmacol. 69 91
    Google Scholar
    92
    Protein folding in the endoplasmic reticulum and the unfolded protein response
    Zhang, K.; Kaufman, R. J.
    Handbook of Experimental Pharmacology (2006), 172 (Molecular Chaperones in Health and Disease), 69-91CODEN: HEPHD2; ISSN:0171-2004. (Springer GmbH)
    A review. In all eukaryotic cells, the endoplasmic reticulum (ER) is an intracellular organelle where folding and assembly occurs for proteins destined to the extracellular space, plasma membrane, and the exo/endocytic compartments. As a protein-folding compartment, the ER is exquisitely sensitive to alterations in homeostasis, and provides stringent quality control systems to ensure that only correctly folded proteins transit to the Golgi app. and unfolded or misfolded proteins are retained and ultimately degraded. A no. of biochem. and physiol. stimuli, such as perturbation in Ca homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, altered glycosylation, and overloading of cholesterol can disrupt ER homeostasis, impose stress to the ER, and subsequently lead to accumulation of unfolded or misfolded proteins in the ER lumen. The ER has evolved highly specific signaling pathways called the unfolded protein response (UPR) to cope with the accumulation of unfolded or misfolded proteins. Elucidation of the mol. mechanisms by which accumulation of unfolded proteins in the ER transmits a signal to the cytoplasm and nucleus has led to major new insights into the diverse cellular and physiol. processes that are regulated by the UPR. Here, the authors summarize how cells respond to the accumulation of unfolded proteins in the cell and the relevance of these signaling pathways to human physiol. and disease.
  93. 93
    Paton, A. W., Beddoe, T., Thorpe, C. M., Whisstock, J. C., Wilce, M. C., Rossjohn, J., Talbot, U. M., and Paton, J. C.2006 AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP Nature 443 548 552
    Google Scholar
    There is no corresponding record for this reference.
  94. 94
    Cribb, A. E., Peyrou, M., Muruganandan, S., and Schneider, L.2005 The endoplasmic reticulum in xenobiotic toxicity Drug Metab. Rev. 37 405 442
    Google Scholar
    94
    The endoplasmic reticulum in xenobiotic toxicity
    Cribb, Alastair E.; Peyrou, Mathieu; Muruganandan, Shanmugam; Schneider, Laetitia
    Drug Metabolism Reviews (2005), 37 (3), 405-442CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis, Inc.)
    A review. The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P 450 enzymes involved in xenobiotic metab., as well as a no. of conjugating enzymes. In addn. to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and the folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-assocd. apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in mol. events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity is discussed.
  95. 95
    Ellgaard, L., and Ruddock, L. W.2005 The human protein disulphide isomerase family: Substrate interactions and functional properties EMBO Rep. 6 28 32
    Google Scholar
    There is no corresponding record for this reference.
  96. 96
    Sitia, R., and Molteni, S. N.2004 Stress, protein (mis)folding, and signaling: The redox connection Sci. STKE pe27
    Google Scholar
    There is no corresponding record for this reference.
  97. 97
    Wilkinson, B., and Gilbert, H. F.2004 Protein disulfide isomerase Biochim. Biophys. Acta 1699 35 44
    Google Scholar
    There is no corresponding record for this reference.
  98. 98
    West, J. D., and Marnett, L. J.2005 Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal Chem. Res. Toxicol. 18 1642 1653
    Google Scholar
    There is no corresponding record for this reference.
  99. 99
    Carbone, D. L., Doorn, J. A., Kiebler, Z., and Petersen, D. R.2005 Cysteine modification by lipid peroxidation products inhibits protein disulfide isomerase Chem. Res. Toxicol. 18 1324 1331
    Google Scholar
    There is no corresponding record for this reference.
  100. 100
    Bruderer, R. M., Brasseur, C., and Meyer, H. H.2004 The AAA ATPase p97/VCP interacts with its alternative co-factors, Ufd1-Npl4 and p47, through a common bipartite binding mechanism J. Biol. Chem. 279 49609 49616
    Google Scholar
    There is no corresponding record for this reference.
  101. 101
    Cao, K., Nakajima, R., Meyer, H. H., and Zheng, Y.2003 The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis Cell 115 355 367
    Google Scholar
    There is no corresponding record for this reference.
  102. 102
    Hetzer, M., Meyer, H. H., Walther, T. C., Bilbao-Cortes, D., Warren, G., and Mattaj, I. W.2001 Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly Nat. Cell Biol. 3 1086 1091
    Google Scholar
    102
    Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly
    Hetzer, Martin; Meyer, Hemmo H.; Walther, Tobias C.; Bilbao-Cortes, Daniel; Warren, Graham; Mattaj, Iain W.
    Nature Cell Biology (2001), 3 (12), 1086-1091CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
    Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the mol. mechanisms and specificity of fusion events involved in NE formation.
  103. 103
    Patel, S., and Latterich, M.1998 The AAA team: related ATPases with diverse functions Trends Cell Biol. 8 65 71
    Google Scholar
    103
    The AAA team: related ATPases with diverse functions
    Patel, Sheetal; Latterich, Martin
    Trends in Cell Biology (1998), 8 (2), 65-71CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Science Ltd.)
    A review with 56 refs. A new family of related ATPases has emerged, characterized by a highly conserved AAA motif. This motif forms a 230-amino-acid domain that contains Walker homol. sequences and imparts ATPase activity. Homol. between AAA-family members is confined mostly to the AAA domain, although addnl. homol. outside the AAA motif is present among closely related proteins. AAA proteins act in a variety of cellular functions, including cell-cycle regulation, protein degrdn., organelle biogenesis and vesicle-mediated protein transport. The AAA domain is required for protein function, but its exact role and the specific activity that it confers on AAA proteins is still unclear. This review describes current understanding of the AAA protein family.
  104. 104
    Ye, Y., Meyer, H. H., and Rapoport, T. A.2001 The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol Nature 414 652 656
    Google Scholar
    There is no corresponding record for this reference.
  105. 105
    Wang, Q., Song, C., and Li, C. C.2004 Molecular perspectives on p97-VCP: Progress in understanding its structure and diverse biological functions J. Struct. Biol. 146 44 57
    Google Scholar
    There is no corresponding record for this reference.
  106. 106
    Dai, R. M., and Li, C. C.2001 Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation Nat. Cell Biol. 3 740 744
    Google Scholar
    106
    Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation
    Dai, Ren Ming; Li, Chou-Chi H.
    Nature Cell Biology (2001), 3 (8), 740-744CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
    The ubiquitin-proteasome (Ub-Pr) degrdn. pathway regulates many cellular activities, but how ubiquitinated substrates are targeted to the proteasome is not understood. We have shown previously that valosin-contg. protein (VCP) phys. and functionally targets the ubiquitinated nuclear factor κB inhibitor, IκBα to the proteasome for degrdn. VCP is an abundant and a highly conserved member of the AAA (ATPases assocd. with a variety of cellular activities) family. Besides acting as a chaperone in membrane fusions, VCP has been shown to have a role in a no. of seemingly unrelated cellular activities. Here we report that loss of VCP function results in an inhibition of Ub-Pr-mediated degrdn. and an accumulation of ubiquitinated proteins. VCP assocs. with ubiquitinated proteins through the direct binding of its amino-terminal domain to the multi-ubiquitin chains of substrates. Furthermore, its N-terminal domain is required in Ub-Pr-mediated degrdn. We conclude that VCP is a multi-ubiquitin chain-targeting factor that is required in the degrdn. of many Ub-Pr pathway substrates, and provide a common mechanism that underlies many of the functions of VCP.
  107. 107
    Qiu, Y., Benet, L. Z., and Burlingame, A. L.1998 Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry J. Biol. Chem. 273 17940 17953
    Google Scholar
    There is no corresponding record for this reference.
  108. 108
    Doorn, J. A., Hurley, T. D., and Petersen, D. R.2006 Inhibition of human mitochondrial aldehyde dehydrogenase by 4-hydroxynon-2-enal and 4-oxonon-2-enal Chem. Res. Toxicol. 19 102 110
    Google Scholar
    There is no corresponding record for this reference.
  109. 109
    Dietze, E. C., Schafer, A., Omichinski, J. G., and Nelson, S. D.1997 Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide Chem. Res. Toxicol. 10 1097 1103
    Google Scholar
    There is no corresponding record for this reference.
  110. 110
    Uchida, K., and Stadtman, E. R.1993 Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction J. Biol. Chem. 268 6388 6393
    Google Scholar
    110
    Covalent attachment of 4-hydroxynonenal to glyceraldehyde 3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction
    Uchida, Koji; Stadtman, Earl R.
    Journal of Biological Chemistry (1993), 268 (9), 6388-93CODEN: JBCHA3; ISSN:0021-9258.
    In the present study, the detailed mechanism of 4-hydroxynonenal (HNE) modification of a key enzyme in intermediary metab., glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is studied mainly focusing on the formation of HNE-amino acid adducts in the enzyme. When GAPDH (1 mg/mL) was treated with 0-2 mM HNE in sodium phosphate buffer (pH 7.2) for 2 h at 37°, the enzyme was inactivated by HNE in a concn.-dependent manner. The loss of enzyme activity was assocd. with the loss of free sulfhydryl groups. Following its redn. with NaBH4, amino acid anal. of the HNE-modified enzyme demonstrated that histidine and lysine residues were also modified. At concns. lower than 0.5 mM, HNE reacts preferentially with cysteine and lysine residues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HNE-modified enzyme suggested the formation of intra- and intermol. cross-links of the enzyme subunit. The HNE-dependent loss of amino acid residues was accompanied by the generation of protein-linked carbonyl derivs. as assessed by redn. with NaB[3H]H4 and reaction with 2,4-dinitrophenylhydrazine. Thus, the conjugation of all the amino acids appears to involve Michael addn. type reactions in which the carbonyl function of HNE would be preserved. The modified histidine residues were quant. recovered as the HNE-histidine adduct. However, only 28% of the missing lysine could be accounted for as the HNE-lysine deriv., and only 15.6% of the modified cysteine could be accounted for as the HNE-cysteine thioether deriv. It is proposed that the carbonyl groups of the HNE-derived Michael addn. products may undergo secondary reactions with the amino acid groups of lysine residues to yield inter- and intrasubunit cross-links.
  111. 111
    Pumford, N. R., Halmes, N. C., Martin, B. M., Cook, R. J., Wagner, C., and Hinson, J. A.1997 Covalent binding of acetaminophen to N-10-formyltetrahydrofolate dehydrogenase in mice J. Pharmacol. Exp. Ther. 280 501 505
    Google Scholar
    111
    Covalent binding of acetaminophen to N-10-formyl-tetrahydrofolate dehydrogenase in mice
    Pumford, Neil R.; Halmes, N. Christine; Martin, Brian M.; Cook, Robert J.; Wagner, Conrad; Hinson, Jack A.
    Journal of Pharmacology and Experimental Therapeutics (1997), 280 (1), 501-505CODEN: JPETAB; ISSN:0022-3565. (Williams & Wilkins)
    The analgesic acetaminophen is frequently used as a model chem. to study hepatotoxicity; however, the crit. mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatog. and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were homologous with the deduced amino acid sequence from the cDNA of N-10-formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity at 2 h. The covalent binding of acetaminophen to proteins such as N-10-formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.
  112. 112
    Oleinik, N. V., and Krupenko, S. A.2003 Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis Mol. Cancer Res. 1 577 588
    Google Scholar
    112
    Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis
    Oleinik, Natalia V.; Krupenko, Sergey A.
    Molecular Cancer Research (2003), 1 (8), 577-588CODEN: MCROC5; ISSN:1541-7786. (American Association for Cancer Research)
    We have recently shown that transient expression of 10-formyltetrahydrofolate dehydrogenase (FDH) strongly inhibits proliferation of several cancer cell lines and ultimately results in cell death. In the present studies using Tet-On system, we have generated a stable A549 lung carcinoma cell line capable of inducible FDH expression. Using this system, we were able to express FDH at different levels depending on concn. of the inducer, doxycycline, and we have obsd. that inhibition of proliferation depends on FDH intracellular levels. We have further shown that induction of FDH expression results in initiation of apoptosis beginning 24 h post-induction. Apoptotic cells revealed cleavage of poly-(ADP-ribose) polymerase and general caspase inhibitor zVAD-fmk protected cells against FDH-induced apoptosis. FDH-expressing cells showed accumulation of cells in G0-G1 phase and a sharp decrease of cells in S phase. Accumulation of intracellular FDH was followed by accumulation of the tumor suppressor protein p53 and its downstream target p21. These results indicate that FDH antiproliferative effects on A549 cells include both G1 cell cycle arrest and caspase-dependent apoptosis.
  113. 113
    Cho, S. G., Lee, Y. H., Park, H. S., Ryoo, K., Kang, K. W., Park, J., Eom, S. J., Kim, M. J., Chang, T. S., Choi, S. Y., Shim, J., Kim, Y., Dong, M. S., Lee, M. J., Kim, S. G., Ichijo, H., and Choi, E. J.2001 Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1 J. Biol. Chem. 276 12749 12755
    Google Scholar
    113
    Glutathione S-transferase Mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1
    Cho, Ssang-Goo; Lee, Yong Hee; Park, Hee-Sae; Ryoo, Kanghyun; Kang, Keon Wook; Park, Jihyun; Eom, Soo-Jung; Kim, Myung Jin; Chang, Tong-Shin; Choi, Soo-Yeon; Shim, Jaekyung; Kim, Youngho; Dong, Mi-Sook; Lee, Min-Jae; Kim, Sang Geon; Ichijo, Hidenori; Choi, Eui-Ju
    Journal of Biological Chemistry (2001), 276 (16), 12749-12755CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways. It plays a crit. role in cytokine- and stress-induced apoptosis. To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast two-hybrid method. The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metab. of drugs and xenobiotics, interacted with ASK1. We subsequently confirmed that mGSTM1-1 phys. assocd. with ASK1 both in vivo and in vitro. The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells. MGSTM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metab.
  114. 114
    Ryoo, K., Huh, S. H., Lee, Y. H., Yoon, K. W., Cho, S. G., and Choi, E. J.2004 Negative regulation of MEKK1-induced signaling by glutathione S-transferase Mu J. Biol. Chem. 279 43589 43594
    Google Scholar
    There is no corresponding record for this reference.
  115. 115
    Dorion, S., Lambert, H., and Landry, J.2002 Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1 J. Biol. Chem. 277 30792 30797
    Google Scholar
    There is no corresponding record for this reference.
  116. 116
    Nerland, D. E., Cai, J., Pierce, W. M., Jr., and and Benz, F. W.2001 Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo Chem. Res. Toxicol. 14 799 806
    Google Scholar
    There is no corresponding record for this reference.
  117. 117
    Hsieh, J. C., Huang, S. C., Chen, W. L., Lai, Y. C., and Tam, M. F.1991 Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3 Biochem. J. 278Part 1 293 297
    Google Scholar
    There is no corresponding record for this reference.
  118. 118
    Chen, W. L., Hsieh, J. C., Hong, J. L., Tsai, S. P., and Tam, M. F.1992 Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3 Biochem. J. 286Part 1 205 210
    Google Scholar
    There is no corresponding record for this reference.
  119. 119
    Ozdemirler, G., Aykac, G., Uysal, M., and Oz, H.1994 Liver lipid peroxidation and glutathione-related defence enzyme systems in mice treated with paracetamol J. Appl. Toxicol. 14 297 299
    Google Scholar
    119
    Liver lipid peroxidation and glutathione-related defense enzyme systems in mice treated with paracetamol
    Ozdemirler, Gul; Aykac, Gulcin; Uysal, Mujdat; Oz, Hikmet
    Journal of Applied Toxicology (1994), 14 (4), 297-9CODEN: JJATDK; ISSN:0260-437X.
    Glutathione levels were found to be decreased while lipid peroxide levels were increased in total liver homogenates 6 h following paracetamol treatment (500 mg kg-1 i.p.). Furthermore, it has been detd. that cytosolic glutathione S-transferase (GST) activity was decreased and glutathione peroxidase (GSH-Px) activity remained unchanged. On the other hand, a decrease in liver microsomal lipid peroxide levels and an increase in GST and GSH-Px activity has been obsd. The authors concluded that decreased lipid peroxide levels in microsomes could be a consequence of increased GSH-Px and GST enzyme activities. In this way, these glutathione-related defense enzyme systems may play an important role in protecting microsomes from lipid peroxidn.
  120. 120
    Vega, M. C., Walsh, S. B., Mantle, T. J., and Coll, M.1998 The three-dimensional structure of Cys-47-modified mouse liver glutathione S-transferase P1-1. Carboxymethylation dramatically decreases the affinity for glutathione and is associated with a loss of electron density in the alphaB-310B region J. Biol. Chem. 273 2844 2850
    Google Scholar
    There is no corresponding record for this reference.
  121. 121
    Koen, Y. M., Yue, W., Galeva, N. A., Williams, T. D., and Hanzlik, R. P.2006 Site-specific arylation of rat glutathione s-transferase A1 and A2 by bromobenzene metabolites in vivo Chem. Res. Toxicol. 19 1426 1434
    Google Scholar
    There is no corresponding record for this reference.
  122. 122
    Mitchell, A. E., Morin, D., Lame, M. W., and Jones, A. D.1995 Purification, mass spectrometric characterization, and covalent modification of murine glutathione S-transferases Chem. Res. Toxicol. 8 1054 1062
    Google Scholar
    There is no corresponding record for this reference.
  123. 123
    Berhane, K., Widersten, M., Engstrom, A., Kozarich, J. W., and Mannervik, B.1994 Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases Proc. Natl. Acad. Sci. U.S.A. 91 1480 1484
    Google Scholar
    There is no corresponding record for this reference.
  124. 124
    Hartley, D. P., Ruth, J. A., and Petersen, D. R.1995 The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione S-transferase Arch. Biochem. Biophys. 316 197 205
    Google Scholar
    There is no corresponding record for this reference.
  125. 125
    Chelikani, P., Fita, I., and Loewen, P. C.2004 Diversity of structures and properties among catalases Cell Mol. Life Sci. 61 192 208
    Google Scholar
    125
    Diversity of structures and properties among catalases
    Chelikani, P.; Fita, I.; Loewen, P. C.
    Cellular and Molecular Life Sciences (2004), 61 (2), 192-208CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review. More than 300 catalase sequences are now available, divided among monofunctional catalases (>225), bifunctional catalase-peroxidases (>50) and Mn-contg. catalases (>25). When combined with the recent appearance of crystal structures from at least 2 representatives from each of these groups (9 from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large no. of modifications unique to catalases, a result of interacting with reactive O species (ROS). Biochem. and physiol. characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in microorganisms generally is controlled either by sensors of ROS or by growth phase regulons, although the detailed mechanisms vary considerably.
  126. 126
    Kahl, R., Kampkotter, A., Watjen, W., and Chovolou, Y.2004 Antioxidant enzymes and apoptosis Drug Metab. Rev. 36 747 762
    Google Scholar
    126
    Antioxidant enzymes and apoptosis
    Kahl, Regine; Kampkoetter, Andreas; Waetjen, Wim; Chovolou, Yvonni
    Drug Metabolism Reviews (2004), 36 (3-4), 747-762CODEN: DMTRAR; ISSN:0360-2532. (Marcel Dekker, Inc.)
    A review. A review on the relation between apoptosis and the two antioxidant enzymes, manganese superoxide dismutase (MnSOD) and catalase. Two schemes for the involvement of MnSOD and catalase in the regulation of apoptosis are established. First, both MnSOD and catalase inhibit apoptosis by removing superoxide anion radicals or H2O2, resp., because these reactive oxygen species are mediators required for the apoptotic program to inhibit a survival pathway. Second, an increase in H2O2 by downregulation or inhibition of catalase activity and/or upregulation of MnSOD activity inhibits apoptosis while a decrease in H2O2 by upregulation of catalase activity and/or downregulation of MnSOD activity supports apoptosis, possibly because of a supportive role of H2O2 in a survival pathway.
  127. 127
    Gonzalez, F. J., Peters, J. M., and Cattley, R. C.1998 Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activator receptor alpha J. Natl. Cancer Inst. 90 1702 1709
    Google Scholar
    127
    Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activated receptor α
    Gonzalez, Frank J.; Peters, Jeffrey M.; Cattley, Russell C.
    Journal of the National Cancer Institute (1998), 90 (22), 1702-1709CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)
    A review with 77 refs. Peroxisome proliferators are a diverse group of chems. that include several therapeutically used drugs (e.g., hypolipidemic agents), plasticizers and org. solvents used in the chem. industry, herbicides, and naturally occurring hormones. As the name implies, peroxisome proliferators cause an increase in the no. and size of peroxisomes in the liver, kidney, and heart tissue of susceptible species, such as rats and mice. Long-term administration of peroxisome proliferators can cause liver cancer in these animals, a response that has been the central issue of research on peroxisome proliferators for many years. Peroxisome proliferators are representative of the class of nongenotoxic carcinogens that cause cancer through mechanisms that do not involve direct DNA damage. The fact that humans are frequently exposed to these agents makes them of particular concern to government regulatory agencies responsible for assuring human safety. Whether frequent exposure to peroxisome proliferators represents a hazard to humans is unknown; however, increased cancer risk has not been shown to be assocd. with long-term therapeutic administration of the hypolipidemic drugs gemfibrozil, fenofibrate, and clofibrate. To make sound judgments regarding the safety of peroxisome proliferators, the validity of extrapolating results from rodent bioassays to humans must be based on the agents' mechanism of action and species differences in biol. activity and carcinogenicity. The peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor superfamily, has been found to mediate the activity of peroxisome proliferators in mice. Gene-knockout mice lacking PPARα are refractory to peroxisome proliferation and peroxisome proliferator-induced changes in gene expression. Furthermore, PPARα-null mice are resistant to hepatocarcinogenesis when fed a diet contg. a potent nongenotoxic carcinogen WY-14,643. Recent studies have revealed that humans have considerably lower levels of PPARα in liver than rodents, and this difference may, in part, explain the species differences in the carcinogenic response to peroxisome proliferators.
  128. 128
    Reddy, J. K.2004 Peroxisome proliferators and peroxisome proliferator-activated receptor alpha: Biotic and xenobiotic sensing Am. J. Pathol. 164 2305 2321
    Google Scholar
    128
    Peroxisome proliferators and peroxisome proliferator-activator receptor α: Biotic and xenobiotic sensing
    Reddy, Janardan K.
    American Journal of Pathology (2004), 164 (6), 2305-2321CODEN: AJPAA4; ISSN:0002-9440. (American Society for Investigative Pathology)
    A review discusses factual and conceptual advances made in the field of peroxisome proliferator-activated receptors (PPARs). It focuses on the PPAR family of nuclear receptors and PPARα as a sensor for xenobiotic peroxisome proliferators.
  129. 129
    Qiu, Y., Benet, L. Z., and Burlingame, A. L.2001 Identification of hepatic protein targets of the reactive metabolites of the non-hepatotoxic regioisomer of acetaminophen, 3′-hydroxyacetanilide, in the mouse in vivo using two-dimensional gel electrophoresis and mass spectrometry Adv. Exp. Med. Biol. 500 663 673
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!
Citation Statements
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 58 publications.

