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Monocyclic β-Lactams Are Selective, Mechanism-Based Inhibitors of Rhomboid Intramembrane Proteases

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† MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
‡ Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.
*[email protected]
Cite this: ACS Chem. Biol. 2011, 6, 4, 325–335
Publication Date (Web):December 22, 2010
https://doi.org/10.1021/cb100314y

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Abstract

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Rhomboids are relatively recently discovered intramembrane serine proteases that are conserved throughout evolution. They have a wide range of biological functions, and there is also much speculation about their potential medical relevance. Although rhomboids are weakly inhibited by some broad-spectrum serine protease inhibitors, no potent and specific inhibitors have been identified for these enzymes, which are mechanistically distinct from and evolutionarily unrelated to the classical soluble serine proteases. Here we report a new biochemical assay for rhomboid function based on the use of quenched fluorescent substrate peptides. We have developed this assay into a high-throughput format and have undertaken an inhibitor and activator screen of approximately 58,000 small molecules. This has led to the identification of a new class of rhomboid inhibitors, a series of monocyclic β-lactams, which are more potent than any previous inhibitor. They show selectivity, both for rhomboids over the soluble serine protease chymotrypsin and also, importantly, between different rhomboids; they can inhibit mammalian as well as bacterial rhomboids; and they are effective both in vitro and in vivo. These compounds represent important templates for further inhibitor development, which could have an impact both on biological understanding of rhomboid function and potential future drug development.

§ Author Contributions

These authors contributed equally to this work

Intramembrane proteases are relative newcomers to the long list of proteases that control many biologically and medically important cellular processes. The four families of intramembrane proteases comprise site-2 protease, (1) presenilin/γ-secretase, (2) signal peptide peptidase, (3) and rhomboids. (4) The last are intramembrane serine proteases but, like all the other families, are evolutionarily unrelated to their well-known soluble counterparts. Rhomboids exist in most sequenced organisms and, despite ignorance of many of their functions, are already known to participate in processes as diverse as growth factor signaling, (4-6) mitochondrial function, (7-9) invasion of host cells by apicomplexan parasites, (10-12) and bacterial protein export. (13)
One of the key differences between rhomboids and other serine proteases is that their active site resides in the lipid bilayer of membranes, where it is formed by residues contributed by transmembrane helices. (4, 14) This characteristic membrane environment establishes biophysical constraints on any potential inhibitors and also raises questions about the potential in vivo availability of inhibitors that might work in vitro in a detergent micelle system. There are no selective inhibitors yet reported for any rhomboids, although they are (weakly) inhibited by isocoumarins, broad-spectrum inhibitors of serine proteases, and a few other serine protease inhibitors. (4, 15)
There are strong incentives to identify selective rhomboid inhibitors. As research tools, such inhibitors could dramatically accelerate progress in discovery of the physiological role of rhomboids, especially in higher organisms such as mammals, where a genetic approach is slow and difficult. Furthermore, although they are not yet fully validated disease targets, there are very strong indications that rhomboid inhibitors are likely to have medical utility. (10, 12, 16-18) Being proteolytic enzymes, it seems likely that rhomboids are inherently amenable to small molecule inhibitor screening.
Here we describe the development of a new in vitro assay of rhomboid activity based on the use of fluorogenic peptides that span the recognition motif and cleavage site of known substrates. We have further developed this assay into a high-throughput format and undertaken a screen of a library of small molecules to look for compounds that can inhibit (or activate) rhomboid activity. This approach has led to the identification of a series of monocyclic β-lactams, the first potent and selective rhomboid inhibitors.

Results and Discussion

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Development of an in Vitro HTS Rhomboid assay

Previously reported in vitro assays of rhomboid activity have relied on incubating detergent-solubilized rhomboids with radioactive or non-radioactive protein substrates that encompassed the full transmembrane domain (TMD) and flanking regions. (15, 19-21) These approaches were not readily adaptable to a high-throughput screening (HTS) format, so we investigated the possibility of generating suitable fluorogenic peptidic substrates for rhomboids. Although natural substrates are full-length TMDs, rhomboids typically rely on a short recognition motif to determine cleavage. (19) We therefore tested the ability of short peptides derived from known rhomboid substrates to be cleaved by purified bacterial rhomboid proteases in vitro (Figure 1A,B). HPLC and mass spectrometry analysis showed that synthetic peptides derived from Providencia stuartii TatA and Drosophila Gurken were cleaved by P. stuartii and E. coli rhomboids AarA and GlpG at their natural cleavage sites, (19) albeit with varying efficiencies (Figure 1B).

Figure 1

Figure 1. Fluorescent assay design. (A) Schematic representation of rhomboid enzyme AarA cleaving a natural rhomboid substrate or a substrate-based peptide. (B) Comparison of AarA activity on three peptides derived from known rhomboid substrates. Rhomboid cleavage sites (asterisks) were determined by HPLC using peptide standards or MALDI-TOF mass spectrometry. The Gurken-derived peptide was modified with QSY21 dark quencher and Chromis-645 fluorophore to yield fluorogenic peptide KSp21. (C) Time course of cleavage of 1 μM KSp21 by 0.4 μM AarA (wt or S150A) or 0.2 μM chymotrypsin, at 25 °C. (D) Fluorescent activity assay adapted to high-throughput screen (HTS) format. Z′ factor = 0.77 (signal/background = 10, coefficients of variation = 6%) (see Methods). High control = AarA + DMSO; low control = no enzyme + DMSO.

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The ability of rhomboids to cleave short peptides allowed us to develop fluorogenic peptide substrates, suitable for high-throughput screening. We introduced several Förster resonance energy transfer (FRET) donor−acceptor pairs into the TatA and Gurken peptides, modifying residues that had been shown not to be part of the recognition motif of the substrate. (19) We tested a range of fluorescent dyes (MCA, TAMRA-550, AlexaFluor-647, Chromis-645), and the best results were obtained with a Gurken-derived 15-mer peptide, labeled by the Chromis-645/QSY21 FRET pair at the C-terminus and P5 position, respectively (Figure 1B). This peptide, KSp21, had better aqueous solubility than the other FRET-labeled peptides and was the most efficient fluorogenic substrate for AarA, the rhomboid whose substrate specificity is best understood. Progress curves of KSp21 cleavage by AarA, recorded continuously, were exponential, with a linear initial phase (Figure 1C). The initial reaction rate increased linearly with enzyme concentration up to 200 nM (Figure S1A) and to substrate concentration up to 1 μM (data not shown). At higher substrate concentrations (>2 μM), KSp21 precipitated, which prevented us from determining its apparent KM and Vmax. The solubility of KSp21 was improved by increasing DMSO concentration in the assay buffer, which also enhanced AarA activity. In a miniaturized format (20 μL, 384-well plate) adapted to HTS, the assay showed an excellent robustness with Z′ score (statistical indicator of assay reproducibility and sensitivity (22)) of ∼0.8 and a large assay window (Figure 1D). We concluded that the KSp21-based fluorescent assay, using the rhomboid AarA, provided an effective platform for a high-throughput screen for rhomboid inhibitors and activators.

A High-Throughput Screen

A flowchart summarizing the different steps of the screening campaign is illustrated in Figure 2A.

Figure 2

Figure 2. Screening campaign summary. (A) Screen flowchart. (B) Fluorescent signal of 162 initial hits and 190 high controls (DMSO + AarA), both denoted by blue diamonds, and 32 low controls (DMSO, no enzyme), denoted by magenta squares. DMSO high controls or inactive compounds cluster around the 100,000 activity units mark, inhibitors are represented by points below 100,000 activity units, and activators are above. (C) Plot of correlation between initial hits from high-throughput screen and secondary screen; 7 hits (6 false inhibitors, 1 false activator) were not confirmed as they showed ∼100% activity in the second screen (dotted line). (D) Chemical structure and apparent IC50 values of hits selected for chemistry optimization: series 1, bis(trifluoromethyl)-amide (bis-CF3), based on compound F1; and series 2, monocyclic β-lactam, based on compound L1.

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Pilot and Full Screens

The KSp21-based assay was validated in a pilot screen of the NINDS collection of 1,040 bioactive compounds (ICCB Longwood, Harvard Medical School). We obtained Z′ and Z (Z, the ratio of separation band to the signal dynamic range, allows the comparison between HTS assays (22)) factors of 0.9 and 0.6−0.8, respectively, and a correlation between two runs of R2 = 0.79, with a hit rate of 2.6% (cutoff as defined in Methods). These statistics strongly validated the technical quality of the screen. (22) We therefore ran a full primary screen for inhibitors and activators of AarA from a library of 57,703 molecules, comprising a set of diverse compounds from a variety of commercial suppliers, two small libraries of protease and kinase inhibitors, and a collection of 2,000 natural compounds (Analyticon). Compounds were screened at 20 μM, and AarA activity was measured by reading fluorescence after stopping the reaction. The statistics of the screen were comparable to the pilot (Z′ = 0.7−0.9), but hit rates were lower: 0.1−0.2% (synthetic molecules from the diversity set and focused libraries) and 1.3% (natural compounds). Interestingly, the 53 compounds of the Biomol classic protease inhibitors set (Enzo Life Sciences) did not give any hits, except the broad range serine protease inhibitor dichloroisocoumarin (DCI), a known rhomboid inhibitor. Combined with previous data that most serine protease inhibitors do not affect rhomboids, (4, 15) this illustrates that rhomboids are indeed novel targets.

Hit Confirmation and Counter-screen

We picked 162 compounds from the full screen and confirmed 97% of them in the same fluorescent assay (Figure 2B,C). In a counter-screen, the compounds were added postreaction to identify any false positives that interfere with the assay technology itself (e.g., precipitation-inducers, quenchers, or fluorescent compounds). No interfering compounds were detected, but one false activator was discarded (data not shown).

IC50/EC50 Measurements and Hit Ranking

A subset of 110 hits that inhibited AarA activity to less than 80% or increased it to more than 120% of the control and showed liability of 0 to 2 (defined as the frequency that the same compound has been hit in 16 previous screens of the same library against unrelated enzymes, thus excluding any obviously promiscuous and unspecific effectors), were characterized further. The potency of these compounds, including both inhibitors and activators, was determined by titrating their effect on AarA activity in the fluorescent assay. Subsequent kinetics revealed that the best activators were rather weak (EC50 > 0.1 mM), although they did produce up to 30-fold increase in initial rate at the highest concentration tested (100 μM; data not shown). Despite their potential interest, these activators were not pursued further in this study.
Continuous recording of reaction time courses was used to rank inhibitors by potency and selectivity over chymotrypsin, an unrelated serine protease. After plotting the initial rate of reaction versus compound concentration, we used a 4-parameter logistic equation to fit our data (Sigma Plot 11, Systat Software, Inc.) and calculate the apparent IC50 values (for simplicity, IC50 henceforth). We defined the rhomboid selectivity as the ratio of IC50 against chymotrypsin:IC50 against AarA. Of the 33 inhibitors with IC50 lower than 30 μM, 15 were synthetic small molecules (S1−S15, Table S1a) and 18 were natural products or semisynthetic analogues thereof (N1−N18, Table S1b). The natural products were highly heterogeneous in structure and not amenable to chemical optimization or rapid analogue synthesis and so were not pursued further. The synthetic compounds showed more promise. Strikingly, five related bis(trifluoromethyl) amide compounds (“bis-CF3”, prefixed “F”, Table S1a and Figure 2D) formed a distinctive series of inhibitors with a best IC50 ≤ 20 μM. Despite their low liability, these initial hits also inhibited chymotrypsin (selectivity 0.5−1). We also chose to pursue the monocyclic β-lactam S12/L1 (β-lactam series prefixed “L”, Figure 2D) which, despite being slightly less potent (IC50 = 23 μM) than the bis-CF3 compounds, had other encouraging attributes. Despite inhibiting chymotrypsin (selectivity 0.7), L1 had a liability of zero and, apart from cephaloridine (a broad spectrum antibiotic with cytotoxicity; promiscuous, with liability >2 in our screen), was uniquely positive among 87 other β-lactams in the library (not shown). Furthermore, the β-lactam motif present in L1 is a commonly occurring substructure within the protease inhibitor field, and several are well established as drugs. No other hits survived the triage process or brief exploratory chemistry to be selected for further analysis.

Structure−Activity Relationships and Selectivity of Hit Analogues

In the previous section, we described the selection of two classes of hit from the primary screen for optimization by chemical modification: the bis-CF3 compounds (F1−F5) and the monocyclic β-lactam L1. Each was used as a starting point for the chemical synthesis of related compounds, which were then assayed for potency and selectivity against AarA using the fluorescent peptide assay. Subsequently, active hits were validated in an independent in vitro assay, using a whole membrane protein substrate instead of a fluorescent peptide, and tested for selectivity against a different rhomboid showing similar substrate specificity, E. coli GlpG. (19)

Bis-CF3 Compounds (F-Series)

A total of 108 chemical structures related to compound F1 were either purchased or synthesized (structures and detailed results in Table S2). Modification or replacement of the bis(trifluoromethyl) group was attempted in the following ways: steric replacement of the fluorine atoms (F6, F9−11, F18, F23), “acidity”/chemical modifications (F15, F17, F24−26) including sulfone (F16), classical protease warhead-type replacements (F8, F12−14, F22), and moving/deleting the fluorines (F4−5, F7, F19). All of these changes to the bis-CF3 moiety completely destroyed inhibitory activity in the fluorescent assay. On the left-hand side of the molecule (Figure 2D), a range of substituents on the aniline ring were tolerated (Table S2), but replacement with a pyridine ring (F27−F29) was not. The best compounds were up to 17-fold more potent than F1, with IC50 values in the low micromolar range (F70−F73, Figure 3A). Despite this improvement, the structure−activity relationship analysis (SAR) of the aniline portion was rather flat, although the degree of selectivity over chymotrypsin was significantly improved (Figure 3A, Table S2). A number of analogues showed a net preference for AarA over chymotrypsin; among them, compounds F61 and F102 combined good potency with excellent selectivity.

Figure 3

Figure 3. In vitro effects of selected bis-CF3 analogues with modifications of the aniline moiety. (A) Results of fluorescent assay with 600 nM KSp21 peptide and either 100 nM AarA or 37 nM chymotrypsin. (B) Gel-based assay with 320 nM GlpG and 3 μM Gurken chimeric substrate and increasing concentration (μM) of inhibitor F72. Enzyme (E) substrate (S), and product (P) bands are labeled on the right-hand side of the gel. High molecular weight protein aggregates appear (vertical line) as inhibitor concentration increases.

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Despite this quite promising improvement of the bis-CF3 compounds, when tested against a full-length substrate in the gel-based assay, they raised significant concerns. Even at low micromolar levels they triggered formation of high molecular weight protein complexes, combined with the disappearance of the GlpG (Figure 3B), suggesting that they may cause aggregation of both enzyme and substrate. Indeed, in the presence of bis-CF3 compounds, substrate aggregated in the absence of enzyme, and enzyme oligomerized without substrate (data not shown). Although we have not investigated their mechanism of action and their low liability suggests that they might be acting more specifically than simply as unselective protein aggregators, these data were of sufficient concern to make us decide not to pursue this series further.

β-Lactams (L-Series)

A total of 58 analogues of β-lactam L1 were synthesized. Potency and specificity data of key compounds are shown in Table 1, and full details are in Supplementary Table S3; we here describe our main conclusions about structure−activity relationship of this series. As shown in Figure 2D, we introduced structural changes at all four positions on the β-lactam ring: the N-substituent (R1), the carbonyl group at C-2 and substituents at C-3 and C-4.
Table 1. Structure−activity Relationship of Key β-Lactam Compounds Described in the Main Text
1

Endogenous GlpG activity in E. coli NR698 (see Methods). *72-86% AarA activity at 100 μM compound.

•

Deletion of the C-2 carbonyl group (L30−31) suppressed activity, lending support to a mechanism-based mode of inhibition analogous to other β-lactam serine protease inhibitors, in which the rhomboid catalytic serine nucleophilically attacks the carbonyl group at C-2.

•

As the N-sulfonyl substituent is predicted to make the β-lactam ring very reactive, we tested straight-chain analogues to see whether the ring-opened form was responsible for inhibition. The chloroketones TPCK and TLCK were inactive against AarA, as were analogues L36−40, implying that the intact β-lactam ring is required for inhibitory activity.

•

Activity against AarA was suppressed by sulfonyl removal (L6−7) or replacement by urea (L28, L32−33), amide (L8, L12), or carbamate (L29). L34 was atypical as it retained activity against AarA despite the amide modification.

•

An N-aryl sulfonyl substituent was essential (L9 inactive), and lipophilic meta-substituents improved potency (L19−20, L42, L44−45).

•

Although the aryl ring at C-4 was not required for activity, its presence improved potency further, as did small lipophilic meta-substituents (L44−45). Up to 4-fold gain in potency against AarA was achieved in L44, by combining meta-substituents on both aryl rings.

•

The methyl group at C-3 in L1 was not essential (L2, L4−5, L14, L16−20, L35, L41−49 and L58), but the parent (unsubstituted) β-lactam L15 was inactive. Amide derivatives at C-3 reduced (L50−54) or abolished activity (L55−57).

The β-lactams showed very different degrees of selectivity for rhomboids over chymotrypsin. Out of 58 compounds, 19 strongly preferred chymotrypsin, whereas 6 preferred AarA (L14, L25−26, L41−42 and L54). Three of these six compounds combined selectivity for AarA with high potency: L25 and L26, without a substituent at C-4 (inactive on chymotrypsin), and L42 containing a 3′-biaryl sulfonyl group. This potential for high selectivity in favor of rhomboids is important, as it contrasts with other known rhomboid inhibitors (of which isocoumarins are best characterized), which are broad-spectrum serine protease inhibitors. (23)
In conclusion, the potency of these β-lactams against AarA was significantly improved after chemical optimization, so that the best IC50 values in the fluorescent assay were equivalent to that of DCI (L42, L44−45). Importantly, several β-lactams showed much better selectivity than DCI for rhomboid over chymotrypsin.

β-Lactams Show High Potency and Selectivity for GlpG

Unlike the bis-CF3 compounds, the β-lactams did not trigger substrate or enzyme aggregation in the gel-based assay of GlpG (Figure 4A) or AarA (data not shown), which relies on full-length protein substrates. Moreover, many showed high potency against GlpG (Figure 4B, Table 1), the best IC50 values being around 0.4−0.8 μM. We noted that IC50 values against AarA were 2-fold (L58) to 14-fold (L44) higher (Table 1) in the assay based on full-length proteins than in the fluorescent assay; in fact, L35 and L41 did not inhibit AarA at all in this assay, but they did in the fluorescent peptide assay. The greater resistance to inhibition in the full-length protein assay presumably reflects the different nature of the two assays, for example, concentration differences and the possibly higher affinity for the enzyme of a full-length, TMD-containing substrate, compared to a short peptide. We are unable to make a direct comparison of IC50 values against GlpG in the two assays, as GlpG does not cleave the KSp21 fluorescent peptide.

Figure 4

Figure 4. In vitro activity of β-lactams: high potency on GlpG and structure−activity relationship (SAR) against AarA and GlpG. (A) Typical gel showing β-lactam (L35) inhibiting the cleavage of Gurken chimeric substrate (3 μM) by GlpG (320 nM). Enzyme (E), substrate (S), and product (P) bands are indicated. (B) Band intensity was quantified using Image J, and substrate conversion was expressed as % of DMSO control. The apparent IC50 values in brackets were determined by fitting the data to a 4-parameter logistic equation. (C) Diagram summarizing SAR results. Analogues based on the L1 skeleton were obtained by structural modifications of the encircled substituents.

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SAR data show a clear relationship between compound structure and selectivity between the two rhomboids (Tables 1 and S3). For instance, certain substituents at C-4 lead to a complete preference for AarA (L14, L44, and L46) over GlpG. Similarly, L26 inhibited only AarA, in contrast with L25. On the other hand, GlpG was preferably targeted if the N-sulfonyl substituent was replaced (e.g., L8, L29), suggesting that GlpG could tolerate greater structural changes at this position. Curiously, the resynthesized racemic 3,4-cis-L1 (relative cis C-3:C-4 stereochemistry) was very potent on GlpG, whereas only the single enantiomer 3S,4S-L1 (HTS hit) was active on AarA. Many β-lactams preferred GlpG over AarA (Table 1): potency was 67- to 302-fold greater on GlpG than on AarA for L2, L10, L11, L19, L42, and L58. This net preference is probably also true for L4, L16, L17, L18, L20, L25, L45, and L47 as AarA retained 71−86% of DMSO control activity at 100 μM compound (Tables 1 and S3).
Although we cannot directly compare selectivity of compounds against GlpG and chymotrypsin, because they are assayed in two different formats, we can deduce high selectivity for GlpG over chymotrypsin for compounds that already show selectivity for AarA in the fluorescent assay. Examples are compounds L25 and L42 where a net rhomboid preference over chymotrypsin was achieved, with a high selectivity for GlpG (Table 1).
To summarize our conclusions based on in vitro analysis of β-lactam inhibition of rhomboid activity, we found that (i) an intact β-lactam ring is required for activity, suggesting a mechanism of action involving nucleophilic attack at C-2; (ii) β-lactams showed greater potency on GlpG than on AarA; (iii) distinct structural modifications, summarized in Figure 4C, resulted in improved potency and differential selectivity between two bacterial rhomboids, as well as between rhomboid and chymotrypsin (Table 1). The fact that different β-lactams preferentially targeted AarA or GlpG is significant as it validates the concept of developing compounds to target specific rhomboids.

In Vivo Inhibition by β-Lactams

Intramembrane proteases normally work in the lipid bilayer, but in vitro assays typically rely on detergent-solubilized enzyme activity. Are the β-lactam inhibitors we have identified effective in vivo? We tested whether any were able to inhibit endogenous GlpG in E. coli. To this end, the LacYTM2-containing fusion protein substrate of GlpG (21) was expressed in wild-type and GlpG-null strains of E. coli. Upon induction of substrate expression, only the wild-type strain was able to cleave LacYTM2, confirming that its processing is dependent on endogenous GlpG. Initial experiments indicated no inhibitory activity for the most potent β-lactams, and we reasoned that they might not be able to penetrate the bacterial outer membrane, which can form an impermeable barrier to small molecules. We therefore assayed activity using an E. coli mutant strain defective in outer membrane biogenesis (NR698) that has been reported to display increased outer membrane permeability to small molecules. (24) Similar to the wild-type E. coli, NR698 was able to process the LacYTM2 substrate efficiently, and cleavage was completely abolished by glpG disruption (strain KS69) (Figure 5A). In this background, seven out of 16 β-lactams inhibited endogenous GlpG, of which five (L16, L29, L35, L41, and L42) had an EC50 (defined as the concentration of the compound at which it exerted half of its maximal attainable effect (25)) between 5 and 10 μM (Figure 5B, Table 1).

Figure 5

Figure 5. In vivo activity of β-lactams. (A) Inhibition of endogenous GlpG by L29 in E. coli NR698 carrying imp4213 allele and a plasmid expressing the LacYTM2-based rhomboid substrate. Substrate (S) and product (P) were detected by Western blot with anti-Myc antibody, as described in Methods. The E. coli NR698 (+) or its ΔglpG variant (Δ) were grown in the presence of inhibitors at a range of concentrations and 2% DMSO. (B) The in vivo EC50 values (μM, in brackets) of selected β-lactams were obtained from band quantification performed as in Figure 4. Graph shows average data from three experiments, with standard errors up to 20%. (C) Mouse RHBDL2 was inhibited in vivo by L29, but not L35 and L41 (tested at 10 and 30 μM). COS7 cells were transfected with mRHBDL2 and Gurken substrate. Western blot was probed with anti-FLAG (Gurken) or anti-HA (mRHBDL2) antibody. S and P represent the substrate and product band, respectively.