  1. Wei Li, Zixia Hu, Chen Sun, Yuwei Wang, Weiwei Li, Ying Peng, Jiang Zheng. A Metabolic Activation-Based Chemoproteomic Platform to Profile Adducted Proteins Derived from Furan-Containing Compounds. ACS Chemical Biology 2022, 17 (4) , 873-882. https://doi.org/10.1021/acschembio.1c00917
  2. Timon Geib, Madhuranayaki Thulasingam, Jesper Z. Haeggström, Lekha Sleno. Investigation of Clozapine and Olanzapine Reactive Metabolite Formation and Protein Binding by Liquid Chromatography-Tandem Mass Spectrometry. Chemical Research in Toxicology 2020, 33 (9) , 2420-2431. https://doi.org/10.1021/acs.chemrestox.0c00191
  3. Dongju Lin, Weiwei Li, Ying Peng, Chunfeng Jiang, Youjun Xu, Huiyuan Gao, and Jiang Zheng . Role of Metabolic Activation in 8-Epidiosbulbin E Acetate-Induced Liver Injury: Mechanism of Action of the Hepatotoxic Furanoid. Chemical Research in Toxicology 2016, 29 (3) , 359-366. https://doi.org/10.1021/acs.chemrestox.5b00501
  4. Makan Golizeh, André LeBlanc, and Lekha Sleno . Identification of Acetaminophen Adducts of Rat Liver Microsomal Proteins using 2D-LC-MS/MS. Chemical Research in Toxicology 2015, 28 (11) , 2142-2150. https://doi.org/10.1021/acs.chemrestox.5b00317
  5. Sarah C. Shuck, Orrette R. Wauchope, Kristie L. Rose, Philip J. Kingsley, Carol A. Rouzer, Steven M. Shell, Norie Sugitani, Walter J. Chazin, Irene Zagol-Ikapitte, Olivier Boutaud, John A. Oates, James J. Galligan, William N. Beavers, and Lawrence J. Marnett . Protein Modification by Adenine Propenal. Chemical Research in Toxicology 2014, 27 (10) , 1732-1742. https://doi.org/10.1021/tx500218g
  6. Jinghua Yu, Pan Deng, Dafang Zhong, and Xiaoyan Chen . Identification of Amino Acid and Glutathione N-Conjugates of Toosendanin: Bioactivation of the Furan Ring Mediated by CYP3A4. Chemical Research in Toxicology 2014, 27 (9) , 1598-1609. https://doi.org/10.1021/tx5002145
  7. Lisa A. Peterson . Reactive Metabolites in the Biotransformation of Molecules Containing a Furan Ring. Chemical Research in Toxicology 2013, 26 (1) , 6-25. https://doi.org/10.1021/tx3003824
  8. S. Cyrus Khojasteh, Dylan P. Hartley, Kevin A. Ford, Hirdesh Uppal, Shimako Oishi, and Sidney D. Nelson . Characterization of Rat Liver Proteins Adducted by Reactive Metabolites of Menthofuran. Chemical Research in Toxicology 2012, 25 (11) , 2301-2309. https://doi.org/10.1021/tx300144d
  9. Yuqin Shang, Ray Mernaugh, and Xiangqun Zeng . Characterization of the Native and Denatured Herceptin by Enzyme Linked Immunosorbent Assay and Quartz Crystal Microbalance Using a High-Affinity Single Chain Fragment Variable Recombinant Antibody. Analytical Chemistry 2012, 84 (19) , 8164-8170. https://doi.org/10.1021/ac301235a
  10. Nicholas M. Njuguna, Collen Masimirembwa, and Kelly Chibale . Identification and Characterization of Reactive Metabolites in Natural Products-Driven Drug Discovery. Journal of Natural Products 2012, 75 (3) , 507-513. https://doi.org/10.1021/np200786j
  11. Anthony Lozama, Christopher W. Cunningham, Michael J. Caspers, Justin T. Douglas, Christina M. Dersch, Richard B. Rothman, and Thomas E. Prisinzano . Opioid Receptor Probes Derived from Cycloaddition of the Hallucinogen Natural Product Salvinorin A. Journal of Natural Products 2011, 74 (4) , 718-726. https://doi.org/10.1021/np1007872
  12. Thomas E. Prisinzano. Natural Products as Tools for Neuroscience: Discovery and Development of Novel Agents to Treat Drug Abuse. Journal of Natural Products 2009, 72 (3) , 581-587. https://doi.org/10.1021/np8005748
  13. Keisuke Ikehata, Tatyana G. Duzhak, Nadezhda A. Galeva, Tao Ji, Yakov M. Koen and Robert P. Hanzlik . Protein Targets of Reactive Metabolites of Thiobenzamide in Rat Liver in Vivo. Chemical Research in Toxicology 2008, 21 (7) , 1432-1442. https://doi.org/10.1021/tx800093k
  14. Sajid Iqbal, Faisal Ahmad, Abdul Haleem, Bushra Rehman, Salma Mohammed Aljahdali, Sajjad Ahmad, Muniba Jadoon, Qurban Ali, Talmeez Ur Rehman, Sajjad Ahmed Khan, Safia Ahmed. Bioprospecting potential, metabolic profiling, and molecular docking studies of metabolites from endophytic fungi associated with Teucrium stocksianum. Journal of Biomolecular Structure and Dynamics 2025, 2 , 1-16. https://doi.org/10.1080/07391102.2025.2513571
  15. Fernando Bessone, Geraldine L. Hillotte, Natalia Ahumada, Fernanda Jaureguizahar, Anabela C. Medeot, Marcelo G. Roma. UDCA for Drug-Induced Liver Disease: Clinical and Pathophysiological Basis. Seminars in Liver Disease 2024, 44 (01) , 001-022. https://doi.org/10.1055/s-0044-1779520
  16. Zahra Sadeghi, Jun-Li Yang, Alessandro Venditti, Mahdi Moridi Farimani. A review of the phytochemistry, ethnopharmacology and biological activities of Teucrium genus (Germander). Natural Product Research 2022, 36 (21) , 5647-5664. https://doi.org/10.1080/14786419.2021.2022669
  17. Min Tian, Ying Peng, Jiang Zheng. Metabolic Activation and Hepatotoxicity of Furan-Containing Compounds. Drug Metabolism and Disposition 2022, 50 (5) , 655-670. https://doi.org/10.1124/dmd.121.000458
  18. Fuad O. Abdullah, Faiq H. S. Hussain, Abdullah Sh. Sardar, Gianluca Gilardoni, Zaw Min Thu, Giovanni Vidari. Bio-Active Compounds from Teucrium Plants Used in the Traditional Medicine of Kurdistan Region, Iraq. Molecules 2022, 27 (10) , 3116. https://doi.org/10.3390/molecules27103116
  19. Wenxiang Fan, Linhong Fan, Zhengtao Wang, Li Yang. Limonoids From the Genus Melia (Meliaceae): Phytochemistry, Synthesis, Bioactivities, Pharmacokinetics, and Toxicology. Frontiers in Pharmacology 2022, 12 https://doi.org/10.3389/fphar.2021.795565
  20. Min-Ran Wang, Jing-Yang He, Ji-Xiang He, Ke-Ke Liu, Jing Yang. A Paal–Knorr agent for chemoproteomic profiling of targets of isoketals in cells. Chemical Science 2021, 12 (43) , 14557-14563. https://doi.org/10.1039/D1SC02230J
  21. Yi-Kun Wang, Wen Qun Li, Shuang Xia, Lin Guo, Yan Miao, Bi-Kui Zhang. Metabolic Activation of the Toxic Natural Products From Herbal and Dietary Supplements Leading to Toxicities. Frontiers in Pharmacology 2021, 12 https://doi.org/10.3389/fphar.2021.758468
  22. Hyelim Cho, Qiong Shen, Lydia H. Zhang, Mikiko Okumura, Akinori Kawakami, Jessi Ambrose, Frederic Sigoillot, Howard R. Miller, Scott Gleim, Amanda Cobos-Correa, Ying Wang, Philippe Piechon, Guglielmo Roma, Fabian Eggimann, Charles Moore, Peter Aspesi, Felipa A. Mapa, Heather Burks, Nathan T. Ross, Philipp Krastel, Marc Hild, Thomas J. Maimone, David E. Fisher, Daniel K. Nomura, John A. Tallarico, Stephen M. Canham, Jeremy L. Jenkins, William C. Forrester. CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism. Cell Chemical Biology 2021, 28 (10) , 1407-1419.e6. https://doi.org/10.1016/j.chembiol.2021.03.004
  23. Qiyan Lin, Huida Guan, Chao Ma, Liangni Chen, Lanlan Cao, Hanze Liu, Xuemei Cheng, Changhong Wang. Biotransformation patterns of dictamnine in vitro/in vivo and its relative molecular mechanism of dictamnine-induced acute liver injury in mice. Environmental Toxicology and Pharmacology 2021, 85 , 103628. https://doi.org/10.1016/j.etap.2021.103628
  24. Maria-Eleni Grafakou, Christina Barda, Helen Skaltsa. Secondary Metabolites of Teucrium Species with Toxic Effects. 2020, 211-230. https://doi.org/10.1007/978-3-030-52159-2_8
  25. Bo Wen, Peter Gorycki. Bioactivation of herbal constituents: mechanisms and toxicological relevance. Drug Metabolism Reviews 2019, 51 (4) , 453-497. https://doi.org/10.1080/03602532.2019.1655570
  26. Rhian B. Cope. Toxicology and Drug Interactions of Nutraceuticals. 2019, 711-727. https://doi.org/10.1007/978-3-030-04624-8_51
  27. Zhuo-Qing Li, Li-Long Jiang, Dong-Sheng Zhao, Jing Zhou, Ling-Li Wang, Zi-Tian Wu, Xian Zheng, Zi-Qi Shi, Ping Li, Hui-Jun Li. The Modulatory Role of CYP3A4 in Dictamnine-Induced Hepatotoxicity. Frontiers in Pharmacology 2018, 9 https://doi.org/10.3389/fphar.2018.01033
  28. Yitong Liu. Incorporation of absorption and metabolism into liver toxicity prediction for phytochemicals: A tiered in silico QSAR approach. Food and Chemical Toxicology 2018, 118 , 409-415. https://doi.org/10.1016/j.fct.2018.05.039
  29. Christopher Brewer, Taosheng Chen. Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450. International Journal of Molecular Sciences 2017, 18 (11) , 2353. https://doi.org/10.3390/ijms18112353
  30. Matthew Y. Bendikov, John O. Miners, Bradley S. Simpson, David J. Elliot, Susan J. Semple, David J. Claudie, Ross A. McKinnon, Elizabeth M. J. Gillam, Matthew J. Sykes. In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes. Xenobiotica 2017, 47 (6) , 461-469. https://doi.org/10.1080/00498254.2016.1203041
  31. Xiaoyan Lu, Cai Ji, Wei Tong, Xueping Lian, Ying Wu, Xiaohui Fan, Yue Gao. Integrated analysis of microRNA and mRNA expression profiles highlights the complex and dynamic behavior of toosendanin-induced liver injury in mice. Scientific Reports 2016, 6 (1) https://doi.org/10.1038/srep34225
  32. Dongju Lin, Kai Wang, Xiucai Guo, Huiyuan Gao, Ying Peng, Jiang Zheng. Lysine- and cysteine-based protein adductions derived from toxic metabolites of 8-epidiosbulbin E acetate. Toxicology Letters 2016, 264 , 20-28. https://doi.org/10.1016/j.toxlet.2016.10.007
  33. Makan Golizeh, Timon Geib, Lekha Sleno. Identification of 4‐hydroxynonenal protein targets in rat, mouse and human liver microsomes by two‐dimensional liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2016, 30 (13) , 1488-1494. https://doi.org/10.1002/rcm.7577
  34. Susanne Ramm, Elisabeth Limbeck, Angela Mally. Functional and cellular consequences of covalent target protein modification by furan in rat liver. Toxicology 2016, 361-362 , 49-61. https://doi.org/10.1016/j.tox.2016.06.018
  35. H. Li, Y. Peng, J. Zheng. Metabolic Activation and Toxicities of Furanoterpenoids. 2016, 55-97. https://doi.org/10.1016/B978-0-12-804700-2.00002-7
  36. T. Eric Ballard, Upendra P. Dahal, Andrew J. Bessire, Richard P. Schneider, Kieran F. Geoghegan, Alfin D. N. Vaz. A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry. Rapid Communications in Mass Spectrometry 2015, 29 (22) , 2175-2183. https://doi.org/10.1002/rcm.7375
  37. J.R. Roede, K.S. Fritz. Hepatotoxicity of Reactive Aldehydes☆. 2015https://doi.org/10.1016/B978-0-12-801238-3.02113-9
  38. Xiaoyan Chen. Profiling and Characterization of Bioactive Substances of Herbal Medicine and their Metabolites Using Mass Spectrometry. 2014, 1-64. https://doi.org/10.1002/9781118541203.xen0032
  39. Robert P. Hanzlik, Yakov M. Koen, Jianwen Fang. Bioinformatic Analysis of 302 Reactive Metabolite Target Proteins. Which Ones Are Important for Cell Death?. Toxicological Sciences 2013, 135 (2) , 390-401. https://doi.org/10.1093/toxsci/kft166
  40. J. Joven, A. Rull, E. Rodriguez-Gallego, J. Camps, M. Riera-Borrull, A. Hernández-Aguilera, V. Martin-Paredero, A. Segura-Carretero, V. Micol, C. Alonso-Villaverde, J.A. Menéndez. Multifunctional targets of dietary polyphenols in disease: A case for the chemokine network and energy metabolism. Food and Chemical Toxicology 2013, 51 , 267-279. https://doi.org/10.1016/j.fct.2012.10.004
  41. . Bioactivation and Natural Products. 2012, 203-224. https://doi.org/10.1002/9783527655748.ch9
  42. Vanessa González‐Pérez, David J. Kroll. ADME of Natural Toxins. 2012, 1-16. https://doi.org/10.1002/9780470921920.edm044
  43. S. Moro, J. K. Chipman, P. Antczak, N. Turan, W. Dekant, F. Falciani, A. Mally. Identification and Pathway Mapping of Furan Target Proteins Reveal Mitochondrial Energy Production and Redox Regulation as Critical Targets of Furan Toxicity. Toxicological Sciences 2012, 126 (2) , 336-352. https://doi.org/10.1093/toxsci/kfs005
  44. B.K. Park, H. Laverty, A. Srivastava, D.J. Antoine, D. Naisbitt, D.P. Williams. Drug bioactivation and protein adduct formation in the pathogenesis of drug-induced toxicity. Chemico-Biological Interactions 2011, 192 (1-2) , 30-36. https://doi.org/10.1016/j.cbi.2010.09.011
  45. Shoude Zhang, Weiqiang Lu, Xiaofeng Liu, Yanyan Diao, Fang Bai, Liyan Wang, Lei Shan, Jin Huang, Honglin Li, Weidong Zhang. Fast and effective identification of the bioactive compounds and their targets from medicinal plants via computational chemical biology approach. MedChemComm 2011, 2 (6) , 471. https://doi.org/10.1039/c0md00245c
  46. Angela Mally, Carmen Graff, Olga Schmal, Sabrina Moro, Carolin Hamberger, Ute M. Schauer, Jens Brück, Sibel Özden, Max Sieber, Ulrich Steger, Dieter Schrenk, Gordon C. Hard, James Kevin Chipman, Wolfgang Dekant. Functional and proliferative effects of repeated low‐dose oral administration of furan in rat liver. Molecular Nutrition & Food Research 2010, 54 (11) , 1556-1567. https://doi.org/10.1002/mnfr.201000064
  47. Andreina Ricci, Simona Piccolella, Antonio Fiorentino, Federico Pepi, Brigida D'Abrosca, Pietro Monaco. A tandem mass spectrometric investigation of the low‐energy collision‐activated fragmentation of neo ‐clerodane diterpenes. Rapid Communications in Mass Spectrometry 2010, 24 (11) , 1543-1556. https://doi.org/10.1002/rcm.4545
  48. Ka‐Wing Cheng, Chi‐Chun Wong, Mingfu Wang, Qing‐Yu He, Feng Chen. Identification and characterization of molecular targets of natural products by mass spectrometry. Mass Spectrometry Reviews 2010, 29 (1) , 126-155. https://doi.org/10.1002/mas.20235
  49. María José Gómez-Lechón, José V. Castell, María Teresa Donato. The Use of Hepatocytes to Investigate Drug Toxicity. 2010, 389-415. https://doi.org/10.1007/978-1-60761-688-7_21
  50. A. Srivastava, J. L. Maggs, D. J. Antoine, D. P. Williams, D. A. Smith, B. K. Park. Role of Reactive Metabolites in Drug-Induced Hepatotoxicity. 2010, 165-194. https://doi.org/10.1007/978-3-642-00663-0_7
  51. J.R. Roede, B.J. Stewart, D.R. Petersen. Hepatotoxicity of Reactive Aldehydes. 2010, 581-594. https://doi.org/10.1016/B978-0-08-046884-6.01025-3
  52. Yang Wang, Yoshiaki Azuma, David B Friedman, Robert J Coffey, Kristi L Neufeld. Novel association of APC with intermediate filaments identified using a new versatile APC antibody. BMC Cell Biology 2009, 10 (1) https://doi.org/10.1186/1471-2121-10-75
  53. Jianwen Fang, Yakov M Koen, Robert P Hanzlik. Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners. BMC Chemical Biology 2009, 9 (1) https://doi.org/10.1186/1472-6769-9-5
  54. Matthew T. Labenski, Ashley A. Fisher, Herng-Hsiang Lo, Terrence J. Monks, Serrine S. Lau. Protein Electrophile-Binding Motifs: Lysine-Rich Proteins Are Preferential Targets of Quinones. Drug Metabolism and Disposition 2009, 37 (6) , 1211-1218. https://doi.org/10.1124/dmd.108.026211
  55. Robert P. Hanzlik, Jianwen Fang, Yakov M. Koen. Filling and mining the reactive metabolite target protein database. Chemico-Biological Interactions 2009, 179 (1) , 38-44. https://doi.org/10.1016/j.cbi.2008.08.016
  56. Denise S. Simpson, Kimberly M. Lovell, Anthony Lozama, Nina Han, Victor W. Day, Christina M. Dersch, Richard B. Rothman, Thomas E. Prisinzano. Synthetic studies of neoclerodane diterpenes from Salvia divinorum: role of the furan in affinity for opioid receptors. Organic & Biomolecular Chemistry 2009, 7 (18) , 3748. https://doi.org/10.1039/b905148a
  57. . Current World Literature. Current Opinion in Clinical Nutrition & Metabolic Care 2009, 95-103. https://doi.org/10.1097/MCO.0b013e32831fd97a
  58. Jeroen Carol-Visser, Marcel van der Schans, Alex Fidder, Albert G. Hulst, Ben L.M. van Baar, Hubertus Irth, Daan Noort. Development of an automated on-line pepsin digestion–liquid chromatography–tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents. Journal of Chromatography B 2008, 870 (1) , 91-97. https://doi.org/10.1016/j.jchromb.2008.06.008
Download PDF
Go to Chemical Research in Toxicology
Get e-Alerts
Get e-Alerts

Chemical Research in Toxicology

Cite this: Chem. Res. Toxicol. 2007, 20, 10, 1393–1408
Click to copy citationCitation copied!
https://doi.org/10.1021/tx7001405
Published September 25, 2007

Copyright © 2007 American Chemical Society. This publication is licensed under these Terms of Use.

Request reuse permissions

Article Views

2334

Altmetric

-

Citations

58
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Scheme 1

    Scheme 1. Proposed Mechanism for the Bioactivation of 3-Substituted Furans and the Formation of Reactive Electrophilic Enedial Metabolites
    High Resolution Image
    Download MS PowerPoint Slide

    Scheme 2

    Scheme 2. Synthesis of the Enedial Derivative of Teucrin A and N-Terminal Biotinylated Peptide Conjugatesa
    High Resolution Image
    Download MS PowerPoint Slide

    Scheme a(a) Dimethyldioxirane (DMDO), acetone. (b) Btn-AKDVY, 0.1 M phosphate buffer, pH 7.4. (c) Btn-ACDVY, 0.2 M phosphate buffer, pH 7.4.

    Figure 1

    Figure 1. 1H NMR and COSY spectra (500 MHz, DMSO-d6) of the conjugate of 1 with Btn-AKDVY peptide (2) with the labeled structure showing the positions of the relevant protons.

    High Resolution Image
    Download MS PowerPoint Slide

    Scheme 3

    Scheme 3. Proposed Mechanism of Pyrroline-2-one Adduct Formation and Epimerization of C12
    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Process of the anti-teucrin A ScFv antibody selection from the rodent phage-displayed library.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Western blot of BSA adducted in vitro with teucrin A enedial using the periplasmic extracts of the selected 2I2 ScFv clone.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Crystal structure of human serum albumin (PDB: 1AO6 ) with the highlighted residues corresponding to the ones significantly adducted in bovine serum albumin (BSA) by teucrin A enedial 1 in vitro (lysines in red, cysteine in yellow, and L233 in white). The right frame is rotated 90° with respect to the left frame.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. Flow chart of the immunoprecipitation procedure with the 2I2 ScFv antibody.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. Representative MS/MS spectrum of a BSA peptide modified on the lysine residue by teucrin A enedial.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 7

    Figure 7. (A) Time course of the plasma alanine aminotrasferase (ALT) activity in Spague–Dawley rats (n = 3) after oral administration of 100 mg/kg of teucrin A. (B) Western blot of the rat liver proteins adducted in vivo by teucrin A metabolite(s) detected with the periplasmic extracts of the selected 2I2 ScFv clone.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 8

    Figure 8. Networks and canonical pathways generated by Ingenuity Pathways Analysis for the targets of teucrin A, called focused genes. (A) The direct interactions between the focused genes (gray) and the transcriptional regulators in network 1. (B) The direct interactions between the focused genes (gray) and the transcriptional regulators in network 2. (C) The cellular pathways, functions, and diseases associated with the focused genes. Abbreviations: A, activation; E, expression; T, transcription; PD, protein–DNA interaction; PP, protein–protein interaction. Network 1: BRCA1, breast cancer 1, early onset; DGKA, diacylglycerol kinase α; PPARA, peroxisome proliferative activated receptor α; PPARG, peroxisome proliferative activated receptor γ; SP1, Sp1 transcription factor; SRC, v-src sarcoma (Schmidt–Ruppin A-2) viral oncogene homologue; SREBF2, sterol regulatory element binding transcription factor 2; TP53, tumor protein 53. Network 2: ABCB11, ATP-binding cassette, subfamily B (MDR/TAP), member 11; AKR1C4, aldo-keto reductase family 1, member C4; CYP2C9, cytochrome P450, family 2, subfamily C, polypeptide 9; ESR1, estrogen receptor 1; FOS, v-fos FBJ murine osteosarcoma viral oncogene homologue; GATA4, GATA binding protein 4; HNF4A, hepatocyte nuclear factor 4α; RXRA, retinoid X receptor α; TCF1, transcription factor 1, hepatic (hepatic nuclear factor HNF1); UGT2B1, UDP glucuronosyltransferase 2 family, polypepetide B1; UGT2B7, UDP glucuronosyltransferase 2 family, polypeptode B7; XBP1, X-box binding protein 1.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 129 other publications.