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Finally, we also tested whether selected β-lactams showed inhibitory activity against a mammalian rhomboid. COS7 cells were transfected with C-terminally KDEL-tagged mouse rhomboid-2 (RHBDL2) and Gurken substrate. In this well-established assay, (26) the intracellular level of Gurken processing by RHBDL2 was assayed by comparing the ratio of product to substrate band intensity between the compound and solvent (DMSO) control. Out of 12 β-lactams tested, some had an effect on protein expression but did not change the product to substrate band intensity ratio, whereas three compounds (L25, L29, and L58) reproducibly displayed detectable inhibition of RHBDL2 in vivo, albeit at relatively modest levels. Notably, L29, the most potent inhibitor in bacteria, was also most potent against RHBDL2 (Figure 5C).
We conclude that selected monocyclic β-lactams not only are potent rhomboid inhibitors in vitro in the detergent micelle environment where our screen was performed but also can inhibit rhomboids in their native lipid membranes. Unlike isocoumarins, the best β-lactams are selective for rhomboids over chymotrypsin. Finally, our data show that monocyclic β-lactams can inhibit diverse prokaryotic and eukaryotic rhomboids and also that their selectivity is tunable by chemical modification.

Mechanism of Action of β-Lactam Rhomboid Inhibitors

Diverse β-lactams are inhibitors of several hydrolases, including some soluble serine proteases (reviewed in ref 23). However, rhomboids are evolutionarily unrelated to other proteases, and much remains unknown about their enzymatic activity, so it is impossible to predict with confidence the mechanism of their inhibition by the compounds we have identified. To begin to address this issue, we first tested whether inhibition was reversible, by following activity recovery after a rapid 100-fold dilution of the preincubated enzyme−inhibitor mixture into the reaction buffer and 30 min recovery period. (27, 28) We tested four β-lactams (L19, L42, L43, and L45) with IC50 < 20 μM against AarA, along with DCI as a positive control. All behaved similarly to L42 (Figure 6A): we observed a partial and significant recovery of activity after 100-fold dilution of enzyme and inhibitor (20−35% activity of the DMSO control) but negligible recovery after 100-fold dilution of the enzyme maintaining the initial inhibitor concentration (2−7% the activity of DMSO control), indicative of partial or slow reversibility of inhibition. We also tested for time-dependence of inhibition, which is typical of irreversible inhibitors. (23) Consistent with this idea, prolonging the preincubation time of AarA and β-lactams led to progressively more potent inhibitory effect (Figure 6B and data not shown). This point is important because it means that the apparent IC50 values of the inhibitors will strongly depend on the length of time of preincubation with rhomboid (Figure S2). The IC50 data reported in this paper all refer to 30 min preincubation.

Figure 6

Figure 6. Mechanism of action of selected β-lactams. (A) Reversibility of L42 inhibition of AarA was tested using the 100-fold dilution technique described in Results. Enzyme was incubated with inhibitor or DMSO for 30 min pre- and postdilution (to 71 nM final concentration); residual activity was then assayed in the presence of 600 nM KSp21 substrate. (B) The L42 compound displays time-dependent inhibition of AarA. AarA (100 nM) was preincubated with 1−100 μM L42 for varying times (8−188 min), after which activity was measured using 600 nM KSp21 fluorogenic substrate. Activities were expressed relative to the solvent (DMSO) control. The inset shows a plot of 1/kobsvs 1/[inhibitor concentration] (31). (C) The inhibitory monocyclic β-lactam L29 forms a 1:1 covalent complex with GlpG through its catalytic serine. GlpG or its S201A mutant (30 μM) were incubated with the indicated compound (100 μM) for 30 min at 25 °C, purified through a C4 ZipTip, and analyzed by MALDI-TOF mass spectrometry.

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Finally, to establish whether the inhibitors form covalent complexes with rhomboid, to assess the stoichiometry of such complexes, and to determine the possible specificity of this reaction for the catalytic serine, we used MALDI-TOF mass spectrometry. We analyzed the influence of two of the most potent β-lactam inhibitors of GlpG (L29 and L1) and an inactive β-lactam (L30) on the mass of purified full-length GlpG and its mutant form lacking the catalytic serine (S201A). In a proof-of-principle experiment we showed that 3-fold stoichiometric excess (100 μM) of the known mechanism-based isocoumarin inhibitor JLK-6 (29) generated a single adduct with wild-type GlpG but not with the S201A mutant (data not shown). Using the same experimental method, the β-lactams L29 and L1 caused a mass shift of the enzyme peak that was approximately equal to their molecular weight when incubated with wild-type GlpG but not with the catalytic serine mutant S201A (Figure 6C and not shown). Conversely, the β-lactam compound L30 that did not inhibit GlpG in vitro had no effect on the mass of either wild-type or catalytic serine mutant of GlpG (Figure 6C). These results demonstrate that monocyclic β-lactams inhibit rhomboid proteases by specific covalent binding to their catalytic serine. At present it is not clear whether the compounds L29 and L1 can also bind to the catalytic histidine: although a doubly bound adduct, with alkylation of the acyl enzyme intermediate through the active site His, has been described for some β-lactams, (23) the chemical structures of L29 and L1 suggest this to be unlikely. Moreover, the slow reversibility of inhibition indicated by the dilution assay rather suggests a single covalent link through the catalytic serine. (23) A definitive answer about the mechanism of action will require more accurate mass data or a crystal structure of the enzyme−inhibitor complex.

Concluding Remarks

Intramembrane proteases like rhomboids are increasingly understood to control many important biological processes, but they are much less well characterized than most protease families. Until now, no rhomboid inhibitors have been identified, beyond a small number of broad-spectrum serine protease inhibitors, notably isocoumarins, which have weak inhibitory activity and high cytotoxicity. The monocyclic β-lactam inhibitors that we have now identified provide exciting starting points for further development of potent and specific rhomboid inhibitors. Moreover, since many β-lactams are already used as pharmaceuticals, these molecules may provide valuable leads for future drug development.

Methods

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Materials

Peptides were synthesized commercially, except for the MCA-DNP modified Gurken, which was made in-house. Fluorescent peptide KSp21 labeled with dark quencher QSY21 and Chromis-645-A-PDA fluorescent dye (50 mg) was synthesized by Cambridge Research Biochemicals (CRB). Dodecyl maltoside was from Glycon Biochemical or Anatrace. E. coli NR698 (MC4100 imp4213) was a generous gift from Dr. Tom Silhavy, Princeton University, NJ, USA.

Recombinant Protein Expression and Purification

Bacterial rhomboids (wild-type and its catalytic serine to alanine mutant) C-terminally His-tagged were expressed in E. coli C43(DE3), (30) isolated from membrane fraction, solubilized in dodecyl maltoside (DDM), and purified on a His-Pure Cobalt (ThermoScientific) affinity column. The column was washed with 10 mM imidazole in 20 mM Hepes-NaOH, 0.3 M NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, pH 7.4. AarA and GlpG were eluted at 70 and 150 mM imidazole, respectively. Gurken or LacY derived chimeric substrates (MBP-TMD-Trx-His6 (19)) were overexpressed in GlpG-deficient E. coli (MC4100 glpG::tet, strain KS52), which had been generated by P1 phage mediated transduction of the glpG::tet allele from E. coli GW338 (21) onto a fresh MC4100 background. Overexpressed chimeric substrates were isolated from the membrane fraction, solubilized in DDM, and purified on a His-Pure column (wash buffer, elution buffer and strip buffer at 25, 70, and 150 mM imidazole, respectively), except that Gurken TMD fusion was further purified on amylose resin (New England Biolabs).

In Vitro Fluorescent Assay, HTS, and Counter-screen

Rhomboid protease assay was performed at 24−25 °C in Nunc White 384-well plates (VWR) in a final reaction volume of 20 μL. Each reaction contained the following final concentration of reagents: 25 mM Hepes pH 7.4, 5 mM EDTA, 5% (v/v) glycerol, 0.5% (w/v) DDM, 20% (v/v) DMSO, 100 nM rhomboid AarA or 37 nM chymotrypsin (Sigma), and 600 nM of rhomboid fluorescent peptide substrate. Importantly, increasing the DMSO concentration from 5% to 20% led to a linear increase in the cleavage rate of KSp21, suggesting that DMSO did not compromise AarA function in this concentration range. Using Biomek FX (Beckman-Coulter) robotics, 8 μL of enzyme mix (reaction buffer, DDM, AarA, or enzyme buffer) was first added in each well, then 2 μL of 200 μM compound or DMSO only. All compounds were preincubated with the rhomboid enzyme for 30 min at 25 °C to accommodate any slow-binding effectors, and the reaction was started by the addition of 10 μL of peptide mix (reaction buffer, DDM, DMSO, peptide) using a Flexdrop liquid dispenser (Perkin-Elmer). After 50−60 min, the reaction was stopped, while still in the linear phase, with 4 μL of 1 M phosphoric acid pH 1.5 on a WellMate (ThermoScientific). Fluorescence intensity was detected using a Pherastar plate reader (BMG-Labtech) with excitation at 640 nm and emission at 680 nm. After screening 57,703 synthetic and natural compounds, we identified 162 hits, applying a cutoff at 80% (or 120%) activity of DMSO high control activity for hit confirmation and counter-screening. Counter-screen consisted of adding the compounds after the reaction was completed to identify compounds that interfered with the fluorescent signal, independent of rhomboid inhibition.

Chemical Synthesis

For variation of the bis-CF3 aryl ring portion, the appropriate aniline was coupled with commercially available 3,3,3-trifluoro-2-(trifluoromethyl)propionic acid under nonbasic conditions. Noncommercial anilines were accessed by standard functional group transformations of nitro compounds, followed by reduction. Replacements for the bis-CF3 group were prepared by coupling with the appropriate acid, followed by further transformations where necessary. Most β-lactams were prepared by N-sulfonylation of commercially available 4-phenylazetidine-2-one. Non-commercial C-4-substituted analogues were accessed by ring closure of the corresponding β-amino acids, followed by N-sulfonylation. Compounds with an amide at C-3 were prepared from N-protected dl-serine by ring closure of the N-methoxyamide, conversion to the N-sulfonyl β-lactam, deprotection of the C-3 amino group, and treatment with the acid chloride or acid under standard conditions. Open chain analogues were prepared by means of standard transformations on the corresponding amino acids.

In Vitro Gel-Based Assay

Activity of the best hits was assessed in an independent in vitro assay using purified recombinant protein substrates (MBP-TMD-Trx-His6, with LacY or Gurken TMD) followed by SDS-PAGE analysis of the cleavage products, as reported previously. (19) A typical 20 μL reaction, run at 37 °C for 60−75 min, contained 25 mM Hepes-NaOH pH 7.4, 5 mM EDTA, 5% (v/v) glycerol, 0.05% (w/v) DDM, 0.4 M (for GlpG) or 0.1 M (for AarA) NaCl, 3 μM chimeric protein substrate, 320 nM GlpG or 700 nM AarA, and 5% (v/v) DMSO. All compounds were preincubated with the enzymes for 30 min at 25 °C.