    1. 1
      Guengerich, F. P., and Liebler, D. C.1985 Enzymatic activation of chemicals to toxic metabolites Crit. Rev. Toxicol. 14 259 307
      1
      Enzymic activation of chemicals to toxic metabolites
      Guengerich, F. Peter; Liebler, Daniel C.
      Critical Reviews in Toxicology (1985), 14 (3), 259-307CODEN: CRTXB2; ISSN:0045-6446.
      A review with 333 refs. on the enzymol. related to the bioactivation of xenobiotics, the key chem. principles which govern the prodn. and fate of reactive compds., and some biol. and biochem. factors which are important in considering potential toxicity.
    2. 2
      Pumford, N. R.1997 Protein targets of xenobiotic reactive intermediates Annu. Rev. Pharmacol. Toxicol. 37 91 117
      There is no corresponding record for this reference.
    3. 3
      Petersen, D. R., and Doorn, J. A.2004 Reactions of 4-hydroxynonenal with proteins and cellular targets Free Radical Biol. Med. 37 937 945
      3
      Reactions of 4-hydroxynonenal with proteins and cellular targets
      Petersen, Dennis R.; Doorn, Jonathan A.
      Free Radical Biology & Medicine (2004), 37 (7), 937-945CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
      A review. Peroxidative degrdn. of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidn., redn., and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidn. and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo prodn. of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alc.-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biol. signaling.
    4. 4
      Carbone, D. L., Doorn, J. A., Kiebler, Z., Ickes, B. R., and Petersen, D. R.2005 Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease J. Pharmacol. Exp. Ther. 315 8 15
      4
      Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease
      Carbone, David L.; Doorn, Jonathan A.; Kiebler, Zachary; Ickes, Brian R.; Petersen, Dennis R.
      Journal of Pharmacology and Experimental Therapeutics (2005), 315 (1), 8-15CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
      Lipid peroxidn. during oxidative stress leads to increased concns. of thiol-reactive α,β-unsatd. aldehyde, including 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepd. from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alc.-treated animals. In vitro chaperoning expts. using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concns. of this aldehyde ranging from 23 to 450 μM. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concns. as low as 6 μM (1:1 M ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by liq. chromatog./tandem mass spectrometry peptide anal. identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alc.-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.
    5. 5
      West, J. D., and Marnett, L. J.2006 Endogenous reactive intermediates as modulators of cell signaling and cell death Chem. Res. Toxicol. 19 173 194
      5
      Endogenous Reactive Intermediates as Modulators of Cell Signaling and Cell Death
      West, James D.; Marnett, Lawrence J.
      Chemical Research in Toxicology (2006), 19 (2), 173-194CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A review. The prodn. of endogenous reactive intermediates, the mechanistic features of cell death, and the signaling pathways that these intermediates ultimately alter to influence cell death processes are discussed.
    6. 6
      Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A.2004 Drug-protein adducts: An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development Chem. Res. Toxicol. 17 3 16
      6
      Drug-Protein Adducts: An Industry Perspective on Minimizing the Potential for Drug Bioactivation in Drug Discovery and Development
      Evans, David C.; Watt, Alan P.; Nicoll-Griffith, Deborah A.; Baillie, Thomas A.
      Chemical Research in Toxicology (2004), 17 (1), 3-16CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A review. It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug-protein adducts and organ toxicity. Because the potential does exist, however, for these processes to be causally related, the general strategy at Merck Research Labs. has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metab. vectors and typically involves the initial use of small mol. trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) prepns. from preclin. species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Labs. The availability of a dedicated Labeled Compd. Synthesis group, coupled to a close working relationship between Drug Metab. and Medicinal Chem., represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.
    7. 7
      Baillie, T. A., Cayen, M. N., Fouda, H., Gerson, R. J., Green, J. D., Grossman, S. J., Klunk, L. J., LeBlanc, B., Perkins, D. G., and Shipley, L. A.2002 Drug metabolites in safety testing Toxicol. Appl. Pharmacol. 182 188 196
      7
      Drug metabolites in safety testing
      Baillie, Thomas A.; Cayen, Mitchell N.; Fouda, Hassan; Gerson, Ronald J.; Green, James D.; Grossman, Scott J.; Klunk, Lewis J.; LeBlanc, Bernard; Perkins, Darcy G.; Shipley, Lisa A.
      Toxicology and Applied Pharmacology (2002), 182 (3), 188-196CODEN: TXAPA9; ISSN:0041-008X. (Elsevier Science)
      A review, summarizing the deliberations of a multidisciplinary committee, sponsored by the Pharmaceutical Research and Manufacturers of America, on current "best practices" within the U.S. pharmaceutical industry in assessing the role of drug metabolites as potential mediators of the toxicity of new drug products. Input to the document was obtained from numerous sources, including members of the pharmaceutical industry, academic investigators, and representatives of regulatory agencies who attended a workshop on the subject in Nov. 2000. The overall goal of the paper is to define practical and scientifically based approaches to the use of metabolite data that address contemporary issues in the safety evaluation of drug candidates. Although there remains a lack of consensus on how best to deal with several aspects of this complex subject, this paper raises a no. of points to consider, which emphasize the need to treat drug metabolite issues on a case-by-case basis. It is hoped that the discussion will promote continued dialog among industrial scientists and regulators charged with ensuring the clin. safety of new therapeutic agents.
    8. 8
      Baillie, T. A., and Kassahun, K.2001 Biological reactive intermediates in drug discovery and development: A perspective from the pharmaceutical industry Adv. Exp. Med. Biol. 500 45 51
      8
      Biological reactive intermediates in drug discovery and development: A perspective from the pharmaceutical industry
      Baillie, Thomas A.; Kassahun, Kelem
      Advances in Experimental Medicine and Biology (2001), 500 (Biological Reactive Intermediates VI), 45-51CODEN: AEMBAP; ISSN:0065-2598. (Kluwer Academic/Plenum Publishers)
      A review. The detection of chem.-reactive, electrophilic metabolites poses a particular problem in the discovery and development of drug candidates in pharmaceutical research, inasmuch as it is not possible to accurately predict the likely toxicol. consequences of these intermediates in animal safety studies or in clin. trials. Advances in anal. instrumentation (notably liq. chromatog.-tandem mass spectrometry [LC-MS/MS]) have facilitated the detection of reactive intermediates through the identification of the glutathione (GSH) adducts to which they normally give rise, while the increased use of radiolabeled tracers in drug development permits an early assessment to be made of the propensity of a drug candidate to undergo covalent binding to cellular macromols. Unfortunately, these advances in anal. methods for the detection and characterization of reactive drug metabolites have far outstripped our understanding of the mechanisms of foreign compd.-mediated toxicities at the mol. level, and of the role of both covalent binding and oxidative stress in the cascade of events that lead ultimately to cellular injury or immune-mediated toxicities. In light of these uncertainties, it seems reasonable to argue that one should attempt to minimize, through structural modification, the extent to which a drug candidate undergoes metab. to reactive intermediates. Therefore, it becomes imperative to have close collaboration between Drug Metab. scientists and their counterparts in Medicinal Chem. during both the discovery and early development phases. Bioactivation of troglitazone and the hepatotoxicity of troglitazone metabolites is discussed as an example.
    9. 9
      Carbone, D. L., Doorn, J. A., and Petersen, D. R.2004 4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase Free Radical Biol. Med. 37 1430 1439
      9
      4-Hydroxynonenal regulates 26S proteasomal degradation of alcohol dehydrogenase
      Carbone, David L.; Doorn, Jonathan A.; Petersen, Dennis R.
      Free Radical Biology & Medicine (2004), 37 (9), 1430-1439CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
      The lipid peroxidn. product 4-hydroxynonenal (4-HNE) was shown to interfere with protein function. The goal of this study was to det. the effects of substrate modification by 4-HNE on protein degrdn. Equine liver alc. dehydrogenase (ADH, EC 1.1.1.1) treated with 2-fold molar excess 4-HNE was degraded by a rabbit reticulocyte lysate (RRL) system approx. 1.5-fold faster than control, while treatment with concns. up to 100-fold molar excess aldehyde were inhibitory to degrdn. Involvement of the 26S proteasome (EC 3.4.99.46) was demonstrated through the use of specific proteasome and ATPase inhibitors, and confirmed by measuring the extent of ADH polyubiquitination. Tryptic digestion and LC/MS anal. of 4-HNE-treated ADH identified modification of two zinc chelating Cys residues. Through mol. modeling expts. a conformational shift in both zinc-contg. regions was predicted, with an approx. doubling of the distance between the structural zinc and its resp. chelating residues. Modification of residues in the active site zinc binding motif resulted in less pronounced alteration in protein structure. The data presented here demonstrate accelerated ubiquitination and proteasomal degrdn. of ADH modified with 4-HNE, and suggest a conformational change after 4-HNE docking as a mechanism behind these observations.
    10. 10
      Thome-Kromer, B., Bonk, I., Klatt, M., Nebrich, G., Taufmann, M., Bryant, S., Wacker, U., and Kopke, A.2003 Toward the identification of liver toxicity markers: A proteome study in human cell culture and rats Proteomics 3 1835 62
      There is no corresponding record for this reference.
    11. 11
      Sampey, B. P., Korourian, S., Ronis, M. J., Badger, T. M., and Petersen, D. R.2003 Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury Alcohol Clin. Exp. Res. 27 1015 1022
      11
      Immunohistochemical Characterization of Hepatic Malondialdehyde and 4-Hydroxynonenal Modified Proteins During Early Stages of Ethanol-Induced Liver Injury
      Sampey, Brante P.; Korourian, Soheila; Ronis, Martin J.; Badger, Thomas M.; Petersen, Dennis R.
      Alcoholism: Clinical and Experimental Research (2003), 27 (6), 1015-1022CODEN: ACRSDM; ISSN:0145-6008. (Lippincott Williams & Wilkins)
      BACKGROUND: Chronic ethanol consumption is assocd. with hepatic lipid peroxidn. and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alc.-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alc.-induced liver injury. METHODS: Female Sprague Dawley rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet contg. ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathol. anal., and induction of cytochrome P 4502E1 (CYP2E1). Immunohistochem. methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quant. image anal. RESULTS: After 36 days of treatment, rats receiving the alc.-contg. diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis assocd. with only minor inflammation and necrosis. Alc. administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quant. immunohistochem. anal. revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepd. from rats treated with alc. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphol. and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells assocd. with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alc.-induced lipid peroxidn. is an early event during alc.-mediated liver injury and may be a sensitizing event resulting in the prodn. of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.
    12. 12
      Koen, Y. M., and Hanzlik, R. P.2002 Identification of seven proteins in the endoplasmic reticulum as targets for reactive metabolites of bromobenzene Chem. Res. Toxicol. 15 699 706
      There is no corresponding record for this reference.
    13. 13
      Koen, Y. M., Williams, T. D., and Hanzlik, R. P.2000 Identification of three protein targets for reactive metabolites of bromobenzene in rat liver cytosol Chem. Res. Toxicol. 13 1326 1335
      There is no corresponding record for this reference.
    14. 14
      Rombach, E. M., and Hanzlik, R. P.1999 Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal protein sulfhydryl groups using specific antibodies Chem. Res. Toxicol. 12 159 163
      There is no corresponding record for this reference.
    15. 15
      Hartley, D. P., Kolaja, K. L., Reichard, J., and Petersen, D. R.1999 4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: Immunochemical detection and lobular localization Toxicol. Appl. Pharmacol. 161 23 33
      15
      4-Hydroxynonenal and Malondialdehyde Hepatic Protein Adducts in Rats Treated with Carbon Tetrachloride: Immunochemical Detection and Lobular Localization
      Hartley, Dylan P.; Kolaja, Kyle L.; Reichard, John; Petersen, Dennis R.
      Toxicology and Applied Pharmacology (1999), 161 (1), 23-33CODEN: TXAPA9; ISSN:0041-008X. (Academic Press)
      The metab. of CCl4 initiates the peroxidn. of polyunsatd. fatty acids producing α,β-unsatd. aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compds. like CCl4. To det. the rate at which CCl4-initiated lipid peroxidn. results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl4 (1.0 mL/kg) and euthanized 0-72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immunohistochem. and immunopptn./Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immunohistochem. in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidn. and hepatocellular necrosis increased. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was obsd. 24 h after CCl4 administration and immunopos. hepatocytes were obsd. in zone 2 as well as zone 3. Immunopos. cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administration, at least four adducted proteins were immunopptd. from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to mol. wts. of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be assocd. with hepatocellular injury following CCl4 exposure. (c) 1999 Academic Press.
    16. 16
      Rombach, E. M., and Hanzlik, R. P.1998 Identification of a rat liver microsomal esterase as a target protein for bromobenzene metabolites Chem. Res. Toxicol. 11 178 184
      There is no corresponding record for this reference.
    17. 17
      Hartley, D. P., and Petersen, D. R.1997 Profiles of hepatic cellular protein adduction by malondialdehyde and 4-hydroxynonenal. Studies with isolated hepatocytes Adv. Exp. Med. Biol. 414 123 131
      17
      Profiles of hepatic cellular protein adduction by malondialdehyde and 4-hydroxynonenal: studies with isolated hepatocytes
      Hartley, Dylan P.; Petersen, Dennis R.
      Advances in Experimental Medicine and Biology (1997), 414 (Enzymology and Molecular Biology of Carbonyl Metabolism 6), 123-131CODEN: AEMBAP; ISSN:0065-2598. (Plenum)
      The prodn. of aldehydic products during a time course of iron-initiated lipid peroxidn. was quantitated in freshly isolated hepatocytes. Aldehyde-adducted proteins were detected by use of polyclonal antibodies produced against malondialdehyde- or 4-hydroxynonenal-protein adducts.
    18. 18
      Hartley, D. P., Kroll, D. J., and Petersen, D. R.1997 Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: Detection of 4-hydroxynonenal- and malondialdehyde-protein adducts Chem. Res. Toxicol. 10 895 905
      There is no corresponding record for this reference.
    19. 19
      Hartley, D. P., Lindahl, R., and Petersen, D. R.1995 Covalent modification of class 2 and class 3 aldehyde dehydrogenase by 4-hydroxynonenal Adv. Exp. Med. Biol. 372 93 101
      19
      Covalent modification of class 2 and class 3 aldehyde dehydrogenase by 4-hydroxynonenal
      Hartley, Dylan P.; Lindahl, Ronald; Petersen, Dennis R.
      Advances in Experimental Medicine and Biology (1995), 372 (Enzymology and Molecular Biology of Carbonyl Metabolism 5), 93-101CODEN: AEMBAP; ISSN:0065-2598. (Plenum)
      The authors used two forms of aldehyde dehydrogenase active site models to assess the role of 4-hydroxynonenal covalent interactions in differential enzymic inactivation of these specific isoforms of aldehyde dehydrogenase.
    20. 20
      Bambal, R. B., and Hanzlik, R. P.1995 Bromobenzene 3,4-oxide alkylates histidine and lysine side chains of rat liver proteins in vivo Chem. Res. Toxicol. 8 729 735
      There is no corresponding record for this reference.
    21. 21
      Slaughter, D. E., and Hanzlik, R. P.1991 Identification of epoxide- and quinone-derived bromobenzene adducts to protein sulfur nucleophiles Chem. Res. Toxicol. 4 349 359
      There is no corresponding record for this reference.
    22. 22
      Narasimhan, N., Weller, P. E., Buben, J. A., Wiley, R. A., and Hanzlik, R. P.1988 Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites Xenobiotica 18 491 499
      There is no corresponding record for this reference.
    23. 23
      De Vincenzi, M., Maialetti, F., and Silano, M.2003 Constituents of aromatic plants: Teucrin A Fitoterapia 74 746 749
      There is no corresponding record for this reference.
    24. 24
      Bedir, E., Manyam, R., and Khan, I. A.2003 Neo-clerodane diterpenoids and phenylethanoid glycosides from Teucrium chamaedrys L Phytochemistry 63 977 983
      There is no corresponding record for this reference.
    25. 25
      Kouzi, S. A., McMurtry, R. J., and Nelson, S. D.1994 Hepatotoxicity of germander (Teucrium chamaedrys L.) and one of its constituent neoclerodane diterpenes teucrin A in the mouse Chem. Res. Toxicol. 7 850 856
      25
      Hepatotoxicity of Germander (Teucrium chamaedrys L.) and One of Its Constituent Neoclerodane Diterpenes Teucrin A in the Mouse
      Kouzi, Samir A.; McMurtry, Randolph J.; Nelson, Sidney D.
      Chemical Research in Toxicology (1994), 7 (6), 850-6CODEN: CRTOEC; ISSN:0893-228X.
      The hepatotoxicity of the herbal plant germander and that of one of its major furanoneoclerodane diterpenes, teucrin A, were investigated in mice. Teucrin A was found to cause the same midzonal hepatic necrosis as obsd. with exts. of the powd. plant material. Evidence that bioactivation of teucrin A by cytochromes P 450 (P 450) to a reactive metabolite(s) is required for initiation of the hepatocellular damage is provided by results of expts. on the induction and inhibition of P 450 and from studies on the effects of glutathione depletion. Pretreatment of mice with the P 450 inducer phenobarbital enhanced the hepatotoxic response, as indicated by an increase in plasma alanine aminotransferase (ALT) levels and hepatic necrosis, while pretreatment with the P 450 inhibitor piperonyl butoxide markedly attenuated the toxic response. Hepatotoxicity of teucrin A also was increased following pretreatment with the inhibitor of glutathione synthesis buthionine sulfoximine. Most importantly, the THF analog of teucrin A, obtained by selective chem. redn. of the furan ring, was not hepatotoxic, a result that provides strong evidence that oxidn. of the furan ring moiety of the neoclerodane diterpenes is involved in the initiation of hepatocellular injury caused by germander.
    26. 26
      Lekehal, M., Pessayre, D., Lereau, J. M., Moulis, C., Fouraste, I., and Fau, D.1996 Hepatotoxicity of the herbal medicine germander: Metabolic activation of its furano diterpenoids by cytochrome P450 3A Depletes cytoskeleton-associated protein thiols and forms plasma membrane blebs in rat hepatocytes Hepatology 24 212 218
      There is no corresponding record for this reference.
    27. 27
      Fau, D., Lekehal, M., Farrell, G., Moreau, A., Moulis, C., Feldmann, G., Haouzi, D., and Pessayre, D.1997 Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes Gastroenterology 113 1334 1346
      There is no corresponding record for this reference.
    28. 28
      Boyd, M. R., Grygiel, J. J., and Minchin, R. F.1983 Metabolic activation as a basis for organ-selective toxicity Clin. Exp. Pharmacol. Physiol. 10 87 99
      28
      Metabolic activation as a basis for organ-selective toxicity
      Boyd, Michael R.; Grygiel, John J.; Minchin, Rodney F.
      Clinical and Experimental Pharmacology and Physiology (1983), 10 (1), 87-99CODEN: CEXPB9; ISSN:0305-1870.
      A discussion with many refs.
    29. 29
      Falzon, M., McMahon, J. B., Schuller, H. M., and Boyd, M. R.1986 Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines Cancer Res. 46 3484 3489
      There is no corresponding record for this reference.
    30. 30
      Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M.1988 Preparation of the 8,9-epoxide of the mycotoxin aflatoxin b1: The ultimate carcinogenic species J. Am. Chem. Soc. 110 7929 7931
      There is no corresponding record for this reference.
    31. 31
      Smela, M. E., Currier, S. S., Bailey, E. A., and Essigmann, J. M.2001 The chemistry and biology of aflatoxin B (1): From mutational spectrometry to carcinogenesis Carcinogenesis 22 535 545
      There is no corresponding record for this reference.
    32. 32
      Baer, B. R., Rettie, A. E., and Henne, K. R.2005 Bioactivation of 4-ipomeanol by CYP4B1: Adduct characterization and evidence for an enedial intermediate Chem. Res. Toxicol. 18 855 864
      32
      Bioactivation of 4-Ipomeanol by CYP4B1: Adduct Characterization and Evidence for an Enedial Intermediate
      Baer, Brian R.; Rettie, Allan E.; Henne, Kirk R.
      Chemical Research in Toxicology (2005), 18 (5), 855-864CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromols. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liq. chromatog./electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping mol. Reaction mixts. contg. equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resoln. mass spectrometry and two-dimensional NMR anal. following large-scale isolation of the biol. derived material provided evidence for an N-substituted cysteinyl pyrrole deriv. of IPO, analogous to that characterized previously in model chem. studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P 450/30 min. A panel of 14 com. available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were obsd. with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s.
    33. 33
      Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P.2002 Biotransformation reactions of five-membered aromatic heterocyclic rings Chem. Res. Toxicol. 15 269 299
      33
      Biotransformation Reactions of Five-Membered Aromatic Heterocyclic Rings
      Dalvie, Deepak K.; Kalgutkar, Amit S.; Khojasteh-Bakht, S. Cyrus; Obach, R. Scott; O'Donnell, John P.
      Chemical Research in Toxicology (2002), 15 (3), 269-299CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A review with 278 refs. is given on the biotransformation pathways of most commonly used 5-membered arom. heterocyclic rings. The effect of physicochem. properties such as the electronic effects of heteroatoms, aromaticity, and acidity/basicity (pKa) of the rings are correlated on their biotransformation. The generation of reactive metabolites following oxidn. or redn. of these heterocycles and the subsequent toxicol. consequences are discussed.
    34. 34
      Thomassen, D., Knebel, N., Slattery, J. T., McClanahan, R. H., and Nelson, S. D.1992 Reactive intermediates in the oxidation of menthofuran by cytochromes P-450 Chem. Res. Toxicol. 5 123 130
      There is no corresponding record for this reference.
    35. 35
      McClanahan, R. H., Thomassen, D., Slattery, J. T., and Nelson, S. D.1989 Metabolic activation of (R)-(+)-pulegone to a reactive enonal that covalently binds to mouse liver proteins Chem. Res. Toxicol. 2 349 355
      There is no corresponding record for this reference.
    36. 36
      Ravindranath, V., Burka, L. T., and Boyd, M. R.1984 Reactive metabolites from the bioactivation of toxic methylfurans Science 224 884 886
      36
      Reactive metabolites from the bioactivation of toxic methylfurans
      Ravindranath, Vijayalakshmi; Burka, Leo T.; Boyd, Michael R.
      Science (Washington, DC, United States) (1984), 224 (4651), 884-6CODEN: SCIEAS; ISSN:0036-8075.
      Acetylacrolein [5729-47-5] and methylbutenedial [4360-53-6] were identified as the principal reactive intermediates of 2-methylfuran (I) [534-22-5] and 3-methylfuran [930-27-8] that were produced. They bound covalently to tissue macromols. in hepatic and pulmonary microsomal systems in vitro. Therefore, the reactive compds. may be the ultimate toxic metabolites responsible for target tissue alkylation and toxicity produced by the parent furans in vivo. Epoxides, even if formed transiently, appear not to play a major role in the covalent binding nor in the toxicity of the furans.
    37. 37
      Chen, L. J., Hecht, S. S., and Peterson, L. A.1995 Identification of cis-2-butene-1,4-dial as a microsomal metabolite of furan Chem. Res. Toxicol. 8 903 906
      37
      Identification of cis-2-Butene-1,4-dial as a Microsomal Metabolite of Furan
      Chen, Ling-Jen; Hecht, Stephen S.; Peterson, Lisa A.
      Chemical Research in Toxicology (1995), 8 (7), 903-6CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      The rat liver microsomal metab. of furan was examd. in the presence of NADPH and semicarbazide. HPLC anal. of incubation mixts. revealed the formation of a metabolite that coeluted with stds. for the bis-semicarbazone adduct of cis-2-butene-1,4-dial. The formation of this compd. required the presence of NADPH, semicarbazide, and microsomes. Preparative isolation and chem. characterization of this metabolite confirmed the structural assignment. These data provide evidence that the reactive aldehyde, cis-2-butene-1,4-dial, is a major metabolic product of furan.
    38. 38
      Druckova, A., and Marnett, L. J.2006 Characterization of the amino acid adducts of the enedial derivative of teucrin A Chem. Res. Toxicol. 19 1330 1340
      There is no corresponding record for this reference.
    39. 39
      Davies, S. S., Talati, M., Wang, X., Mernaugh, R. L., Amarnath, V., Fessel, J., Meyrick, B. O., Sheller, J., and Roberts, L. J.2004 Localization of isoketal adducts in vivo using a single-chain antibody Free Radical Biol. Med. 36 1163 1174
      39
      Localization of isoketal adducts in vivo using a single-chain antibody
      Davies, Sean S.; Talati, Megha; Wang, Xiahong; Mernaugh, Raymond L.; Amarnath, Venkataraman; Fessel, Joshua; Meyrick, Barbara O.; Sheller, James; Roberts, L. Jackson
      Free Radical Biology & Medicine (2004), 36 (9), 1163-1174CODEN: FRBMEH; ISSN:0891-5849. (Elsevier)
      Isoketals are highly reactive γ-ketoaldehydes formed by the oxidn. of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2α). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the no. of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.
    40. 40
      Murray, R. W., and Jeyaraman, R.1985 Dioxiranes––Synthesis and reactions of methyldioxiranes J. Org. Chem. 50 2847 2853
      There is no corresponding record for this reference.
    41. 41
      Pope, T., Embelton, J., and Mernaugh, R. L. (1996) Constructionand use of antibody gene repertories. In Antibody Engineering:A Practical Approach (McCafferty, J., Chiswell, D., and Hoogenboom, H., Eds.) IRL Press, Oxford, England.
      There is no corresponding record for this reference.
    42. 42
      Vinion-Dubiel, A. D., McClain, M. S., Cao, P., Mernaugh, R. L., and Cover, T. L.2001 Antigenic diversity among Helicobacter pylori vacuolating toxins Infect. Immun. 69 4329 4336
      42
      Antigenic diversity among Helicobacter pylori vacuolating toxins
      Vinion-Dubiel, Arlene D.; McClain, Mark S.; Cao, Ping; Mernaugh, Raymond L.; Cover, Timothy L.
      Infection and Immunity (2001), 69 (7), 4329-4336CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)
      Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 IgG1κ anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.
    43. 43
      Lapierre, L. A., Avant, K. M., Caldwell, C. M., Ham, A. J., Hill, S., Williams, J. A., Smolka, A. J., and Goldenring, J. R.2007 Characterization of immunoisolated human gastric parietal cells tubulovesicles: Identification of regulators of apical recycling Am. J. Physiol. Gastrointest. Liver Physiol. 292 (5) G1249 62
      There is no corresponding record for this reference.
    44. 44
      Ham, A.-J. (2005) Proteolytic digestion protocols. In The Encyclopediaof Mass Spectrometry, Volume 2 Biological Applications Part A: Proteinsand Peptides (Caprioli, R. M., and Gross, M. L., Eds.) Vol. 2, pp 10– 17, Elsevier Ltd., Kidlington, Oxford, United Kingdom.
      There is no corresponding record for this reference.
    45. 45
      Cortes, H. J., Pfeiffer, C. D., Richter, B. E., and Stevens, T. S.1987 Porous ceramic bed supports for fused-silica packed capillary columns used in liquid-chromatography J. High Resolut. Chromatogr. Chromatogr. Commun. 10 446 448
      45
      Porous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography
      Cortes, H. J.; Pfeiffer, C. D.; Richter, B. E.; Stevens, T. S.
      HRC & CC, Journal of High Resolution Chromatography and Chromatography Communications (1987), 10 (8), 446-8CODEN: HCJCDB; ISSN:0344-7138.
      Porous ceramic bed supports for fused silica packed capillary columns utilized in liq. chromatog. were prepd. by polymg. solns. contg. potassium silicate in-situ within a column to create a mech. stable, rugged, and easily constructed termination. The effect of the bed support length on efficiency, and comparisons to glass wool bed supports, were considered in terms of column efficiencies and hydrodynamic variables. Results obtained indicate better performance for the ceramic bed support.
    46. 46
      Licklider, L .J., Thoreen, C. C., Peng, J. M., and Gygi, S. P.2002 Automation of nanoscale microcapillary liquid chromatography-tandem mass spectromentry with a vented column Anal. Chem. 74 3076 3083
      There is no corresponding record for this reference.
    47. 47
      Yates, J. R., Eng, J. K., Mccormack, A. L., and Schieltz, D.1995 Method to correlate tandem mass-spectra of modified peptides to amino-acid-sequences in the protein database Anal. Chem. 67 1426 1436
      There is no corresponding record for this reference.
    48. 48
      Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P.2005 Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations Nat. Methods 2 667 675
      48
      Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations
      Elias, Joshua E.; Haas, Wilhelm; Faherty, Brendan K.; Gygi, Steven P.
      Nature Methods (2005), 2 (9), 667-675CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Researchers have several options when designing proteomics expts. Primary among these are choices of exptl. method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, the authors compared triplicate measurements of the yeast proteome by liq. chromatog. tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, the authors selected scoring criteria yielding 1% estd. false pos. identifications at max. sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.
    49. 49
      Chen, L. J., Hecht, S. S., and Peterson, L. A.1997 Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan Chem. Res. Toxicol. 10 866 874
      There is no corresponding record for this reference.
    50. 50
      He, X. M., and Carter, D. C.1992 Atomic structure and chemistry of human serum albumin Nature 358 209 215
      50
      Atomic structure and chemistry of human serum albumin
      He, Xiao Min; Carter, Daniel C.
      Nature (London, United Kingdom) (1992), 358 (6383), 209-15CODEN: NATUAS; ISSN:0028-0836.
      The three-dimensional structure of human serum albumin has been detd. crystallog. to a resoln. of 2.8 Å. It comprises three homologous domains that assemble to form a heart-shaped mol. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chem. The structure explains numerous phys. phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.
    51. 51
      Loeper, J., Descatoire, V., Letteron, P., Moulis, C., Degott, C., Dansette, P., Fau, D., and Pessayre, D.1994 Hepatotoxicity of germander in mice Gastroenterology 106 464 472
      There is no corresponding record for this reference.
    52. 52
      Baillie, T. A.2006 Future of toxicology––Metabolic activation and drug design: Challenges and apportunities in chemical toxicology Chem. Res. Toxicol. 19 889 893
      There is no corresponding record for this reference.
    53. 53
      Pumford, N. R., Halmes, N. C., and Hinson, J. A.1997 Covalent binding of xenobiotics to specific proteins in the liver Drug Metab. Rev. 29 39 57
      53
      Covalent binding of xenobiotics to specific proteins in the liver
      Pumford, Neil R.; Halmes, N. Christine; Hinson, Jack A.
      Drug Metabolism Reviews (1997), 29 (1 & 2), 39-57CODEN: DMTRAR; ISSN:0360-2532. (Dekker)
      A review with 67 refs.
    54. 54
      Guengerich, F. P.2005 Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation Arch. Biochem. Biophys. 433 369 378
      There is no corresponding record for this reference.
    55. 55
      Boyd, M. R.1981 Toxicity mediated by reactive metabolites of furans Adv. Exp. Med. Biol. 136Part B 865 879
      There is no corresponding record for this reference.
    56. 56
      Gordon, W. P., Huitric, A. C., Seth, C. L., McClanahan, R. H., and Nelson, S. D.1987 The metabolism of the abortifacient terpene, (R)-(+)-pulegone, to a proximate toxin, menthofuran Drug Metab. Dispos. 15 589 594
      56
      The metabolism of the abortifacient terpene, (R)-(+)-pulegone, to a proximate toxin, menthofuran
      Gordon, W. Perry; Huitric, Alain C.; Seth, Cynthia L.; McClanahan, Robert H.; Nelson, Sidney D.
      Drug Metabolism and Disposition (1987), 15 (5), 589-94CODEN: DMDSAI; ISSN:0090-9556.
      (R)-(+)-Pulegone (I) is metabolized by hepatic microsomal monooxygenses of the mouse to a hepatotoxin. The formation of a toxic metabolite is apparently mediated by cytochromes P 450 of the phenobarbital class inasmuch as phenobarbital pretreatment of mice increases, whereas β-naphthoflavone pretreatment decreases, the extent of hepatic necrosis caused by pulegone. Furthermore, 2 inhibitors of cytochromes P 450, cobaltous chloride and piperonyl butoxide, block toxicity. An analog of I that was labeled with deuterium in the allylic Me groups was significantly less hepatotoxic than the parent compd. Apparently, oxidn. of an allylic Me group is required for generation of a hepatotoxic metabolite. Menthofuran was identified as a proximate toxic metabolite of I, and investigations with I-d6 and 18O2 strongly indicate that menthofuran is formed by a sequence of reactions that involve: (1) oxidn. of an allylic Me group, (2) intramol. cyclization to form a hemiketal, and (3) dehydration to form the furan.
    57. 57
      Kobayashi, T., Sugihara, J., and Harigaya, S.1987 Mechanism of metabolic cleavage of a furan ring Drug Metab. Dispos. 15 877 881
      There is no corresponding record for this reference.
    58. 58
      Parmar, D., and Burka, L. T.1993 Studies on the interaction of furan with hepatic cytochrome P-450 J. Biochem. Toxicol. 8 1 9
      58
      Studies on the interaction of furan with hepatic cytochrome P-450
      Parmar, Devendra; Burka, Leo T.
      Journal of Biochemical Toxicology (1993), 8 (1), 1-9CODEN: JBTOEB; ISSN:0887-2082.
      In vitro incubation of rat liver microsomes with [14C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was obsd. with microsomes from phenobarbital-treated rats and a four- to five-fold increase was obsd. with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chems. contg. an amine group (semi carbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [14C]-furan to microsomal protein. A decrease in cytochrome P 450 (P 450) content and decreases in the activities of P 450-dependent aniline hydroxylase, 7-ethoxycoumarin O-deethylase (ECD), and 7-ethoxyresorufin O-deethylase (ERD) was obsd. 24 h after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P 450 catalyzed metab. could be binding with nucleophilic groups within the P 450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and ECD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P 450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after in vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P 450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P 450.
    59. 59
      Sahali-Sahly, Y., Balani, S. K., Lin, J. H., and Baillie, T. A.1996 In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4 Chem. Res. Toxicol. 9 1007 1012
      There is no corresponding record for this reference.
    60. 60
      Racha, J. K., Rettie, A. E., and Kunze, K. L.1998 Mechanism-based inactivation of human cytochrome P450 1A2 by furafylline: Detection of a 1:1 adduct to protein and evidence for the formation of a novel imidazomethide intermediate Biochemistry 37 7407 7419
      There is no corresponding record for this reference.
    61. 61
      Zhang, K. E., Naue, J. A., Arison, B., and Vyas, K. P.1996 Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: Evidence for furan bioactivation Chem. Res. Toxicol. 9 547 554
      There is no corresponding record for this reference.
    62. 62
      Boyd, M. R., and Dutcher, J. S.1981 Renal toxicity due to reactive metabolites formed in situ in the kidney: Investigations with 4-ipomeanol in the mouse J. Pharmacol. Exp. Ther. 216 640 646
      62
      Renal toxicity due to reactive metabolites formed in situ in the kidney: investigations with 4-ipomeanol in the mouse