In Vivo Assay in Bacteria

The glpG::tet disruption allele from E. coli GW338 (21) was transferred into E. coli NR698 genome using P1 phage transduction to generate strain KS69 (ΔglpG), which was selected and maintained on 5 μg/mL tetracycline. Both NR698 and KS69 were electroporated with pGW93 encoding β-lactamase-LacYTM2-MBP-His6-Myc chimera (21) and selected on 50 μg/mL spectinomycin. NR698 and KS59 carrying pGW93 were grown overnight at 37 °C with 50 μg/mL spectinomycin and 5 μg/mL tetracycline. The next day, 1.5 mL of fresh LB medium was inoculated with 1/100 (NR698) or 1/33 (KS69) of overnight culture in the presence of spectinomycin. After ∼2 h at 37 °C or when OD600 nm reached 0.3−0.4 absorbance unit, 150 μL was transferred to clean tubes, and 3 μL of DMSO or 1−100 μM β-lactam was added (2% (v/v) DMSO throughout). After 15 min at RT, each tube was supplemented with 1 mM cAMP and 1 mM IPTG, and culture was maintained at 25 °C, 1200 rpm, for 1 h for recombinant protein expression. (21) Bacterial cells were collected by centrifugation, and pellets were dissolved in SDS-PAGE sample buffer. After electrophoresis on 4−20% Bis-Tris gradient gel (Invitrogen), samples were transferred to PVDF Immobilon-P membrane (Millipore), and protein loading and transfer efficiency were checked by Ponceau red staining. Myc-tagged proteins were visualized by anti-Myc primary (1:900 c-Myc (A-14):sc-789, Santa-Cruz Biotechnology, Inc.) and anti rabbit-HRP conjugate secondary antibodies and detected by enhanced chemiluminescence (GE Healthcare).

In Vivo Assay in Mammalian Cells

COS7 cells were transfected in 24-well plates with 20 ng of HA-tagged mouse RHBDL2-KDEL (RHBDL2) plasmid, 50 ng of FLAG-tagged Gurken substrate construct, and pcDNA3 vector up to a total amount of 200 ng DNA per well using Fugene6 (Roche). Inhibitors (dissolved in DMSO) were added 2 h post transfection to final concentrations of 10 and 30 μM and 1% (v/v) DMSO. Twelve hours post transfection, cells were lysed in 1X SDS-PAGE sample buffer, and proteins were separated on a 4−20% Tris-glycine gradient gel (Invitrogen). Processing of Gurken by RHBDL2 was analyzed by Western Blot using HRP-conjugated anti-FLAG (Sigma) antibody for Gurken detection and HRP-conjugated anti-HA antibody (Covance) for RHBDL2 detection.

Mass Spectrometry

Purified full-length C-terminally His-tagged GlpG (20) and its active site mutant S201A (30 μM) were incubated with 100 μM inhibitor for 30 min at 25 °C in a buffer of pH 7.5 containing 20 mM HEPES, 0.05% (w/v) dodecylmaltoside, 100 mM NaCl, 10% (v/v) glycerol, and 5% (v/v) dimethylsulfoxide (DMSO). Samples were adjusted to 0.1% (v/v) trifluoroacetic acid (TFA) and purified using a C4 ZipTip (Millipore) according to manufacturer’s instructions, eluting into 5 μL of 10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA, Sigma) matrix dissolved in 50% (v/v) acetonitrile in water and 0.05% (v/v) TFA. The ZipTip eluates were spotted onto a thin layer of CHCA and immediately analyzed by MALDI-TOF mass spectrometry on a Voyager-DE PRO in linear positive mode with external mass calibration on bovine insulin, E. coli thioredoxin, and horse apomyoglobin (Calibration Mixture 3, Applied Biosystems).

Supporting Information

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Author Information

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  • Corresponding Author
    • Matthew Freeman - MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.;  Email: [email protected]
  • Authors
    • Olivier A. Pierrat - MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
    • Kvido Strisovsky - MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
    • Yonka Christova - MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
    • Jonathan Large - Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.
    • Keith Ansell - Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.
    • Nathalie Bouloc - Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.
    • Ela Smiljanic - Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.

Acknowledgment

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We thank our colleagues Debbie Taylor for valuable strategic input, Simon Osborne for expert chemical advice, David Whalley and Puneet Khurana for help with the HTS robotics, David Owen for peptide synthesis and Elaine Stephens for advice on mass spectrometry. We are also grateful to Yoshinori Akiyama (Kyoto) for plasmids and glpG knock-out strains of E. coli and to Tom Silhavy (Princeton) for E. coli strain NR698. O.A.P. was partly funded by the MRC Technology Development Gap Fund. K.S. acknowledges support from a Marie Curie Intraeuropean Fellowship, EMBO Long-Term Fellowship, and Medical Research Council UK Career Development Fellowship.

References

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This article references 31 other publications.

  1. 1
    Rawson, R. B., Zelenski, N. G., Nijhawan, D., Ye, J., Sakai, J., Hasan, M. T., Chang, T. Y., Brown, M. S., and Goldstein, J. L. (1997) Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs Mol. Cell 1, 47– 57
    Google Scholar
    1
    Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs
    Rawson, Robert B.; Zelenski, Nikolai G.; Nijhawan, Deepak; Ye, Jin; Sakai, Juro; Hasan, Mazahir T.; Chang, T. Y.; Brown, Michael S.; Goldstein, Joseph L.
    Molecular Cell (1997), 1 (1), 47-57CODEN: MOCEFL ISSN:. (Cell Press)
    We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element-binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metab. The active NH2-terminal domains of SREBPS are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HEXXH sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlt1Knsw%253D%253D&md5=be99d137a9703a0d8efe5928eb25c492
  2. 2
    De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., Von Figura, K., and Van Leuven, F. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein Nature 391, 387– 390
    Google Scholar
    2
    Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein
    De Strooper, Bart; Saftig, Paul; Craessaerts, Katleen; Vanderstichele, Hugo; Guhde, Gundula; Annaert, Wim; Von Figura, Kurt; Van Leuven, Fred
    Nature (London) (1998), 391 (6665), 387-390CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
    Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the prodn. of the amyloidogenic peptide amyloid-β (1-42) by proteolytic processing of the amyloid precursor protein (APP). Here the authors investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by α- and β-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by γ-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the prodn. of amyloid peptide. Pulse-chase expts. indicated that PS1 deficiency specifically decreased the turnover of the membrane-assocd. fragments of APP. As in the regulation of cholesterol metab. by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. The results indicate that mutations in PS1 that manifest clin. cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXot1GrtQ%253D%253D&md5=4327e88b436a201cd4c01938ae01e9f0
  3. 3
    Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K., and Martoglio, B. (2002) Identification of signal peptide peptidase, a presenilin-type aspartic protease Science 296, 2215– 2218
    Google Scholar
    3
    Identification of signal peptide peptidase, a presenilin-type aspartic protease
    Weihofen, Andreas; Binns, Kathleen; Lemberg, Marius K.; Ashman, Keith; Martoglio, Bruno
    Science (Washington, DC, United States) (2002), 296 (5576), 2215-2218CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biol. important peptides.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XkvFGhtrg%253D&md5=65ab559d2949577e92d7e9da70031698
  4. 4
    Urban, S., Lee, J. R., and Freeman, M. (2001) Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases Cell 107, 173– 182
    Google Scholar
    4
    Drosophila Rhomboid-1 defines a family of putative intramembrane serine proteases
    Urban, Sinisa; Lee, Jeffrey R.; Freeman, Matthew
    Cell (Cambridge, MA, United States) (2001), 107 (2), 173-182CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFα-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but the authors' anal. suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to the authors' knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and the results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotVSntrk%253D&md5=ebc50d63048fdfa6ec971e2f5b618316
  5. 5
    Urban, S., Lee, J. R., and Freeman, M. (2002) A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands EMBO J. 21, 4277– 4286
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Lee, J. R., Urban, S., Garvey, C. F., and Freeman, M. (2001) Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila Cell 107, 161– 171
    Google Scholar
    6
    Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila
    Lee, Jeffrey R.; Urban, Sinisa; Garvey, Clare F.; Freeman, Matthew
    Cell (Cambridge, MA, United States) (2001), 107 (2), 161-171CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The membrane proteins Star and Rhomboid-1 have been genetically defined as the primary regulators of EGF receptor activation in Drosophila, but their mol. mechanisms have been elusive. Both Star and Rhomboid-1 have been assumed to work at the cell surface to control ligand activation. Here, the authors demonstrate that they control receptor signaling by regulating intracellular trafficking and proteolysis of the ligand Spitz. Star is present throughout the secretory pathway and is required to export Spitz from the endoplasmic reticulum to the Golgi app. Rhomboid-1 is localized in the Golgi, where it promotes the cleavage of Spitz. This defines a novel growth factor release mechanism that is distinct from metalloprotease-dependent shedding from the cell surface.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotVSntrg%253D&md5=daeefd89709ee564ba4e6d8973925cc2
  7. 7
    Herlan, M., Vogel, F., Bornhovd, C., Neupert, W., and Reichert, A. S. (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA J. Biol. Chem. 278, 27781– 27788
    Google Scholar
    7
    Processing of Mgm1 by the Rhomboid-type Protease Pcp1 Is Required for Maintenance of Mitochondrial Morphology and of Mitochondrial DNA
    Herlan, Mark; Vogel, Frank; Bornhoevd, Carsten; Neupert, Walter; Reichert, Andreas S.
    Journal of Biological Chemistry (2003), 278 (30), 27781-27788CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, assocd. with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341-352; Wong, E. D., Wagner, J. A., Scott, S. V., Okreglak, V., Holewinske, T. J., Cassidy-Stone, A., and Nunnari, J. (2003) J. Cell Biol. 160, 303-311; Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in press). Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, we have characterized these isoforms. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphol. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Δpcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Δmgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphol. and maintenance of mitochondrial DNA.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlsFKktbw%253D&md5=1644c04e57cdedb3d14c707e84fc3516
  8. 8
    McQuibban, G. A., Saurya, S., and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease Nature 423, 537– 541
    Google Scholar
    8
    Mitochondrial membrane remodelling regulated by a conserved rhomboid protease
    McQuibban, G. Angus; Saurya, Saroj; Freeman, Matthew
    Nature (London, United Kingdom) (2003), 423 (6939), 537-541CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signaling in Drosophila. Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have addnl. roles. Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p. RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria. We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion. Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Δ mitochondrial phenotype. Our data indicate that mitochondrial membrane remodeling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signaling. In addn., mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homolog, PARL, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktVygtLY%253D&md5=36bbe704b629a548b08da2152f5147ca
  9. 9
    McQuibban, G. A., Lee, J. R., Zheng, L., Juusola, M., and Freeman, M. (2006) Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function Curr. Biol. 16, 982– 989
    Google Scholar
    9
    Normal Mitochondrial Dynamics Requires Rhomboid-7 and Affects Drosophila Lifespan and Neuronal Function
    McQuibban, G. Angus; Lee, Jeffrey R.; Zheng, Lei; Juusola, Mikko; Freeman, Matthew
    Current Biology (2006), 16 (10), 982-989CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    In addn. to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biol. The authors have recently identified a novel and conserved mitochondrial rhomboid intramembrane protease that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane. The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. In Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. The authors also find that mutations in Drosophila optic atrophy 1-like (Opa1-like), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. Rhomboid-7 mutant flies have severe neurol. defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. Rhomboid-7 mutant flies also have a greatly reduced lifespan.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVKjtro%253D&md5=85e73636998083919a36877e7e079fe1
  10. 10
    O’Donnell, R. A., Hackett, F., Howell, S. A., Treeck, M., Struck, N., Krnajski, Z., Withers-Martinez, C., Gilberger, T. W., and Blackman, M. J. (2006) Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite J. Cell Biol. 174, 1023– 1033
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Baker, R. P., Wijetilaka, R., and Urban, S. (2006) Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria PLoS Pathog. 2, e113
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Srinivasan, P., Coppens, I., and Jacobs-Lorena, M. (2009) Distinct roles of Plasmodium rhomboid 1 in parasite development and malaria pathogenesis PLoS Pathog. 5, e1000262
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Stevenson, L. G., Strisovsky, K., Clemmer, K. M., Bhatt, S., Freeman, M., and Rather, P. N. (2007) Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase Proc. Natl. Acad. Sci. U.S.A. 104, 1003– 1008
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Wang, Y., Zhang, Y., and Ha, Y. (2006) Crystal structure of a rhomboid family intramembrane protease Nature 444, 179– 180
    Google Scholar
    14
    Crystal structure of a rhomboid family intramembrane protease
    Wang, Yongcheng; Zhang, Yingjiu; Ha, Ya
    Nature (London, United Kingdom) (2006), 444 (7116), 179-180CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and γ-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 Å resoln. crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water mols. are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFyqt7jO&md5=a4e1925460417fb1b990beb7fc01b262
  15. 15
    Urban, S. and Wolfe, M. S. (2005) Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity Proc. Natl. Acad. Sci. U.S.A. 102, 1883– 1888
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Baxt, L. A., Rastew, E., Bracha, R., Mirelman, D., and Singh, U. (2010) Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis Eukaryotic Cell 9, 1283– 1293
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Freeman, M. (2008) Rhomboid proteases and their biological functions Annu. Rev. Genet. 42, 191– 210
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Urban, S. (2009) Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms Nat. Rev. Microbiol. 7, 411– 423
    Google Scholar
    18
    Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms
    Urban, Sinisa
    Nature Reviews Microbiology (2009), 7 (6), 411-423CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Proteolysis in cellular membranes to liberate effector domains from their transmembrane anchors is a well-studied regulatory mechanism in animal biol. and disease. By contrast, the function of intramembrane proteases in unicellular organisms has received little attention. Recent progress has now established that intramembrane proteases execute pivotal roles in a range of pathogens, from regulating Mycobacterium tuberculosis envelope compn., cholera toxin prodn., bacterial adherence and conjugation, to malaria parasite invasion, fungal virulence, immune evasion by parasitic amoebae and hepatitis C virus assembly. These advances raise the exciting possibility that intramembrane proteases may serve as targets for combating a wide range of infectious diseases. This Review focuses on summarizing the advances, evaluating the limitations and highlighting the promise of this newly emerging field.
    >> More from SciFinder ®
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  19. 19
    Strisovsky, K., Sharpe, H. J., and Freeman, M. (2009) Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates Mol. Cell 36, 1048– 1059
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Lemberg, M. K., Menendez, J., Misik, A., Garcia, M., Koth, C. M., and Freeman, M. (2005) Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases EMBO J. 24, 464– 472
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Maegawa, S., Ito, K., and Akiyama, Y. (2005) Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane Biochemistry 44, 13543– 13552
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays J. Biomol. Screening 4, 67– 73
    Google Scholar
    22
    A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
    Zhang; Chung; Oldenburg
    Journal of biomolecular screening (1999), 4 (2), 67-73 ISSN:.
    The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
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  23. 23
    Powers, J. C., Asgian, J. L., Ekici, O. D., and James, K. E. (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases Chem. Rev. 102, 4639– 4750
    Google Scholar
    23
    Irreversible inhibitors of serine, cysteine, and threonine proteases
    Powers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen Ellis
    Chemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
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  24. 24
    Ruiz, N., Falcone, B., Kahne, D., and Silhavy, T. J. (2005) Chemical conditionality: a genetic strategy to probe organelle assembly Cell 121, 307– 317
    Google Scholar
    24
    Chemical conditionality: A genetic strategy to probe organelle assembly
    Ruiz, Natividad; Falcone, Brian; Kahne, Daniel; Silhavy, Thomas J.
    Cell (Cambridge, MA, United States) (2005), 121 (2), 307-317CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The assembly of the Escherichia coli outer membrane (OM) is poorly understood. Although insight into fundamental cellular processes is often obtained from studying mutants, OM-defective mutants have not been very informative because they generally have nonspecific permeability defects. Here, the authors show that toxic small mols. can be used in selections employing strains with permeability defects to create particular chem. conditions that demand specific suppressor mutations. Suppressor phenotypes are correlated with the phys. properties of the small mols., but the mutations are not in their target genes. Instead, mutations allow survival by partially restoring membrane impermeability. Using "chem. conditionality," the authors identified mutations in yfgL and showed that YfgL is part of a multiprotein complex involved in the assembly of OM β barrel proteins. They posit that panels of toxic small mols. will be useful for generating chem. conditionalities that enable identification of genes required for organelle assembly in other organisms.
    >> More from SciFinder ®
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  25. 25
    Neubig, R. R., Spedding, M., Kenakin, T., and Christopoulos, A. (2003) International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology Pharmacol. Rev. 55, 597– 606
    Google Scholar
    25
    International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology
    Neubig, Richard R.; Spedding, Michael; Kenakin, Terry; Christopoulos, Arthur
    Pharmacological Reviews (2003), 55 (4), 597-606CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
    A review. The recommendations that follow have been updated from the proposals of a Tech. Subcommittee set up by the International Union of Pharmacol. Committee on Receptor Nomenclature and Drug Classification. International Union of Pharmacol. Committee on Receptor Nomenclature and Drug Classification. IX. Recommendations on terms and symbols in quant. pharmacol.
    >> More from SciFinder ®
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  26. 26
    Urban, S., Schlieper, D., and Freeman, M. (2002) Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids Curr. Biol. 12, 1507– 1512
    Google Scholar
    26
    Conservation of Intramembrane Proteolytic Activity and Substrate Specificity in Prokaryotic and Eukaryotic Rhomboids
    Urban, Sinisa; Schlieper, Daniel; Freeman, Matthew
    Current Biology (2002), 12 (17), 1507-1512CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands. Although nothing is known about the function of the ∼100 currently known rhomboid genes conserved throughout evolution, a recent anal. suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the prodn. of a quorum-sensing factor. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans. However, the function of prokaryotic Rhomboids is unknown. We have examd. the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-pos. and four Gram-neg. species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor α (TGFα) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homol., the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.
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  27. 27