      Boyd, Micheal R.; Dutcher, John S.
      Journal of Pharmacology and Experimental Therapeutics (1981), 216 (3), 640-6CODEN: JPETAB; ISSN:0022-3565.
      The in vitro metab. and covalent binding of 4-ipomeanol (I) [32954-58-8] was mediated by O-requiring, NADPH-dependent, CO-inhibitable microsomal enzymes present in the livers, lungs and kidneys of adult male mice. These activities were inhibitable by piperonyl butoxide and they were markedly enhanced in hepatic microsomes from C57/6J mice, but not DBA/2J mice, pretreated with 3-methylcholanthrene. The i.p. administration of I to adult male mice resulted in the covalent binding of large amts. of its metabolite(s) in the lungs and kidneys. The material bound in the kidneys was located predominantly in the proximal renal cortical tubules. The covalent binding and toxicity of I to the renal tubules could be prevented by pretreatment of the animals with piperonyl butoxide. The hepatic covalent binding and toxicity of I were enhanced and the pulmonary and renal covalent binding and toxicity were decreased in C57BL/6J mice pretreated with 3-methylcholanthrene; however, this pretreatment did not significantly alter the tissue covalent binding or toxicity of I in noninducible DBA/2J mice. These results support the view that renal damage by I in the mouse is caused by reactive I metabolite(s) formed in situ in the kidney.
    63. 63
      Smothers, J. F., Henikoff, S., and Carter, P.2002 Tech.Sight. Phage display. Affinity selection from biological libraries Science 298 621 622
      There is no corresponding record for this reference.
    64. 64
      Smothers, J. F., and Henikoff, S.2001 Predicting in vivo protein peptide interactions with random phage display Comb. Chem. High Throughput Screening 4 585 591
      64
      Predicting in vivo protein-peptide interactions with random phage display
      Smothers, James F.; Henikoff, Steven
      Combinatorial Chemistry and High Throughput Screening (2001), 4 (7), 585-591CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers)
      A review. Binding sites in protein complexes occasionally map to small peptides within one or more proteins. Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest. The natural substrates for the protein are typically known in advance. However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two-hybrid screening. Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo. Here, we review an approach that addresses this problem. First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display. Next, searching programs detect similar blocks of conserved sequences within naturally-occurring proteins to predict partner proteins. Finally, the significance of an interaction is tested using site-specific mutagenesis, binding competition or co-immunopptn. expts. This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biol.
    65. 65
      Yip, Y. L., and Ward, R. L.1999 Epitope discovery using monoclonal antibodies and phage peptide libraries Comb. Chem. High Throughput Screening 2 125 138
      65
      Epitope discovery using monoclonal antibodies and phage peptide libraries
      Yip, Yum L.; Ward, Robyn L.
      Combinatorial Chemistry and High Throughput Screening (1999), 2 (3), 125-138CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers)
      A review with 148 refs. Phage display is a biol. system which facilitates the cloning and rapid selection of peptides from large combinatorial libraries. In comparison to the chem. combinatorial approach, the advantages of phage display lie in its simplicity and replicability. While phage display has many diverse applications, this review focuses on the use of phage peptide libraries to discover epitopes recognized by monoclonal antibodies. As monoclonal antibodies are useful tools for the detection of proteins and for the investigation of mol. interactions, the identification of their epitopes will serve to elucidate the structure and function of proteins, as well as aid in the discovery of new drugs and the development of vaccines.
    66. 66
      Wang, L. F., and Yu, M.2004 Epitope identification and discovery using phage display libraries: Applications in vaccine development and diagnostics Curr. Drug Targets 5 1 15
      66
      Epitope identification and discovery using phage display libraries: Applications in vaccine development and diagnostics
      Wang, Lin-fa; Yu, Meng
      Current Drug Targets (2004), 5 (1), 1-15CODEN: CDTUAU; ISSN:1389-4501. (Bentham Science Publishers Ltd.)
      A review. Antigenic epitopes are the part (contact points) of an antigen involved in specific interaction with the antigen-binding site (the paratope) of an antibody or a T-cell receptor. Detailed anal. of epitopes is important both for the understanding of immunol. events and for the development of more effective vaccine and diagnostic tools for various diseases. Identification and characterization of epitopes is a complex process. Although various methods have been developed in this area, there still lacks a simple common approach which can be applied to all epitopes. Since its first introduction more than a decade ago, phage display technol. has made a major impact in this area of research. With the exponential growth in this area, it is impractical to review the entire literature detailing all possible applications. Instead, this review aims to focus on specific applications related to the discovery and identification of epitopes which have potential as vaccine candidates or can be used in disease diagnosis.
    67. 67
      Mancini, N., Carletti, S., Perotti, M., Canducci, F., Mammarella, M., Sampaolo, M., and Burioni, R.2004 Phage display for the production of human monoclonal antibodies against human pathogens New Microbiol. 27 315 328
      67
      Phage display for the production of human monoclonal antibodies against human pathogens
      Mancini, N.; Carletti, S.; Perotti, M.; Canducci, F.; Mammarella, M.; Sampaolo, M.; Burioni, R.
      New Microbiologica (2004), 27 (4), 315-328CODEN: NMEIB2; ISSN:1121-7138. (Edizioni Internazionali srl, Div. EDIMES)
      A review. In the last decade an increasing no. of antibodies have made their way from the research benchtops into the clinics and many more are currently under clin. trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the mol. dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.
    68. 68
      Azzazy, H. M., and Highsmith, W. E., Jr2002 Phage display technology: Cclinical applications and recent innovations Clin. Biochem. 35 425 445
      68
      Phage display technology: clinical applications and recent innovations
      Azzazy, Hassan M. E.; Highsmith, W. Edward, Jr.
      Clinical Biochemistry (2002), 35 (6), 425-445CODEN: CLBIAS; ISSN:0009-9120. (Elsevier Science Inc.)
      A review. Phage display is a mol. diversity technol. that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technol. is the direct link that exists between the exptl. phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized mols. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technol.
    69. 69
      Szapacs, M. E., Riggins, J. N., Zimmerman, L. J., and Liebler, D. C.2006 Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions Biochemistry 45 10521 10528
      There is no corresponding record for this reference.
    70. 70
      Ivanov, A. I., Christodoulou, J., Parkinson, J. A., Barnham, K. J., Tucker, A., Woodrow, J., and Sadler, P. J.1998 Cisplatin binding sites on human albumin J. Biol. Chem. 273 14721 14730
      70
      Cisplatin binding sites on human albumin
      Ivanov, Andrei I.; Christodoulou, John; Parkinson, John A.; Barnham, Kevin J.; Tucker, Alan; Woodrow, John; Sadler, Peter J.
      Journal of Biological Chemistry (1998), 273 (24), 14721-14730CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Reactions of cisplatin with albumin are thought to play an important role in the metab. of this anticancer drug. These reactions were investigated via (1) labeling of cisplatin with 15N and use of 2-dimensional [1H,15N]NMR spectroscopy, (2) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (3) chem. modification of Cys, Met, and His residues, (4) reactions of bound Pt with thiourea, and (5) gel filtration chromatog. In contrast to previous reports, it was shown that the major S-contg. binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Addnl. monofunctional adducts involving other Met residues and Cys-34 were also obsd. During the later stages of reactions of cisplatin with albumin, release of NH3 occurred due to the strong trans influence of Met S, which weakens the Pt-NH3 bonds, and protein crosslinking was obsd. The consequences of these findings for the biol. activity of cisplatin-albumin complexes are discussed.
    71. 71
      Fabisiak, J. P., Sedlov, A., and Kagan, V. E.2002 Quantification of oxidative/nitrosative modification of CYS(34) in human serum albumin using a fluorescence-based SDS-PAGE assay Antioxid. Redox Signaling 4 855 865
      71
      Quantification of Oxidative/Nitrosative Modification of CYS34 in Human Serum Albumin Using a Fluorescence-Based SDS-PAGE Assay
      Fabisiak, James P.; Sedlov, Andrey; Kagan, Valerian E.
      Antioxidants & Redox Signaling (2002), 4 (5), 855-865CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)
      The SH group represented by cysteine in proteins is fundamental to the redox regulation of protein structure and function. Albumin is the most abundant serum protein whose redox modification modulates its physiol. function, as well as serves as a biomarker of oxidative stress. Measurement of selective Cys modification (S-oxidn./nitrosation, electrophilic substitution) on specific proteins, however, is problematic within complex biol. mixts. such as plasma. We have utilized a maleimide fluorogenic SH reagent, ThioGlo-1, to develop a fluorescence-based quant. assay of SH modification of human serum albumin (hSA) using SDS-PAGE. Fully reduced native albumin contg. one free SH (Cys34) per mol. was utilized as a model protein to characterize the kinetics of ThioGlo-1 reaction using a soln.-based spectrofluorometric assay. Optimum labeling of hSA Cys34 was achieved within 10 min at 60°C using a threefold molar excess of ThioGlo-1 relative to hSA and required SDS. Comparison of the soln. spectrofluorometric assay to fluorescent image anal. of hSA bands localized by SDS-PAGE revealed that SH groups in hSA could be quantified after gel electrophoresis. The soln.- and gel-based methods were in excellent concordance in their ability to quantify SH modification of hSA following exposure to phenoxyl radicals and nitric oxide. The application of ThioGlo-1 staining and SDS-PAGE quantified the degree of hSA modification in complex human plasma exposed to oxidative or nitrosative stress and revealed that hSA is more sensitive to S modification than other SH-contg. plasma proteins.
    72. 72
      Shen, B., and English, A. M.2005 Mass spectrometric analysis of nitroxyl-mediated protein modification: Comparison of products formed with free and protein-based cysteines Biochemistry 44 14030 14044
      72
      Mass Spectrometric Analysis of Nitroxyl-Mediated Protein Modification: Comparison of Products Formed with Free and Protein-Based Cysteines
      Shen, Biao; English, Ann M.
      Biochemistry (2005), 44 (42), 14030-14044CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Although biol. active, nitroxyl (HNO) remains one of the most poorly studied NOx. Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addn. product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 μM), human calbindin D28k (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivs. in incubates contg. 0.9-2.5 mM AS. Ions at M + n × 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramol. sulfinamide crosslinking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were obsd. for HCalB, which addnl. formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramol. disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3- → HNO + NO2-), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.
    73. 73
      Talib, J., Beck, J. L., and Ralph, S. F.2006 A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]- to human serum albumin J. Biol. Inorg. Chem. 11 559 570
      73
      A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]- to human serum albumin
      Talib, Jihan; Beck, Jennifer L.; Ralph, Stephen F.
      JBIC, Journal of Biological Inorganic Chemistry (2006), 11 (5), 559-570CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)
      Electrospray ionization (ESI) mass spectrometry was used to examine the reactions of the clin. used antiarthritic agent [Au(S2O3)2]3-, and AuPEt3Cl, a deriv. of another clin. used agent auranofin, with human serum albumin (HSA) obtained from a human volunteer. Both compds. reacted readily with HSA to form complexes contg. one or more covalently attached gold fragments. In the case of AuPEt3Cl, binding was accompanied by the loss of the chloride ligand, while for [Au(S2O3)2]3- the mass spectral data indicated binding of Au(S2O3) groups. Expts. performed using HSA with Cys34 blocked by reaction with iodoacetamide were consistent with reaction of both gold compds. with this amino acid. Sep. blocking expts. using diethylpyrocarbonate and AuPEt3Cl also provided evidence for histidine residues acting as lower-affinity binding sites for this gold compd. ESI mass spectra of solns. contg. [Au(S2O3)2]3- or [Au(CN)2]-, and HSA, provided evidence for the formation of protein complexes in which intact gold mols. were noncovalently bound. In the case of [Au(S2O3)2]3-, these noncovalent complexes proved to be transitory in nature. However, for [Au(CN)2]- a noncovalent complex contg. a single gold mol. bound to HSA was found to be stable, and constituted the main adduct formed in solns. contg. low-to-medium Au-to-HSA ratios. Evidence was also obtained for the formation of a covalent adduct in which a single Au(CN) moiety was bonded to Cys34 of the protein. AuPEt3Cl reacted to a much lower extent with HSA that had Cys34 modified by formation of a disulfide bond to added cysteine, than with unmodified HSA. This suggests that the extent of modification of the protein in vivo may have an important influence on the transport and bioavailability of gold antiarthritic drugs.
    74. 74
      Ascoli, G. A., Domenici, E., and Bertucci, C.2006 Drug binding to human serum albumin: Abridged review of results obtained with high-performance liquid chromatography and circular dichroism Chirality 18 667 679
      There is no corresponding record for this reference.
    75. 75
      Bertucci, C., and Domenici, E.2002 Reversible and covalent binding of drugs to human serum albumin: Methodological approaches and physiological relevance Curr. Med. Chem. 9 1463 1481
      75
      Reversible and covalent binding of drugs to human serum albumin: methodological approaches and physiological relevance
      Bertucci, Carlo; Domenici, Enrico
      Current Medicinal Chemistry (2002), 9 (15), 1463-1481CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers)
      A review. Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concn. of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metab., distribution and excretion. The free concn. of a drug can also be affected by interaction with coadministered drugs or by pathol. conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clin. impacts for drugs that have a relatively narrow therapeutic index. This article reviews the physiol. role of HSA in in the human body and the pharmacol. consequences of drug-HSA binding; it then focuses on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatog. on immobilized HSA has been shown to be a rapid and effective tool for the characterization of binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. Also discussed is the potential offered by the combined use of CD on the same protein/drug system in soln., not only for the detn. of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs is addressed. The effect of reversible or covalent binding of drugs is also discussed and examples of physiol. relevance reported.
    76. 76
      De Berardinis, V., Moulis, C., Maurice, M., Beaune, P., Pessayre, D., Pompon, D., and Loeper, J.2000 Human microsomal epoxide hydrolase is mthe target of germander-induced autoantibodies on the surface of human hepatocytes Mol. Pharmacol. 58 542 551
      There is no corresponding record for this reference.
    77. 77
      Loeper, J., De Berardinis, V., Moulis, C., Beaune, P., Pessayre, D., and Pompon, D.2001 Human epoxide hydrolase is the target of germander autoantibodies on the surface of human hepatocytes: Enzymatic implications Adv. Exp. Med. Biol. 500 121 124
      77
      Human epoxide hydrolase is the target of germander autoantibodies on the surface of human hepatocytes: Enzymatic implications
      Loeper, Jacqueline; De Berardinis, Veronique; Moulis, Claude; Beaune, Philippe; Pessayre, Dominique; Pompon, Denis
      Advances in Experimental Medicine and Biology (2001), 500 (Biological Reactive Intermediates VI), 121-124CODEN: AEMBAP; ISSN:0065-2598. (Kluwer Academic/Plenum Publishers)
      Wild germander (Teuchrium chamaedrys L.) was traditionally used as a folk medicine tor its choleretic and antiseptic properties. In 1991, germander consumed to treat obesity, caused an epidemic of cytolytic hepatitis. Thirty cases of hepatotoxicity were first reported including cases with pos. rechallenge. For these patients, an early recurrence was obsd. despite lack of other features of hypersensitivity (Castot and Larrey, 1992). In mice, germander toxicity required CYP3A-dependent metab. (Loeper, and al., 1994), more specifically, the metabolic activation of the furan ring of the diterpenoid teucrin A (TA) (Kouzi, and al., 1994). TA-toxicity was via CYP3A-generated electrophilic metabolites that were detoxified by glutathione conjugation, depleted cellular thiols and caused apoptosis in isolated rat hepatocytes (Lekehal, and al., 1996; Fau and al., 1997). To explain this immune process, patient's sera consuming germander tea in great quantity were tested by Western blot anal. They contained autoantibodies directed against human microsomal epoxide hydrolase (hmEH), that was located both, in the endoplasmic reticulum and the plasma membrane (PM) of human hepatocyte and hmEH-expressing yeast. Germander-induced autoantibodles (GIAA) were shown to recognize hmEH on the cell surface. To implicate hmEH in the metabolic activation of TA we used a humanized yeast strain expressing human P 450-reductase and cytochrome b5 transformed with CYP3A4 and/or hmEH cDNAs. TA was metabolized by CYP3A4 into a metabolite which concn. diminished in presence of hmEH. Incubations of CYP3A4 and hmEH with TA inactivated hmEH in a time-dependent-manner, in agreement with formation of reactive teuchrin A-metabolite that could covalently alter and inhibit hmEH. This modified enzyme may bypass the immunol. tolerance that normally exists for hmEH. In conclusion for the first time, we have shown that anti-hmEH autoantibodies can develop in plant-induced hepatitis. The hmEH is present and functional in the human hepatocyte PM and hmEH epitopes are exposed on the outer surface of the PM, allowing the autoantibodies to possibly participate in the immune destruction of hepatocytes. CYP3A4-mediated metabolic activation of TA into a reactive epoxide could modify and inactivate hmEH. This altered protein may bypass immune tolerance and trigger the immune response against hmEH.