    Harper, J. W., Hemmi, K., and Powers, J. C. (1985) Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor Biochemistry 24, 1831– 1841
    Google Scholar
    27
    Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor
    Harper, J. Wade; Hemmi, Keiji; Powers, James C.
    Biochemistry (1985), 24 (8), 1831-41CODEN: BICHAW; ISSN:0006-2960.
    The mechanism-based inactivation of a no. of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated, inactivated all of the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or β-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1, where [I] is the concn. of inhibitor); the inhibited enzyme was quite stable to reactivation (rate const. of deacylation = 2 × 10-5 s-1), whereas enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation decreased dramatically in the presence of reversible inhibitors or substrates; UV spectral measurements indicated that the isocoumarin ring structure was lost upon inactivation. Chymotrypsin Aγ was totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and ∼1 equiv of H+ was released on inactivation. Thus, these compds. react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the 1st mechanism-based inhibitors reported for many of the enzymes tested; 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.
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  28. 28
    Harper, J. W. and Powers, J. C. (1985) Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors Biochemistry 24, 7200– 7213
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Vinothkumar, K. R., Strisovsky, K., Andreeva, A., Christova, Y., Verhelst, S., and Freeman, M. (2010) The structural basis for catalysis and substrate specificity of a rhomboid protease EMBO J. 29, 3797– 3809
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Miroux, B. and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels J. Mol. Biol. 260, 289– 298
    Google Scholar
    30
    Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels
    Miroux, Bruno; Walker, John E.
    Journal of Molecular Biology (1996), 260 (3), 289-298CODEN: JMOBAK; ISSN:0022-2836. (Academic)
    We have investigated the over-prodn. of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expressing of the target membrane protein was induced, most of the BL21 (DE3) host cells died. Similar effects were also obsd. with expression vectors for ten globular proteins. Therefore, protein over-prodn. in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high satn. cell d., and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior t BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells contg. the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H+ symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit B was lower in C41(DE3) and C43(DE3), resp., than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts to overcome other toxic effects assocd. with over-expression.
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  31. 31
    Kitz, R. and Wilson, I. B. (1962) Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase J. Biol. Chem. 237, 3245– 3249
    Google Scholar
    31
    Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase
    Kitz, R.; Wilson, Irwin B.
    Journal of Biological Chemistry (1962), 237 (), 3245-49CODEN: JBCHA3; ISSN:0021-9258.
    Nine esters (anhydrides) of methanesulfonic acid, analogs to well known carbamate (e.g. neostigmine) and phosphate (e.g. paraoxon) inhibitors, were examd. as possible irreversible inhibitors of acetylcholinesterase. Five were active. Of these, 4 formed initial reversible complexes. The progressive development of irreversible inhibition was slowed by reversible inhibitors, indicating that the active site is involved. The inhibited enzyme is not reactivated by water or by hydroxylamine, hydrazine, acetate, or pyridine-2-aldoxime methiodide. It is reactivated by pyridine-3-aldoxime methiodide. On the basis of the evidence presented, it may be concluded safely that the active methanesulfonates form methanesulfonyl enzyme derivs. and, therefore, belong to the class of acid-transferring or oxydiaphoric inhibitors.
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  1. Hester A. Beard, Marta Barniol-Xicota, Jian Yang, Steven H. L. Verhelst. Discovery of Cellular Roles of Intramembrane Proteases. ACS Chemical Biology 2019, 14 (11) , 2372-2388. https://doi.org/10.1021/acschembio.9b00404
  2. Parul Goel, Thorsten Jumpertz, David C. Mikles, Anežka Tichá, Minh T. N. Nguyen, Steven Verhelst, Martin Hubalek, Darren C. Johnson, Daniel A. Bachovchin, Isabella Ogorek, Claus U. Pietrzik, Kvido Strisovsky, Boris Schmidt, and Sascha Weggen . Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases. Biochemistry 2017, 56 (51) , 6713-6725. https://doi.org/10.1021/acs.biochem.7b01066
  3. Minh T. N. Nguyen, Tim Van Kersavond, and Steven H. L. Verhelst . Chemical Tools for the Study of Intramembrane Proteases. ACS Chemical Biology 2015, 10 (11) , 2423-2434. https://doi.org/10.1021/acschembio.5b00693
  4. Eliane V. Wolf, Annett Zeissler, and Steven H. L. Verhelst . Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based Protein Profiling Reveals Inhibitor Selectivity and Rhomboid Autoprocessing. ACS Chemical Biology 2015, 10 (10) , 2325-2333. https://doi.org/10.1021/acschembio.5b00514
  5. Benito Alcaide, Pedro Almendros, Sara Cembellín, and Teresa Martínez del Campo . Gold as Catalyst for the Hydroarylation and Domino Hydroarylation/N1–C4 Cleavage of β-Lactam-Tethered Allenyl Indoles. The Journal of Organic Chemistry 2015, 80 (9) , 4650-4660. https://doi.org/10.1021/acs.joc.5b00535
  6. Neta Uritsky, Michael Shokhen, and Amnon Albeck . The Catalytic Machinery of Rhomboid Proteases: Combined MD and QM Simulations. Journal of Chemical Theory and Computation 2012, 8 (11) , 4663-4671. https://doi.org/10.1021/ct3003767
  7. Allison R. Sherratt, David R. Blais, Houman Ghasriani, John Paul Pezacki, and Natalie K. Goto . Activity-Based Protein Profiling of the Escherichia coli GlpG Rhomboid Protein Delineates the Catalytic Core. Biochemistry 2012, 51 (39) , 7794-7803. https://doi.org/10.1021/bi301087c
  8. Jian Yang, Luís A. R. Carvalho, Shanping Ji, Suyuan Chen, Rui Moreira, Steven H. L. Verhelst. 4‐Oxo‐β‐Lactams as Novel Inhibitors for Rhomboid Proteases. ChemBioChem 2023, 24 (21) https://doi.org/10.1002/cbic.202300418
  9. Samantha L. Hodges, Alexandra A. Bouza, Lori L. Isom, . Therapeutic Potential of Targeting Regulated Intramembrane Proteolysis Mechanisms of Voltage-Gated Ion Channel Subunits and Cell Adhesion Molecules. Pharmacological Reviews 2022, 74 (4) , 1030-1050. https://doi.org/10.1124/pharmrev.121.000340
  10. Zhe Zhuang, Shuang Liu, Jin‐Tang Cheng, Kap‐Sun Yeung, Jennifer X. Qiao, Nicholas A. Meanwell, Jin‐Quan Yu. Ligand‐Enabled β‐C(sp 3 )−H Lactamization of Tosyl‐Protected Aliphatic Amides Using a Practical Oxidant. Angewandte Chemie 2022, 134 (34) https://doi.org/10.1002/ange.202207354
  11. Zhe Zhuang, Shuang Liu, Jin‐Tang Cheng, Kap‐Sun Yeung, Jennifer X. Qiao, Nicholas A. Meanwell, Jin‐Quan Yu. Ligand‐Enabled β‐C(sp 3 )−H Lactamization of Tosyl‐Protected Aliphatic Amides Using a Practical Oxidant. Angewandte Chemie International Edition 2022, 61 (34) https://doi.org/10.1002/anie.202207354
  12. William H. Parsons, Nicholas T. Rutland, Jennifer A. Crainic, Joaquin M. Cardozo, Alyssa S. Chow, Charlotte L. Andrews, Brendan K. Sheehan. Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL. Bioorganic & Medicinal Chemistry Letters 2021, 49 , 128290. https://doi.org/10.1016/j.bmcl.2021.128290
  13. Tim Van Kersavond, Raphael Konopatzki, Merel A. T. van der Plassche, Jian Yang, Steven H. L. Verhelst. Rapid synthesis of internal peptidyl α-ketoamides by on resin oxidation for the construction of rhomboid protease inhibitors. RSC Advances 2021, 11 (7) , 4196-4199. https://doi.org/10.1039/D0RA10614C
  14. Franco Faucher, John M. Bennett, Matthew Bogyo, Scott Lovell. Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases. Cell Chemical Biology 2020, 27 (8) , 937-952. https://doi.org/10.1016/j.chembiol.2020.07.008
  15. Marta Barniol-Xicota, Steven H.L. Verhelst. Isolation of intramembrane proteases in membrane-like environments. Biochimica et Biophysica Acta (BBA) - Biomembranes 2020, 1862 (4) , 183193. https://doi.org/10.1016/j.bbamem.2020.183193
  16. Anežka Tichá, Blanka Collis, Kvido Strisovsky. The Rhomboid Superfamily: Structural Mechanisms and Chemical Biology Opportunities. Trends in Biochemical Sciences 2018, 43 (9) , 726-739. https://doi.org/10.1016/j.tibs.2018.06.009
  17. Sandra Paschkowsky, Felix Oestereich, Lisa Marie Munter. Embedded in the Membrane: How Lipids Confer Activity and Specificity to Intramembrane Proteases. The Journal of Membrane Biology 2018, 251 (3) , 369-378. https://doi.org/10.1007/s00232-017-0008-5
  18. Jian Yang, Marta Barniol-Xicota, Minh T.N. Nguyen, Anezka Ticha, Kvido Strisovsky, Steven H.L. Verhelst. Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases. Bioorganic & Medicinal Chemistry Letters 2018, 28 (8) , 1423-1427. https://doi.org/10.1016/j.bmcl.2017.12.056
  19. Parul Goel, Thorsten Jumpertz, Anežka Tichá, Isabella Ogorek, David C. Mikles, Martin Hubalek, Claus U. Pietrzik, Kvido Strisovsky, Boris Schmidt, Sascha Weggen. Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors. Bioorganic & Medicinal Chemistry Letters 2018, 28 (8) , 1417-1422. https://doi.org/10.1016/j.bmcl.2018.02.017
  20. Anežka Tichá, Stancho Stanchev, Kutti R. Vinothkumar, David C. Mikles, Petr Pachl, Jakub Began, Jan Škerle, Kateřina Švehlová, Minh T.N. Nguyen, Steven H.L. Verhelst, Darren C. Johnson, Daniel A. Bachovchin, Martin Lepšík, Pavel Majer, Kvido Strisovsky. General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases. Cell Chemical Biology 2017, 24 (12) , 1523-1536.e4. https://doi.org/10.1016/j.chembiol.2017.09.007
  21. Stefan Düsterhöft, Ulrike Künzel, Matthew Freeman. Rhomboid proteases in human disease: Mechanisms and future prospects. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2017, 1864 (11) , 2200-2209. https://doi.org/10.1016/j.bbamcr.2017.04.016
  22. Steven H. L. Verhelst. Intramembrane proteases as drug targets. The FEBS Journal 2017, 284 (10) , 1489-1502. https://doi.org/10.1111/febs.13979
  23. Anežka Tichá, Stancho Stanchev, Jan Škerle, Jakub Began, Marek Ingr, Kateřina Švehlová, Lucie Polovinkin, Martin Růžička, Lucie Bednárová, Romana Hadravová, Edita Poláchová, Petra Rampírová, Jana Březinová, Václav Kašička, Pavel Majer, Kvido Strisovsky. Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases. Journal of Biological Chemistry 2017, 292 (7) , 2703-2713. https://doi.org/10.1074/jbc.M116.762849
  24. K. Strisovsky. Mechanism and Inhibition of Rhomboid Proteases. 2017, 279-293. https://doi.org/10.1016/bs.mie.2016.10.014
  25. E. Arutyunova, K. Strisovsky, M.J. Lemieux. Activity Assays for Rhomboid Proteases. 2017, 395-437. https://doi.org/10.1016/bs.mie.2016.11.002
  26. Kvido Strisovsky. Rhomboid protease inhibitors: Emerging tools and future therapeutics. Seminars in Cell & Developmental Biology 2016, 60 , 52-62. https://doi.org/10.1016/j.semcdb.2016.08.021
  27. Pinchas Zer Aviv, Moran Shubely, Yoni Moskovits, Olga Viskind, Amnon Albeck, Didier Vertommen, Sharon Ruthstein, Michael Shokhen, Arie Gruzman. A New Oxopiperazin‐Based Peptidomimetic Molecule Inhibits Prostatic Acid Phosphatase Secretion and Induces Prostate Cancer Cell Apoptosis. ChemistrySelect 2016, 1 (15) , 4658-4667. https://doi.org/10.1002/slct.201600987
  28. Eliane V. Wolf, Steven H.L. Verhelst. Inhibitors of rhomboid proteases. Biochimie 2016, 122 , 38-47. https://doi.org/10.1016/j.biochi.2015.07.007
  29. Sangwoo Cho, Seth W. Dickey, Siniša Urban. Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis. Molecular Cell 2016, 61 (3) , 329-340. https://doi.org/10.1016/j.molcel.2015.12.022
  30. Neta Uritsky, Michael Shokhen, Amnon Albeck. Stepwise Versus Concerted Mechanisms in General‐Base Catalysis by Serine Proteases. Angewandte Chemie 2016, 128 (5) , 1712-1716. https://doi.org/10.1002/ange.201507772
  31. Neta Uritsky, Michael Shokhen, Amnon Albeck. Stepwise Versus Concerted Mechanisms in General‐Base Catalysis by Serine Proteases. Angewandte Chemie International Edition 2016, 55 (5) , 1680-1684. https://doi.org/10.1002/anie.201507772
  32. Eliane V. Wolf, Martin Seybold, Romana Hadravová, Kvido Strisovsky, Steven H. L. Verhelst. Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes. ChemBioChem 2015, 16 (11) , 1616-1621. https://doi.org/10.1002/cbic.201500213
  33. Xinfang Xu, Yongming Deng, David N. Yim, Peter Y. Zavalij, Michael P. Doyle. Enantioselective cis-β-lactam synthesis by intramolecular C–H functionalization from enoldiazoacetamides and derivative donor–acceptor cyclopropenes. Chemical Science 2015, 6 (4) , 2196-2201. https://doi.org/10.1039/C4SC03991B
  34. Sebastian Zoll, Stancho Stanchev, Jakub Began, Jan Škerle, Martin Lepšík, Lucie Peclinovská, Pavel Majer, Kvido Strisovsky. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures. The EMBO Journal 2014, 33 (20) , 2408-2421. https://doi.org/10.15252/embj.201489367
  35. Matthew Freeman. The Rhomboid-Like Superfamily: Molecular Mechanisms and Biological Roles. Annual Review of Cell and Developmental Biology 2014, 30 (1) , 235-254. https://doi.org/10.1146/annurev-cellbio-100913-012944
  36. Senthil K Radhakrishnan, Willem den Besten, Raymond J Deshaies. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition. eLife 2014, 3 https://doi.org/10.7554/eLife.01856
  37. Nina Bergbold, Marius K. Lemberg. Emerging role of rhomboid family proteins in mammalian biology and disease. Biochimica et Biophysica Acta (BBA) - Biomembranes 2013, 1828 (12) , 2840-2848. https://doi.org/10.1016/j.bbamem.2013.03.025
  38. Cory L. Brooks, M. Joanne Lemieux. Untangling structure–function relationships in the rhomboid family of intramembrane proteases. Biochimica et Biophysica Acta (BBA) - Biomembranes 2013, 1828 (12) , 2862-2872. https://doi.org/10.1016/j.bbamem.2013.05.003
  39. Seth W. Dickey, Rosanna P. Baker, Sangwoo Cho, Siniša Urban. Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity. Cell 2013, 155 (6) , 1270-1281. https://doi.org/10.1016/j.cell.2013.10.053
  40. Beatriz Baeza, Luis Casarrubios, Miguel A. Sierra. Towards a General Synthesis of 3‐Metal‐Substituted β‐Lactams. Chemistry – A European Journal 2013, 19 (35) , 11536-11540. https://doi.org/10.1002/chem.201301114
  41. Eliane V. Wolf, Annett Zeißler, Oliver Vosyka, Evelyn Zeiler, Stephan Sieber, Steven H. L. Verhelst, . A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization. PLoS ONE 2013, 8 (8) , e72307. https://doi.org/10.1371/journal.pone.0072307
  42. Kutti R. Vinothkumar, Olivier A. Pierrat, Jonathan M. Large, Matthew Freeman. Structure of Rhomboid Protease in Complex with β-Lactam Inhibitors Defines the S2′ Cavity. Structure 2013, 21 (6) , 1051-1058. https://doi.org/10.1016/j.str.2013.03.013
  43. Ya Ha, Yoshinori Akiyama, Yi Xue. Structure and Mechanism of Rhomboid Protease. Journal of Biological Chemistry 2013, 288 (22) , 15430-15436. https://doi.org/10.1074/jbc.R112.422378
  44. Kvido Strisovsky. Structural and mechanistic principles of intramembrane proteolysis – lessons from rhomboids. The FEBS Journal 2013, 280 (7) , 1579-1603. https://doi.org/10.1111/febs.12199
  45. Oliver Vosyka, Kutti R. Vinothkumar, Eliane V. Wolf, Arwin J. Brouwer, Rob M. J. Liskamp, Steven H. L. Verhelst. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proceedings of the National Academy of Sciences 2013, 110 (7) , 2472-2477. https://doi.org/10.1073/pnas.1215076110
  46. Kvido Strisovsky. Drosophila Rhomboid-1. 2013, 3563-3567. https://doi.org/10.1016/B978-0-12-382219-2.00790-0
  47. Sinisa Urban. Mammalian Rhomboid Proteases. 2013, 3573-3581. https://doi.org/10.1016/B978-0-12-382219-2.00792-4
  48. Sinisa Urban. Bacterial and Protozoan Rhomboid Proteases. 2013, 3581-3593. https://doi.org/10.1016/B978-0-12-382219-2.00793-6
  49. Refaat B. Hamed, J. Ruben Gomez-Castellanos, Luc Henry, Christian Ducho, Michael A. McDonough, Christopher J. Schofield. The enzymes of β-lactam biosynthesis. Nat. Prod. Rep. 2013, 30 (1) , 21-107. https://doi.org/10.1039/C2NP20065A
  50. Hong‐Jun Liao, Graham Carpenter. Regulated Intramembrane Cleavage of the EGF Receptor. Traffic 2012, 13 (8) , 1106-1112. https://doi.org/10.1111/j.1600-0854.2012.01371.x
  51. Subbiah Nagarajan, Pandian Arjun, Nanjian Raaman, Anup Shah, M. Elizabeth Sobhia, Thangamuthu Mohan Das. Stereoselective synthesis of sugar-based β-lactam derivatives: docking studies and its biological evaluation. Tetrahedron 2012, 68 (14) , 3037-3045. https://doi.org/10.1016/j.tet.2012.02.017
  52. Joana M. Santos, Arnault Graindorge, Dominique Soldati-Favre. New insights into parasite rhomboid proteases. Molecular and Biochemical Parasitology 2012, 182 (1-2) , 27-36. https://doi.org/10.1016/j.molbiopara.2011.11.010
  53. Sinisa Urban, Seth W Dickey. The rhomboid protease family: a decade of progress on function and mechanism. Genome Biology 2011, 12 (10) , 231. https://doi.org/10.1186/gb-2011-12-10-231
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  • Abstract

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    Figure 1

    Figure 1. Fluorescent assay design. (A) Schematic representation of rhomboid enzyme AarA cleaving a natural rhomboid substrate or a substrate-based peptide. (B) Comparison of AarA activity on three peptides derived from known rhomboid substrates. Rhomboid cleavage sites (asterisks) were determined by HPLC using peptide standards or MALDI-TOF mass spectrometry. The Gurken-derived peptide was modified with QSY21 dark quencher and Chromis-645 fluorophore to yield fluorogenic peptide KSp21. (C) Time course of cleavage of 1 μM KSp21 by 0.4 μM AarA (wt or S150A) or 0.2 μM chymotrypsin, at 25 °C. (D) Fluorescent activity assay adapted to high-throughput screen (HTS) format. Z′ factor = 0.77 (signal/background = 10, coefficients of variation = 6%) (see Methods). High control = AarA + DMSO; low control = no enzyme + DMSO.

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    Figure 2

    Figure 2. Screening campaign summary. (A) Screen flowchart. (B) Fluorescent signal of 162 initial hits and 190 high controls (DMSO + AarA), both denoted by blue diamonds, and 32 low controls (DMSO, no enzyme), denoted by magenta squares. DMSO high controls or inactive compounds cluster around the 100,000 activity units mark, inhibitors are represented by points below 100,000 activity units, and activators are above. (C) Plot of correlation between initial hits from high-throughput screen and secondary screen; 7 hits (6 false inhibitors, 1 false activator) were not confirmed as they showed ∼100% activity in the second screen (dotted line). (D) Chemical structure and apparent IC50 values of hits selected for chemistry optimization: series 1, bis(trifluoromethyl)-amide (bis-CF3), based on compound F1; and series 2, monocyclic β-lactam, based on compound L1.

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    Figure 3

    Figure 3. In vitro effects of selected bis-CF3 analogues with modifications of the aniline moiety. (A) Results of fluorescent assay with 600 nM KSp21 peptide and either 100 nM AarA or 37 nM chymotrypsin. (B) Gel-based assay with 320 nM GlpG and 3 μM Gurken chimeric substrate and increasing concentration (μM) of inhibitor F72. Enzyme (E) substrate (S), and product (P) bands are labeled on the right-hand side of the gel. High molecular weight protein aggregates appear (vertical line) as inhibitor concentration increases.

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    Figure 4

    Figure 4. In vitro activity of β-lactams: high potency on GlpG and structure−activity relationship (SAR) against AarA and GlpG. (A) Typical gel showing β-lactam (L35) inhibiting the cleavage of Gurken chimeric substrate (3 μM) by GlpG (320 nM). Enzyme (E), substrate (S), and product (P) bands are indicated. (B) Band intensity was quantified using Image J, and substrate conversion was expressed as % of DMSO control. The apparent IC50 values in brackets were determined by fitting the data to a 4-parameter logistic equation. (C) Diagram summarizing SAR results. Analogues based on the L1 skeleton were obtained by structural modifications of the encircled substituents.

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    Figure 5

    Figure 5. In vivo activity of β-lactams. (A) Inhibition of endogenous GlpG by L29 in E. coli NR698 carrying imp4213 allele and a plasmid expressing the LacYTM2-based rhomboid substrate. Substrate (S) and product (P) were detected by Western blot with anti-Myc antibody, as described in Methods. The E. coli NR698 (+) or its ΔglpG variant (Δ) were grown in the presence of inhibitors at a range of concentrations and 2% DMSO. (B) The in vivo EC50 values (μM, in brackets) of selected β-lactams were obtained from band quantification performed as in Figure 4. Graph shows average data from three experiments, with standard errors up to 20%. (C) Mouse RHBDL2 was inhibited in vivo by L29, but not L35 and L41 (tested at 10 and 30 μM). COS7 cells were transfected with mRHBDL2 and Gurken substrate. Western blot was probed with anti-FLAG (Gurken) or anti-HA (mRHBDL2) antibody. S and P represent the substrate and product band, respectively.

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    Figure 6

    Figure 6. Mechanism of action of selected β-lactams. (A) Reversibility of L42 inhibition of AarA was tested using the 100-fold dilution technique described in Results. Enzyme was incubated with inhibitor or DMSO for 30 min pre- and postdilution (to 71 nM final concentration); residual activity was then assayed in the presence of 600 nM KSp21 substrate. (B) The L42 compound displays time-dependent inhibition of AarA. AarA (100 nM) was preincubated with 1−100 μM L42 for varying times (8−188 min), after which activity was measured using 600 nM KSp21 fluorogenic substrate. Activities were expressed relative to the solvent (DMSO) control. The inset shows a plot of 1/kobsvs 1/[inhibitor concentration] (31). (C) The inhibitory monocyclic β-lactam L29 forms a 1:1 covalent complex with GlpG through its catalytic serine. GlpG or its S201A mutant (30 μM) were incubated with the indicated compound (100 μM) for 30 min at 25 °C, purified through a C4 ZipTip, and analyzed by MALDI-TOF mass spectrometry.

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  • References

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    This article references 31 other publications.