    78. 78
      Boitier, E., and Beaune, P.2000 Xenobiotic-metabolizing enzymes as autoantigens in human autoimmune disorders. An update Clin. Rev. Allergy Immunol. 18 215 239
      78
      Xenobiotic-metabolizing enzymes as autoantigens in human autoimmune disorders; An update
      Boitier, Eric; Beaune, Philippe
      Clinical Reviews in Allergy & Immunology (2000), 18 (2), 215-239CODEN: CRAIF2; ISSN:1080-0549. (Humana Press Inc.)
      A review with 146 refs. Topics discussed include drug-induced autoimmune diseases in which autoantibodies against cytochromes P 450 or xenobiotic-metabolizing enzymes; tienilic acid-induced hepatitis; dihydralazine-induced hepatitis; halothane-induced hepatitis; mol. targets of autoimmune disease nonassocd. with a toxic chem.; uridine diphosphate-glucouronosyltransferase as autoantibody target; glutathione-S-transferase as autoantibody target; pathogenesis of autoimmune disease; and mol. mimicry.
    79. 79
      Strassburg, C. P., Obermayer-Straub, P., and Manns, M. P.2000 Autoimmunity in liver diseases Clin. Rev. Allergy Immunol. 18 127 139
      79
      Autoimmunity in liver diseases
      Strassburg, Christian P.; Obermayer-Straub, Petra; Manns, Michael P.
      Clinical Reviews in Allergy & Immunology (2000), 18 (2), 127-139CODEN: CRAIF2; ISSN:1080-0549. (Humana Press Inc.)
      A review with 31 refs. Topics discussed include the role of hepatotropic virus infection in genuine autoimmune hepatitis, the characterization and evaluation of novel autoantigens in hepatic disease as part of autoimmune polyendocrine syndrome type 1, and the investigation of autoimmune features in hepatitis D infection.
    80. 80
      Boitier, E., and Beaune, P.1999 Cytochromes P450 as targets to autoantibodies in immune mediated diseases Mol. Aspects Med. 20 84 137
      80
      Cytochromes P450 as targets to autoantibodies in immune mediated diseases
      Boitier E; Beaune P
      Molecular aspects of medicine (1999), 20 (1-2), 84-137 ISSN:0098-2997.
      There is no expanded citation for this reference.
    81. 81
      Mizutani, T., Shinoda, M., Tanaka, Y., Kuno, T., Hattori, A., Usui, T., Kuno, N., and Osaka, T.2005 Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2 Drug Metab. Rev. 37 235 252
      81
      Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2
      Mizutani, Takaharu; Shinoda, Masakazu; Tanaka, Yuta; Kuno, Takuya; Hattori, Asuka; Usui, Toru; Kuno, Nayumi; Osaka, Takashi
      Drug Metabolism Reviews (2005), 37 (1), 235-252CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis, Inc.)
      A review. Autoimmune hepatitis (AIH) is a disease of unknown etiol., characterized by liver-related autoantibodies. Autoimmune hepatitis is subdivided into two major types: AIH type 1 is characterized by the detection of ANA, SMA, ANCA, anti-ASGP-R, and anti-SLA/LP. Autoimmune hepatitis type 2 is characterized to be mainly related with drug-metabolizing enzymes as autoantigens, such as anti-LKM (liver-kidney microsomal antigen)-1 against CYP2D6, anti-LKM-2 against CYP2C9-tienilic acid, anti-LKM-3 against UGT1A, and anti-LC1 (liver cytosol antigen)-1 and anti-APS (autoimmune polyglandular syndrome type-1) against CYP1A2, CYP2A6, and others. Anti-LKM-1 sera inhibited CYP2D6 activity in vitro but did not inhibit cellular drug metab. in vivo. CYP2D6 is the major target autoantigen of LKM-1 and expressed on plasma membrane (PM) of hepatocytes, suggesting a pathogenic role for anti-LKM-1 in liver injury as a trigger. Anti-CYP1A2 was obsd. in dihydralazine-induced hepatitis, and radiolabeled CYP1A2 disappeared from the PM with a half-life of less than 30 min, whereas microsomal CYP1A2 was stably radiolabeled for several hours. Main antigenic epitopes on CYP2D6 are as 193-212, as 257-269, and as 321-351; and D263 is essential. The third epitope is located on the surface of the protein CYP2D6 and displays a hydrophobic patch that is situated between an arom. residue (W316) and histidine (H326). Some drugs such as anticonvulsants (phenobarbital, phenytoin, and carbamazepine) and halothane are suggested to induce hepatitis with anti-CYP3A and anti-CYP2E1, resp. Autoantibodies against CYP11A1, CYP17, and/or CYP21 involved in the synthesis of steroid hormones are also detected in patients with adrenal failure, gonadal failure, and/or Addison disease.
    82. 82
      Obermayer-Straub, P., Strassburg, C. P., and Manns, M. P.2000 Target proteins in human autoimmunity: cytochromes P450 and UDP- glucuronosyltransferases Can. J. Gastroenterol. 14 429 439
      82
      Target proteins in human autoimmunity: cytochromes P450 and UDP- glucuronosyltransferases
      Obermayer-Straub P; Strassburg C P; Manns M P
      Canadian journal of gastroenterology = Journal canadien de gastroenterologie (2000), 14 (5), 429-39 ISSN:0835-7900.
      Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are targets of autoantibodies in several hepatic and extrahepatic autoimmune diseases. Autoantibodies directed against hepatic CYPs and UGTs were first detected by indirect immunofluorescence as antiliver and/or kidney microsomal antibodies. In autoimmune hepatitis (AIH) type 2, liver and/or kidney microsomal (LKM) type 1 autoantibodies are detected and are directed against CYP2D6. About 10% of AIH-2 sera further contain LKM-3 autoantibodies directed against family 1 UGTs. Chronic infections by hepatitis C virus and hepatitis delta virus may induce several autoimmune phenomena, and multiple autoantibodies are detected. Anti-CYP2D6 autoantibodies are detected in up to 4% of patients with chronic hepatitis C, and anti-CYP2A6 autoantibodies are detected in about 2% of these patients. In contrast, 14% of patients with chronic hepatitis delta virus infections generate anti-UGT autoantibodies. In a small minority of patients, certain drugs are known to induce immune-mediated, idiosyncratic drug reactions, also known as 'druginduced hepatitis'. Drug-induced hepatitis is often associated with autoantibodies directed against hepatic CYPs or other hepatic proteins. Typical examples are tienilic acid-induced hepatitis with anti-CYP2C9, dihydralazine hepatitis with anti-CYP1A2, halothane hepatitis with anti-CYP2E1 and anticonvulsant hepatitis with anti-CYP3A. Recent data suggest that alcoholic liver disease may be induced by mechanisms similar to those that are active in drug-induced hepatitis. Autoantibodies directed against several CYPs are further detected in sera from patients with the autoimmune polyglandular syndrome type 1. Patients with autoimmune polyglandular syndrome type 1 with hepatitis often develop anti-CYP1A2; patients with adrenal failure develop anti-CYP21, anti- CYP11A1 or CYP17; and patients with gonadal failure develop anti-CYP11A1 or CYP17. In idiopathic Addison disease, CYP21 is the major autoantigen.
    83. 83
      Mottaran, E., Stewart, S. F., Rolla, R., Vay, D., Cipriani, V., Moretti, M., Vidali, M., Sartori, M., Rigamonti, C., Day, C. P., and Albano, E.2002 Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease Free Radical Biol. Med. 32 38 45
      83
      Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease
      Mottaran, Elisa; Stewart, Stephen F.; Rolla, Roberta; Vay, Daria; Cipriani, Valentina; Moretti, MariaGrazia; Vidali, Matteo; Sartori, Massimo; Rigamonti, Cristina; Day, Christopher P.; Albano, Emanuele
      Free Radical Biology & Medicine (2001), 32 (1), 38-45CODEN: FRBMEH; ISSN:0891-5849. (Elsevier Science Inc.)
      Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alc. liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hydroxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alc. cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alc. as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead obsd. in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition expts. showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidn. contribute to the development of immune responses assocd. with alc. liver disease.
    84. 84
      Stewart, S. F., Vidali, M., Day, C. P., Albano, E., and Jones, D. E.2004 Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease Hepatology 39 197 203
      There is no corresponding record for this reference.
    85. 85
      Corcos, L., and Lagadic-Gossmann, D.2001 Gene induction by phenobarbital: An update on an old question that receives key novel answers Pharmacol. Toxicol. 89 113 122
      85
      Gene induction by phenobarbital: an update on an old question that receives key novel answers
      Corcos, Laurent; Lagadic-Gossmann, Dominique
      Pharmacology & Toxicology (Copenhagen, Denmark) (2001), 89 (3), 113-122CODEN: PHTOEH; ISSN:0901-9928. (Munksgaard International Publishers Ltd.)
      A review is given. Phenobarbital has long been used as a sedative and antiepileptic drug. The drug is the representative of a myriad of lipophilic mols. able to evoke a pleiotropic response in the liver and also in prokaryotes and flies. A great deal of novel information was obtained in recent years regarding the mechanism of cytochrome P 450 (CYP) gene induction by phenobarbital. Most importantly, a nuclear orphan receptor, the constitutive androstane receptor was identified as a primary determinant of the transcriptional activation of CYP genes in response to phenobarbital-like inducers in mammals. Another nuclear receptor, the pregnane X receptor can also mediate some of the phenobarbital response, but the functional overlap of the 2 inductive pathways is only partial. The response of mammalian CYP2B genes to phenobarbital was abolished in the liver of mice carrying a null allele of the constitutive androstane receptor gene, whereas that of CYP3A genes was lost in pregnane X receptor knock-out mice.
    86. 86
      Czekaj, P.2000 Phenobarbital-induced expression of cytochrome P450 genes Acta Biochim. Pol. 47 1093 1105
      There is no corresponding record for this reference.
    87. 87
      Gibson, G. G., Plant, N. J., Swales, K. E., Ayrton, A., and El-Sankary, W.2002 Receptor-dependent transcriptional activation of cytochrome P4503A genes: Induction mechanisms, species differences and interindividual variation in man Xenobiotica 32 165 206
      There is no corresponding record for this reference.
    88. 88
      Joannard, F., Galisteo, M., Corcos, L., Guillouzo, A., and Lagadic-Gossmann, D.2000 Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: Involvement of several serine/threonine protein kinases and phosphatases Cell. Biol. Toxicol. 16 325 337
      88
      Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases
      Joannard, F.; Galisteo, M.; Corcos, L.; Guillouzo, A.; Lagadic-Gossmann, D.
      Cell Biology and Toxicology (2000), 16 (5), 325-337CODEN: CBTOE2; ISSN:0742-2091. (Kluwer Academic Publishers)
      We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P 450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compds. one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot anal. of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the neg. role of the cAMP/protein kinase A pathway and the pos. role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca2+/calmodulin-dependent protein kinases II (independently of Ca2+) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function resp. as pos. and neg. regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A.
    89. 89
      Haas, I. G.1994 BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum Experientia 50 1012 1020
      There is no corresponding record for this reference.
    90. 90
      Kleizen, B., and Braakman, I.2004 Protein folding and quality control in the endoplasmic reticulum Curr. Opin. Cell Biol. 16 343 349
      90
      Protein folding and quality control in the endoplasmic reticulum
      Kleizen, Bertrand; Braakman, Ineke
      Current Opinion in Cell Biology (2004), 16 (4), 343-349CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)
      A review and discussion. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least 2 main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasizes more than ever that chaperones act in concert with co-factors and with each other.
    91. 91
      Sommer, T., and Jarosch, E.2002 BiP binding keeps ATF6 at bay Dev. Cell 3 1 2
      91
      BiP binding keeps ATF6 at bay
      Sommer, Thomas; Jarosch, Ernst
      Developmental Cell (2002), 3 (1), 1-2CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
      A study by Shen et al. in this issue of Developmental Cell shows that transport to the Golgi complex and subsequent proteolytic activation of the stress-regulated transcription factor ATF6 is initiated by the dissocn. of the ER chaperone BiP from ATF6. This demonstrates that BiP is a key element in sensing the folding capacity within the ER and provides mechanistic insights on how the activation of membrane-bound transcription factors can be regulated.
    92. 92
      Zhang, K., and Kaufman, R. J.2006 Protein folding in the endoplasmic reticulum and the unfolded protein response Handb. Exp. Pharmacol. 69 91
      92
      Protein folding in the endoplasmic reticulum and the unfolded protein response
      Zhang, K.; Kaufman, R. J.
      Handbook of Experimental Pharmacology (2006), 172 (Molecular Chaperones in Health and Disease), 69-91CODEN: HEPHD2; ISSN:0171-2004. (Springer GmbH)
      A review. In all eukaryotic cells, the endoplasmic reticulum (ER) is an intracellular organelle where folding and assembly occurs for proteins destined to the extracellular space, plasma membrane, and the exo/endocytic compartments. As a protein-folding compartment, the ER is exquisitely sensitive to alterations in homeostasis, and provides stringent quality control systems to ensure that only correctly folded proteins transit to the Golgi app. and unfolded or misfolded proteins are retained and ultimately degraded. A no. of biochem. and physiol. stimuli, such as perturbation in Ca homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, altered glycosylation, and overloading of cholesterol can disrupt ER homeostasis, impose stress to the ER, and subsequently lead to accumulation of unfolded or misfolded proteins in the ER lumen. The ER has evolved highly specific signaling pathways called the unfolded protein response (UPR) to cope with the accumulation of unfolded or misfolded proteins. Elucidation of the mol. mechanisms by which accumulation of unfolded proteins in the ER transmits a signal to the cytoplasm and nucleus has led to major new insights into the diverse cellular and physiol. processes that are regulated by the UPR. Here, the authors summarize how cells respond to the accumulation of unfolded proteins in the cell and the relevance of these signaling pathways to human physiol. and disease.
    93. 93
      Paton, A. W., Beddoe, T., Thorpe, C. M., Whisstock, J. C., Wilce, M. C., Rossjohn, J., Talbot, U. M., and Paton, J. C.2006 AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP Nature 443 548 552
      There is no corresponding record for this reference.
    94. 94
      Cribb, A. E., Peyrou, M., Muruganandan, S., and Schneider, L.2005 The endoplasmic reticulum in xenobiotic toxicity Drug Metab. Rev. 37 405 442
      94
      The endoplasmic reticulum in xenobiotic toxicity
      Cribb, Alastair E.; Peyrou, Mathieu; Muruganandan, Shanmugam; Schneider, Laetitia
      Drug Metabolism Reviews (2005), 37 (3), 405-442CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis, Inc.)
      A review. The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P 450 enzymes involved in xenobiotic metab., as well as a no. of conjugating enzymes. In addn. to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and the folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-assocd. apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in mol. events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity is discussed.
    95. 95
      Ellgaard, L., and Ruddock, L. W.2005 The human protein disulphide isomerase family: Substrate interactions and functional properties EMBO Rep. 6 28 32
      There is no corresponding record for this reference.
    96. 96
      Sitia, R., and Molteni, S. N.2004 Stress, protein (mis)folding, and signaling: The redox connection Sci. STKE pe27
      There is no corresponding record for this reference.
    97. 97
      Wilkinson, B., and Gilbert, H. F.2004 Protein disulfide isomerase Biochim. Biophys. Acta 1699 35 44
      There is no corresponding record for this reference.
    98. 98
      West, J. D., and Marnett, L. J.2005 Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal Chem. Res. Toxicol. 18 1642 1653
      There is no corresponding record for this reference.
    99. 99
      Carbone, D. L., Doorn, J. A., Kiebler, Z., and Petersen, D. R.2005 Cysteine modification by lipid peroxidation products inhibits protein disulfide isomerase Chem. Res. Toxicol. 18 1324 1331
      There is no corresponding record for this reference.
    100. 100
      Bruderer, R. M., Brasseur, C., and Meyer, H. H.2004 The AAA ATPase p97/VCP interacts with its alternative co-factors, Ufd1-Npl4 and p47, through a common bipartite binding mechanism J. Biol. Chem. 279 49609 49616
      There is no corresponding record for this reference.
    101. 101
      Cao, K., Nakajima, R., Meyer, H. H., and Zheng, Y.2003 The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis Cell 115 355 367
      There is no corresponding record for this reference.
    102. 102
      Hetzer, M., Meyer, H. H., Walther, T. C., Bilbao-Cortes, D., Warren, G., and Mattaj, I. W.2001 Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly Nat. Cell Biol. 3 1086 1091
      102
      Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly
      Hetzer, Martin; Meyer, Hemmo H.; Walther, Tobias C.; Bilbao-Cortes, Daniel; Warren, Graham; Mattaj, Iain W.
      Nature Cell Biology (2001), 3 (12), 1086-1091CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the mol. mechanisms and specificity of fusion events involved in NE formation.
    103. 103
      Patel, S., and Latterich, M.1998 The AAA team: related ATPases with diverse functions Trends Cell Biol. 8 65 71
      103
      The AAA team: related ATPases with diverse functions
      Patel, Sheetal; Latterich, Martin
      Trends in Cell Biology (1998), 8 (2), 65-71CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Science Ltd.)