    1. 1
      Rawson, R. B., Zelenski, N. G., Nijhawan, D., Ye, J., Sakai, J., Hasan, M. T., Chang, T. Y., Brown, M. S., and Goldstein, J. L. (1997) Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs Mol. Cell 1, 47– 57
      1
      Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs
      Rawson, Robert B.; Zelenski, Nikolai G.; Nijhawan, Deepak; Ye, Jin; Sakai, Juro; Hasan, Mazahir T.; Chang, T. Y.; Brown, Michael S.; Goldstein, Joseph L.
      Molecular Cell (1997), 1 (1), 47-57CODEN: MOCEFL ISSN:. (Cell Press)
      We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element-binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metab. The active NH2-terminal domains of SREBPS are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HEXXH sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.
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    2. 2
      De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., Von Figura, K., and Van Leuven, F. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein Nature 391, 387– 390
      2
      Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein
      De Strooper, Bart; Saftig, Paul; Craessaerts, Katleen; Vanderstichele, Hugo; Guhde, Gundula; Annaert, Wim; Von Figura, Kurt; Van Leuven, Fred
      Nature (London) (1998), 391 (6665), 387-390CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
      Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the prodn. of the amyloidogenic peptide amyloid-β (1-42) by proteolytic processing of the amyloid precursor protein (APP). Here the authors investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by α- and β-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by γ-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the prodn. of amyloid peptide. Pulse-chase expts. indicated that PS1 deficiency specifically decreased the turnover of the membrane-assocd. fragments of APP. As in the regulation of cholesterol metab. by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. The results indicate that mutations in PS1 that manifest clin. cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.
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    3. 3
      Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K., and Martoglio, B. (2002) Identification of signal peptide peptidase, a presenilin-type aspartic protease Science 296, 2215– 2218
      3
      Identification of signal peptide peptidase, a presenilin-type aspartic protease
      Weihofen, Andreas; Binns, Kathleen; Lemberg, Marius K.; Ashman, Keith; Martoglio, Bruno
      Science (Washington, DC, United States) (2002), 296 (5576), 2215-2218CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biol. important peptides.
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    4. 4
      Urban, S., Lee, J. R., and Freeman, M. (2001) Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases Cell 107, 173– 182
      4
      Drosophila Rhomboid-1 defines a family of putative intramembrane serine proteases
      Urban, Sinisa; Lee, Jeffrey R.; Freeman, Matthew
      Cell (Cambridge, MA, United States) (2001), 107 (2), 173-182CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFα-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but the authors' anal. suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to the authors' knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and the results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.
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    5. 5
      Urban, S., Lee, J. R., and Freeman, M. (2002) A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands EMBO J. 21, 4277– 4286
      There is no corresponding record for this reference.
    6. 6
      Lee, J. R., Urban, S., Garvey, C. F., and Freeman, M. (2001) Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila Cell 107, 161– 171
      6
      Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila
      Lee, Jeffrey R.; Urban, Sinisa; Garvey, Clare F.; Freeman, Matthew
      Cell (Cambridge, MA, United States) (2001), 107 (2), 161-171CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The membrane proteins Star and Rhomboid-1 have been genetically defined as the primary regulators of EGF receptor activation in Drosophila, but their mol. mechanisms have been elusive. Both Star and Rhomboid-1 have been assumed to work at the cell surface to control ligand activation. Here, the authors demonstrate that they control receptor signaling by regulating intracellular trafficking and proteolysis of the ligand Spitz. Star is present throughout the secretory pathway and is required to export Spitz from the endoplasmic reticulum to the Golgi app. Rhomboid-1 is localized in the Golgi, where it promotes the cleavage of Spitz. This defines a novel growth factor release mechanism that is distinct from metalloprotease-dependent shedding from the cell surface.
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    7. 7
      Herlan, M., Vogel, F., Bornhovd, C., Neupert, W., and Reichert, A. S. (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA J. Biol. Chem. 278, 27781– 27788
      7
      Processing of Mgm1 by the Rhomboid-type Protease Pcp1 Is Required for Maintenance of Mitochondrial Morphology and of Mitochondrial DNA
      Herlan, Mark; Vogel, Frank; Bornhoevd, Carsten; Neupert, Walter; Reichert, Andreas S.
      Journal of Biological Chemistry (2003), 278 (30), 27781-27788CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, assocd. with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341-352; Wong, E. D., Wagner, J. A., Scott, S. V., Okreglak, V., Holewinske, T. J., Cassidy-Stone, A., and Nunnari, J. (2003) J. Cell Biol. 160, 303-311; Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in press). Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, we have characterized these isoforms. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphol. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Δpcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Δmgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphol. and maintenance of mitochondrial DNA.
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    8. 8
      McQuibban, G. A., Saurya, S., and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease Nature 423, 537– 541
      8
      Mitochondrial membrane remodelling regulated by a conserved rhomboid protease
      McQuibban, G. Angus; Saurya, Saroj; Freeman, Matthew
      Nature (London, United Kingdom) (2003), 423 (6939), 537-541CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signaling in Drosophila. Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have addnl. roles. Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p. RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria. We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion. Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Δ mitochondrial phenotype. Our data indicate that mitochondrial membrane remodeling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signaling. In addn., mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homolog, PARL, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases.
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    9. 9
      McQuibban, G. A., Lee, J. R., Zheng, L., Juusola, M., and Freeman, M. (2006) Normal mitochondrial dynamics requires rhomboid-7 and affects Drosophila lifespan and neuronal function Curr. Biol. 16, 982– 989
      9
      Normal Mitochondrial Dynamics Requires Rhomboid-7 and Affects Drosophila Lifespan and Neuronal Function
      McQuibban, G. Angus; Lee, Jeffrey R.; Zheng, Lei; Juusola, Mikko; Freeman, Matthew
      Current Biology (2006), 16 (10), 982-989CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      In addn. to being energy generators, mitochondria control many cellular processes including apoptosis. They are dynamic organelles, and the machinery of membrane fusion and fission is emerging as a key regulator of mitochondrial biol. The authors have recently identified a novel and conserved mitochondrial rhomboid intramembrane protease that controls membrane fusion in Saccharomyces cerevisiae by processing the dynamin-like GTPase, Mgm1, thereby releasing it from the membrane. The genetics of mitochondrial membrane dynamics has until now focused primarily on yeast. In Drosophila, the mitochondrial rhomboid (Rhomboid-7) is required for mitochondrial fusion during fly spermatogenesis and muscle maturation, both tissues with unusual mitochondrial dynamics. The authors also find that mutations in Drosophila optic atrophy 1-like (Opa1-like), the ortholog of yeast mgm1, display similar phenotypes, suggesting a shared role for Rhomboid-7 and Opa1-like, as with their yeast orthologs. Loss of human OPA1 leads to dominant optic atrophy, a mitochondrial disease leading to childhood onset blindness. Rhomboid-7 mutant flies have severe neurol. defects, evidenced by compromised signaling across the first visual synapse, as well as light-induced neurodegeneration of photoreceptors that resembles the human disease. Rhomboid-7 mutant flies also have a greatly reduced lifespan.
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    10. 10
      O’Donnell, R. A., Hackett, F., Howell, S. A., Treeck, M., Struck, N., Krnajski, Z., Withers-Martinez, C., Gilberger, T. W., and Blackman, M. J. (2006) Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite J. Cell Biol. 174, 1023– 1033
      There is no corresponding record for this reference.
    11. 11
      Baker, R. P., Wijetilaka, R., and Urban, S. (2006) Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria PLoS Pathog. 2, e113
      There is no corresponding record for this reference.
    12. 12
      Srinivasan, P., Coppens, I., and Jacobs-Lorena, M. (2009) Distinct roles of Plasmodium rhomboid 1 in parasite development and malaria pathogenesis PLoS Pathog. 5, e1000262
      There is no corresponding record for this reference.
    13. 13
      Stevenson, L. G., Strisovsky, K., Clemmer, K. M., Bhatt, S., Freeman, M., and Rather, P. N. (2007) Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase Proc. Natl. Acad. Sci. U.S.A. 104, 1003– 1008
      There is no corresponding record for this reference.
    14. 14
      Wang, Y., Zhang, Y., and Ha, Y. (2006) Crystal structure of a rhomboid family intramembrane protease Nature 444, 179– 180
      14
      Crystal structure of a rhomboid family intramembrane protease
      Wang, Yongcheng; Zhang, Yingjiu; Ha, Ya
      Nature (London, United Kingdom) (2006), 444 (7116), 179-180CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and γ-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 Å resoln. crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water mols. are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.
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    15. 15
      Urban, S. and Wolfe, M. S. (2005) Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity Proc. Natl. Acad. Sci. U.S.A. 102, 1883– 1888
      There is no corresponding record for this reference.
    16. 16
      Baxt, L. A., Rastew, E., Bracha, R., Mirelman, D., and Singh, U. (2010) Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis Eukaryotic Cell 9, 1283– 1293
      There is no corresponding record for this reference.
    17. 17
      Freeman, M. (2008) Rhomboid proteases and their biological functions Annu. Rev. Genet. 42, 191– 210
      There is no corresponding record for this reference.
    18. 18
      Urban, S. (2009) Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms Nat. Rev. Microbiol. 7, 411– 423
      18
      Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms
      Urban, Sinisa
      Nature Reviews Microbiology (2009), 7 (6), 411-423CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Proteolysis in cellular membranes to liberate effector domains from their transmembrane anchors is a well-studied regulatory mechanism in animal biol. and disease. By contrast, the function of intramembrane proteases in unicellular organisms has received little attention. Recent progress has now established that intramembrane proteases execute pivotal roles in a range of pathogens, from regulating Mycobacterium tuberculosis envelope compn., cholera toxin prodn., bacterial adherence and conjugation, to malaria parasite invasion, fungal virulence, immune evasion by parasitic amoebae and hepatitis C virus assembly. These advances raise the exciting possibility that intramembrane proteases may serve as targets for combating a wide range of infectious diseases. This Review focuses on summarizing the advances, evaluating the limitations and highlighting the promise of this newly emerging field.
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    19. 19
      Strisovsky, K., Sharpe, H. J., and Freeman, M. (2009) Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates Mol. Cell 36, 1048– 1059
      There is no corresponding record for this reference.
    20. 20
      Lemberg, M. K., Menendez, J., Misik, A., Garcia, M., Koth, C. M., and Freeman, M. (2005) Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases EMBO J. 24, 464– 472
      There is no corresponding record for this reference.
    21. 21
      Maegawa, S., Ito, K., and Akiyama, Y. (2005) Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane Biochemistry 44, 13543– 13552
      There is no corresponding record for this reference.
    22. 22
      Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays J. Biomol. Screening 4, 67– 73
      22
      A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
      Zhang; Chung; Oldenburg
      Journal of biomolecular screening (1999), 4 (2), 67-73 ISSN:.
      The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
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    23. 23
      Powers, J. C., Asgian, J. L., Ekici, O. D., and James, K. E. (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases Chem. Rev. 102, 4639– 4750
      23
      Irreversible inhibitors of serine, cysteine, and threonine proteases
      Powers, James C.; Asgian, Juliana L.; Ekici, Oezlem Dogan; James, Karen Ellis
      Chemical Reviews (Washington, DC, United States) (2002), 102 (12), 4639-4750CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Topics discussed include alkylating, acylating, phosphonylating, and sulfonylating agents.
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    24. 24
      Ruiz, N., Falcone, B., Kahne, D., and Silhavy, T. J. (2005) Chemical conditionality: a genetic strategy to probe organelle assembly Cell 121, 307– 317
      24
      Chemical conditionality: A genetic strategy to probe organelle assembly
      Ruiz, Natividad; Falcone, Brian; Kahne, Daniel; Silhavy, Thomas J.
      Cell (Cambridge, MA, United States) (2005), 121 (2), 307-317CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The assembly of the Escherichia coli outer membrane (OM) is poorly understood. Although insight into fundamental cellular processes is often obtained from studying mutants, OM-defective mutants have not been very informative because they generally have nonspecific permeability defects. Here, the authors show that toxic small mols. can be used in selections employing strains with permeability defects to create particular chem. conditions that demand specific suppressor mutations. Suppressor phenotypes are correlated with the phys. properties of the small mols., but the mutations are not in their target genes. Instead, mutations allow survival by partially restoring membrane impermeability. Using "chem. conditionality," the authors identified mutations in yfgL and showed that YfgL is part of a multiprotein complex involved in the assembly of OM β barrel proteins. They posit that panels of toxic small mols. will be useful for generating chem. conditionalities that enable identification of genes required for organelle assembly in other organisms.
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    25. 25
      Neubig, R. R., Spedding, M., Kenakin, T., and Christopoulos, A. (2003) International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology Pharmacol. Rev. 55, 597– 606
      25
      International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology
      Neubig, Richard R.; Spedding, Michael; Kenakin, Terry; Christopoulos, Arthur
      Pharmacological Reviews (2003), 55 (4), 597-606CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
      A review. The recommendations that follow have been updated from the proposals of a Tech. Subcommittee set up by the International Union of Pharmacol. Committee on Receptor Nomenclature and Drug Classification. International Union of Pharmacol. Committee on Receptor Nomenclature and Drug Classification. IX. Recommendations on terms and symbols in quant. pharmacol.
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    26. 26
      Urban, S., Schlieper, D., and Freeman, M. (2002) Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids Curr. Biol. 12, 1507– 1512
      26
      Conservation of Intramembrane Proteolytic Activity and Substrate Specificity in Prokaryotic and Eukaryotic Rhomboids
      Urban, Sinisa; Schlieper, Daniel; Freeman, Matthew
      Current Biology (2002), 12 (17), 1507-1512CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands. Although nothing is known about the function of the ∼100 currently known rhomboid genes conserved throughout evolution, a recent anal. suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the prodn. of a quorum-sensing factor. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans. However, the function of prokaryotic Rhomboids is unknown. We have examd. the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-pos. and four Gram-neg. species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor α (TGFα) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homol., the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.
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    27. 27
      Harper, J. W., Hemmi, K., and Powers, J. C. (1985) Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor Biochemistry 24, 1831– 1841
      27
      Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor
      Harper, J. Wade; Hemmi, Keiji; Powers, James C.
      Biochemistry (1985), 24 (8), 1831-41CODEN: BICHAW; ISSN:0006-2960.
      The mechanism-based inactivation of a no. of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated, inactivated all of the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or β-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1, where [I] is the concn. of inhibitor); the inhibited enzyme was quite stable to reactivation (rate const. of deacylation = 2 × 10-5 s-1), whereas enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation decreased dramatically in the presence of reversible inhibitors or substrates; UV spectral measurements indicated that the isocoumarin ring structure was lost upon inactivation. Chymotrypsin Aγ was totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and ∼1 equiv of H+ was released on inactivation. Thus, these compds. react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the 1st mechanism-based inhibitors reported for many of the enzymes tested; 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.
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    28. 28
      Harper, J. W. and Powers, J. C. (1985) Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors Biochemistry 24, 7200– 7213
      There is no corresponding record for this reference.
    29. 29
      Vinothkumar, K. R., Strisovsky, K., Andreeva, A., Christova, Y., Verhelst, S., and Freeman, M. (2010) The structural basis for catalysis and substrate specificity of a rhomboid protease EMBO J. 29, 3797– 3809
      There is no corresponding record for this reference.
    30. 30
      Miroux, B. and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels J. Mol. Biol. 260, 289– 298
      30
      Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels
      Miroux, Bruno; Walker, John E.
      Journal of Molecular Biology (1996), 260 (3), 289-298CODEN: JMOBAK; ISSN:0022-2836. (Academic)
      We have investigated the over-prodn. of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expressing of the target membrane protein was induced, most of the BL21 (DE3) host cells died. Similar effects were also obsd. with expression vectors for ten globular proteins. Therefore, protein over-prodn. in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high satn. cell d., and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior t BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells contg. the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H+ symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit B was lower in C41(DE3) and C43(DE3), resp., than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts to overcome other toxic effects assocd. with over-expression.
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    31. 31
      Kitz, R. and Wilson, I. B. (1962) Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase J. Biol. Chem. 237, 3245– 3249
      31
      Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase
      Kitz, R.; Wilson, Irwin B.
      Journal of Biological Chemistry (1962), 237 (), 3245-49CODEN: JBCHA3; ISSN:0021-9258.
      Nine esters (anhydrides) of methanesulfonic acid, analogs to well known carbamate (e.g. neostigmine) and phosphate (e.g. paraoxon) inhibitors, were examd. as possible irreversible inhibitors of acetylcholinesterase. Five were active. Of these, 4 formed initial reversible complexes. The progressive development of irreversible inhibition was slowed by reversible inhibitors, indicating that the active site is involved. The inhibited enzyme is not reactivated by water or by hydroxylamine, hydrazine, acetate, or pyridine-2-aldoxime methiodide. It is reactivated by pyridine-3-aldoxime methiodide. On the basis of the evidence presented, it may be concluded safely that the active methanesulfonates form methanesulfonyl enzyme derivs. and, therefore, belong to the class of acid-transferring or oxydiaphoric inhibitors.
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