      A review with 56 refs. A new family of related ATPases has emerged, characterized by a highly conserved AAA motif. This motif forms a 230-amino-acid domain that contains Walker homol. sequences and imparts ATPase activity. Homol. between AAA-family members is confined mostly to the AAA domain, although addnl. homol. outside the AAA motif is present among closely related proteins. AAA proteins act in a variety of cellular functions, including cell-cycle regulation, protein degrdn., organelle biogenesis and vesicle-mediated protein transport. The AAA domain is required for protein function, but its exact role and the specific activity that it confers on AAA proteins is still unclear. This review describes current understanding of the AAA protein family.
    104. 104
      Ye, Y., Meyer, H. H., and Rapoport, T. A.2001 The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol Nature 414 652 656
      There is no corresponding record for this reference.
    105. 105
      Wang, Q., Song, C., and Li, C. C.2004 Molecular perspectives on p97-VCP: Progress in understanding its structure and diverse biological functions J. Struct. Biol. 146 44 57
      There is no corresponding record for this reference.
    106. 106
      Dai, R. M., and Li, C. C.2001 Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation Nat. Cell Biol. 3 740 744
      106
      Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation
      Dai, Ren Ming; Li, Chou-Chi H.
      Nature Cell Biology (2001), 3 (8), 740-744CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      The ubiquitin-proteasome (Ub-Pr) degrdn. pathway regulates many cellular activities, but how ubiquitinated substrates are targeted to the proteasome is not understood. We have shown previously that valosin-contg. protein (VCP) phys. and functionally targets the ubiquitinated nuclear factor κB inhibitor, IκBα to the proteasome for degrdn. VCP is an abundant and a highly conserved member of the AAA (ATPases assocd. with a variety of cellular activities) family. Besides acting as a chaperone in membrane fusions, VCP has been shown to have a role in a no. of seemingly unrelated cellular activities. Here we report that loss of VCP function results in an inhibition of Ub-Pr-mediated degrdn. and an accumulation of ubiquitinated proteins. VCP assocs. with ubiquitinated proteins through the direct binding of its amino-terminal domain to the multi-ubiquitin chains of substrates. Furthermore, its N-terminal domain is required in Ub-Pr-mediated degrdn. We conclude that VCP is a multi-ubiquitin chain-targeting factor that is required in the degrdn. of many Ub-Pr pathway substrates, and provide a common mechanism that underlies many of the functions of VCP.
    107. 107
      Qiu, Y., Benet, L. Z., and Burlingame, A. L.1998 Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry J. Biol. Chem. 273 17940 17953
      There is no corresponding record for this reference.
    108. 108
      Doorn, J. A., Hurley, T. D., and Petersen, D. R.2006 Inhibition of human mitochondrial aldehyde dehydrogenase by 4-hydroxynon-2-enal and 4-oxonon-2-enal Chem. Res. Toxicol. 19 102 110
      There is no corresponding record for this reference.
    109. 109
      Dietze, E. C., Schafer, A., Omichinski, J. G., and Nelson, S. D.1997 Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide Chem. Res. Toxicol. 10 1097 1103
      There is no corresponding record for this reference.
    110. 110
      Uchida, K., and Stadtman, E. R.1993 Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction J. Biol. Chem. 268 6388 6393
      110
      Covalent attachment of 4-hydroxynonenal to glyceraldehyde 3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction
      Uchida, Koji; Stadtman, Earl R.
      Journal of Biological Chemistry (1993), 268 (9), 6388-93CODEN: JBCHA3; ISSN:0021-9258.
      In the present study, the detailed mechanism of 4-hydroxynonenal (HNE) modification of a key enzyme in intermediary metab., glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is studied mainly focusing on the formation of HNE-amino acid adducts in the enzyme. When GAPDH (1 mg/mL) was treated with 0-2 mM HNE in sodium phosphate buffer (pH 7.2) for 2 h at 37°, the enzyme was inactivated by HNE in a concn.-dependent manner. The loss of enzyme activity was assocd. with the loss of free sulfhydryl groups. Following its redn. with NaBH4, amino acid anal. of the HNE-modified enzyme demonstrated that histidine and lysine residues were also modified. At concns. lower than 0.5 mM, HNE reacts preferentially with cysteine and lysine residues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HNE-modified enzyme suggested the formation of intra- and intermol. cross-links of the enzyme subunit. The HNE-dependent loss of amino acid residues was accompanied by the generation of protein-linked carbonyl derivs. as assessed by redn. with NaB[3H]H4 and reaction with 2,4-dinitrophenylhydrazine. Thus, the conjugation of all the amino acids appears to involve Michael addn. type reactions in which the carbonyl function of HNE would be preserved. The modified histidine residues were quant. recovered as the HNE-histidine adduct. However, only 28% of the missing lysine could be accounted for as the HNE-lysine deriv., and only 15.6% of the modified cysteine could be accounted for as the HNE-cysteine thioether deriv. It is proposed that the carbonyl groups of the HNE-derived Michael addn. products may undergo secondary reactions with the amino acid groups of lysine residues to yield inter- and intrasubunit cross-links.
    111. 111
      Pumford, N. R., Halmes, N. C., Martin, B. M., Cook, R. J., Wagner, C., and Hinson, J. A.1997 Covalent binding of acetaminophen to N-10-formyltetrahydrofolate dehydrogenase in mice J. Pharmacol. Exp. Ther. 280 501 505
      111
      Covalent binding of acetaminophen to N-10-formyl-tetrahydrofolate dehydrogenase in mice
      Pumford, Neil R.; Halmes, N. Christine; Martin, Brian M.; Cook, Robert J.; Wagner, Conrad; Hinson, Jack A.
      Journal of Pharmacology and Experimental Therapeutics (1997), 280 (1), 501-505CODEN: JPETAB; ISSN:0022-3565. (Williams & Wilkins)
      The analgesic acetaminophen is frequently used as a model chem. to study hepatotoxicity; however, the crit. mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatog. and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were homologous with the deduced amino acid sequence from the cDNA of N-10-formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity at 2 h. The covalent binding of acetaminophen to proteins such as N-10-formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.
    112. 112
      Oleinik, N. V., and Krupenko, S. A.2003 Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis Mol. Cancer Res. 1 577 588
      112
      Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis
      Oleinik, Natalia V.; Krupenko, Sergey A.
      Molecular Cancer Research (2003), 1 (8), 577-588CODEN: MCROC5; ISSN:1541-7786. (American Association for Cancer Research)
      We have recently shown that transient expression of 10-formyltetrahydrofolate dehydrogenase (FDH) strongly inhibits proliferation of several cancer cell lines and ultimately results in cell death. In the present studies using Tet-On system, we have generated a stable A549 lung carcinoma cell line capable of inducible FDH expression. Using this system, we were able to express FDH at different levels depending on concn. of the inducer, doxycycline, and we have obsd. that inhibition of proliferation depends on FDH intracellular levels. We have further shown that induction of FDH expression results in initiation of apoptosis beginning 24 h post-induction. Apoptotic cells revealed cleavage of poly-(ADP-ribose) polymerase and general caspase inhibitor zVAD-fmk protected cells against FDH-induced apoptosis. FDH-expressing cells showed accumulation of cells in G0-G1 phase and a sharp decrease of cells in S phase. Accumulation of intracellular FDH was followed by accumulation of the tumor suppressor protein p53 and its downstream target p21. These results indicate that FDH antiproliferative effects on A549 cells include both G1 cell cycle arrest and caspase-dependent apoptosis.
    113. 113
      Cho, S. G., Lee, Y. H., Park, H. S., Ryoo, K., Kang, K. W., Park, J., Eom, S. J., Kim, M. J., Chang, T. S., Choi, S. Y., Shim, J., Kim, Y., Dong, M. S., Lee, M. J., Kim, S. G., Ichijo, H., and Choi, E. J.2001 Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1 J. Biol. Chem. 276 12749 12755
      113
      Glutathione S-transferase Mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1
      Cho, Ssang-Goo; Lee, Yong Hee; Park, Hee-Sae; Ryoo, Kanghyun; Kang, Keon Wook; Park, Jihyun; Eom, Soo-Jung; Kim, Myung Jin; Chang, Tong-Shin; Choi, Soo-Yeon; Shim, Jaekyung; Kim, Youngho; Dong, Mi-Sook; Lee, Min-Jae; Kim, Sang Geon; Ichijo, Hidenori; Choi, Eui-Ju
      Journal of Biological Chemistry (2001), 276 (16), 12749-12755CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways. It plays a crit. role in cytokine- and stress-induced apoptosis. To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast two-hybrid method. The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metab. of drugs and xenobiotics, interacted with ASK1. We subsequently confirmed that mGSTM1-1 phys. assocd. with ASK1 both in vivo and in vitro. The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells. MGSTM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metab.
    114. 114
      Ryoo, K., Huh, S. H., Lee, Y. H., Yoon, K. W., Cho, S. G., and Choi, E. J.2004 Negative regulation of MEKK1-induced signaling by glutathione S-transferase Mu J. Biol. Chem. 279 43589 43594
      There is no corresponding record for this reference.
    115. 115
      Dorion, S., Lambert, H., and Landry, J.2002 Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1 J. Biol. Chem. 277 30792 30797
      There is no corresponding record for this reference.
    116. 116
      Nerland, D. E., Cai, J., Pierce, W. M., Jr., and and Benz, F. W.2001 Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo Chem. Res. Toxicol. 14 799 806
      There is no corresponding record for this reference.
    117. 117
      Hsieh, J. C., Huang, S. C., Chen, W. L., Lai, Y. C., and Tam, M. F.1991 Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3 Biochem. J. 278Part 1 293 297
      There is no corresponding record for this reference.
    118. 118
      Chen, W. L., Hsieh, J. C., Hong, J. L., Tsai, S. P., and Tam, M. F.1992 Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3 Biochem. J. 286Part 1 205 210
      There is no corresponding record for this reference.
    119. 119
      Ozdemirler, G., Aykac, G., Uysal, M., and Oz, H.1994 Liver lipid peroxidation and glutathione-related defence enzyme systems in mice treated with paracetamol J. Appl. Toxicol. 14 297 299
      119
      Liver lipid peroxidation and glutathione-related defense enzyme systems in mice treated with paracetamol
      Ozdemirler, Gul; Aykac, Gulcin; Uysal, Mujdat; Oz, Hikmet
      Journal of Applied Toxicology (1994), 14 (4), 297-9CODEN: JJATDK; ISSN:0260-437X.
      Glutathione levels were found to be decreased while lipid peroxide levels were increased in total liver homogenates 6 h following paracetamol treatment (500 mg kg-1 i.p.). Furthermore, it has been detd. that cytosolic glutathione S-transferase (GST) activity was decreased and glutathione peroxidase (GSH-Px) activity remained unchanged. On the other hand, a decrease in liver microsomal lipid peroxide levels and an increase in GST and GSH-Px activity has been obsd. The authors concluded that decreased lipid peroxide levels in microsomes could be a consequence of increased GSH-Px and GST enzyme activities. In this way, these glutathione-related defense enzyme systems may play an important role in protecting microsomes from lipid peroxidn.
    120. 120
      Vega, M. C., Walsh, S. B., Mantle, T. J., and Coll, M.1998 The three-dimensional structure of Cys-47-modified mouse liver glutathione S-transferase P1-1. Carboxymethylation dramatically decreases the affinity for glutathione and is associated with a loss of electron density in the alphaB-310B region J. Biol. Chem. 273 2844 2850
      There is no corresponding record for this reference.
    121. 121
      Koen, Y. M., Yue, W., Galeva, N. A., Williams, T. D., and Hanzlik, R. P.2006 Site-specific arylation of rat glutathione s-transferase A1 and A2 by bromobenzene metabolites in vivo Chem. Res. Toxicol. 19 1426 1434
      There is no corresponding record for this reference.
    122. 122
      Mitchell, A. E., Morin, D., Lame, M. W., and Jones, A. D.1995 Purification, mass spectrometric characterization, and covalent modification of murine glutathione S-transferases Chem. Res. Toxicol. 8 1054 1062
      There is no corresponding record for this reference.
    123. 123
      Berhane, K., Widersten, M., Engstrom, A., Kozarich, J. W., and Mannervik, B.1994 Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases Proc. Natl. Acad. Sci. U.S.A. 91 1480 1484
      There is no corresponding record for this reference.
    124. 124
      Hartley, D. P., Ruth, J. A., and Petersen, D. R.1995 The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione S-transferase Arch. Biochem. Biophys. 316 197 205
      There is no corresponding record for this reference.
    125. 125
      Chelikani, P., Fita, I., and Loewen, P. C.2004 Diversity of structures and properties among catalases Cell Mol. Life Sci. 61 192 208
      125
      Diversity of structures and properties among catalases
      Chelikani, P.; Fita, I.; Loewen, P. C.
      Cellular and Molecular Life Sciences (2004), 61 (2), 192-208CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review. More than 300 catalase sequences are now available, divided among monofunctional catalases (>225), bifunctional catalase-peroxidases (>50) and Mn-contg. catalases (>25). When combined with the recent appearance of crystal structures from at least 2 representatives from each of these groups (9 from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large no. of modifications unique to catalases, a result of interacting with reactive O species (ROS). Biochem. and physiol. characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in microorganisms generally is controlled either by sensors of ROS or by growth phase regulons, although the detailed mechanisms vary considerably.
    126. 126
      Kahl, R., Kampkotter, A., Watjen, W., and Chovolou, Y.2004 Antioxidant enzymes and apoptosis Drug Metab. Rev. 36 747 762
      126
      Antioxidant enzymes and apoptosis
      Kahl, Regine; Kampkoetter, Andreas; Waetjen, Wim; Chovolou, Yvonni
      Drug Metabolism Reviews (2004), 36 (3-4), 747-762CODEN: DMTRAR; ISSN:0360-2532. (Marcel Dekker, Inc.)
      A review. A review on the relation between apoptosis and the two antioxidant enzymes, manganese superoxide dismutase (MnSOD) and catalase. Two schemes for the involvement of MnSOD and catalase in the regulation of apoptosis are established. First, both MnSOD and catalase inhibit apoptosis by removing superoxide anion radicals or H2O2, resp., because these reactive oxygen species are mediators required for the apoptotic program to inhibit a survival pathway. Second, an increase in H2O2 by downregulation or inhibition of catalase activity and/or upregulation of MnSOD activity inhibits apoptosis while a decrease in H2O2 by upregulation of catalase activity and/or downregulation of MnSOD activity supports apoptosis, possibly because of a supportive role of H2O2 in a survival pathway.
    127. 127
      Gonzalez, F. J., Peters, J. M., and Cattley, R. C.1998 Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activator receptor alpha J. Natl. Cancer Inst. 90 1702 1709
      127
      Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activated receptor α
      Gonzalez, Frank J.; Peters, Jeffrey M.; Cattley, Russell C.
      Journal of the National Cancer Institute (1998), 90 (22), 1702-1709CODEN: JNCIEQ; ISSN:0027-8874. (Oxford University Press)
      A review with 77 refs. Peroxisome proliferators are a diverse group of chems. that include several therapeutically used drugs (e.g., hypolipidemic agents), plasticizers and org. solvents used in the chem. industry, herbicides, and naturally occurring hormones. As the name implies, peroxisome proliferators cause an increase in the no. and size of peroxisomes in the liver, kidney, and heart tissue of susceptible species, such as rats and mice. Long-term administration of peroxisome proliferators can cause liver cancer in these animals, a response that has been the central issue of research on peroxisome proliferators for many years. Peroxisome proliferators are representative of the class of nongenotoxic carcinogens that cause cancer through mechanisms that do not involve direct DNA damage. The fact that humans are frequently exposed to these agents makes them of particular concern to government regulatory agencies responsible for assuring human safety. Whether frequent exposure to peroxisome proliferators represents a hazard to humans is unknown; however, increased cancer risk has not been shown to be assocd. with long-term therapeutic administration of the hypolipidemic drugs gemfibrozil, fenofibrate, and clofibrate. To make sound judgments regarding the safety of peroxisome proliferators, the validity of extrapolating results from rodent bioassays to humans must be based on the agents' mechanism of action and species differences in biol. activity and carcinogenicity. The peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor superfamily, has been found to mediate the activity of peroxisome proliferators in mice. Gene-knockout mice lacking PPARα are refractory to peroxisome proliferation and peroxisome proliferator-induced changes in gene expression. Furthermore, PPARα-null mice are resistant to hepatocarcinogenesis when fed a diet contg. a potent nongenotoxic carcinogen WY-14,643. Recent studies have revealed that humans have considerably lower levels of PPARα in liver than rodents, and this difference may, in part, explain the species differences in the carcinogenic response to peroxisome proliferators.
    128. 128
      Reddy, J. K.2004 Peroxisome proliferators and peroxisome proliferator-activated receptor alpha: Biotic and xenobiotic sensing Am. J. Pathol. 164 2305 2321
      128
      Peroxisome proliferators and peroxisome proliferator-activator receptor α: Biotic and xenobiotic sensing
      Reddy, Janardan K.
      American Journal of Pathology (2004), 164 (6), 2305-2321CODEN: AJPAA4; ISSN:0002-9440. (American Society for Investigative Pathology)
      A review discusses factual and conceptual advances made in the field of peroxisome proliferator-activated receptors (PPARs). It focuses on the PPAR family of nuclear receptors and PPARα as a sensor for xenobiotic peroxisome proliferators.
    129. 129
      Qiu, Y., Benet, L. Z., and Burlingame, A. L.2001 Identification of hepatic protein targets of the reactive metabolites of the non-hepatotoxic regioisomer of acetaminophen, 3′-hydroxyacetanilide, in the mouse in vivo using two-dimensional gel electrophoresis and mass spectrometry Adv. Exp. Med. Biol. 500 663 673
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    Detailed mass spectrometric analysis of the peptide fragments of target proteins. This information is available free of charge via the Internet at http://pubs.acs.org.


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.