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Impact of Autosomal Recessive Juvenile Parkinson’s Disease Mutations on the Structure and Interactions of the Parkin Ubiquitin-like Domain

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Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1
*Phone: 519-661-4021. Fax: 519-661-3175. E-mail: [email protected]. Web: www.biochem.uwo.ca/fac/shaw.
Cite this: Biochemistry 2011, 50, 13, 2603–2610
Publication Date (Web):February 24, 2011
https://doi.org/10.1021/bi200065g

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Abstract

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Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson’s disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.

  Funding Statement

This research was supported by research (FRN 14606) and maintenance (FRN 80148) grants from the Canadian Institutes of Health Research (G.S.S.), an award from the Canada Research Chairs Program (G.S.S.), and a Canadian Institutes of Health Research Doctoral Scholarship (S.S.S.).

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson’s disease. The distinguishing features of ARJP from the more prevalent idiopathic form of the disease include its earlier onset age, slower disease course, occasional dystonia, and long-lasting response to low doses of l-dopa. ARJP is thought to result from mutations in one or more genes, including the gene that encodes the E3 ubiquitin ligase parkin, (1) accounting for approximately 50% of all cases. Parkin, as with all E3 ligases, is an important enzyme in the ubiquitin-mediated proteolysis pathway. It has been shown to covalently link ubiquitin to potential substrates such as Eps15, (2, 3) CDCrel-1, (4) α-synuclein, (5) synphilin-1, (6) AIMP2, (7) and FBP-1. (8) However, confirmation of some of these substrates and their functional outcomes remains controversial. (9, 10) More recently, parkin has been shown to have an important role in mitochondrial function. Parkin ubiquitination of mitochondrial proteins serves to recruit and assemble the autophagy machinery to clear impaired mitochondria. (11) Interestingly, the activity of complex I of the mitochondrial respiratory chain is decreased in the substantia nigra and other tissues in Parkinson’s disease patients. Further, several complex I inhibitors reproduce key features of Parkinson’s disease such as loss of dopaminergic neurons and motor deficits. (12, 13)
Parkin is a multidomain E3 ubiquitin ligase consisting of 465 amino acids with five distinct domains: an N-terminal ubiquitin-like (Ubl) domain followed by a unique parkin-specific domain (UPD), a RING0 domain, and two C-terminal RING domains separated by an in-between RING (IBR) domain. (14) The N-terminal Ubl domain has been shown to be essential for the ligase activity of parkin since deletion or mutation of this domain results in impaired E3 ligase activity. (5, 15) The Ubl domain has also been shown to participate in a variety of protein interactions. For example, binding of the Ubl domain to the S5a proteasomal subunit has been suggested to position parkin and its bound substrates near the degradation machinery. (16) Alternatively, the parkin Ubl domain has been shown to interact with the endocytotic protein, Eps15 (2, 3, 16), in order to facilitate Eps15 ubiquitination used in the Akt signaling cascade.
Early onset PD has been linked to homozygous and compound heterozygous mutations in the parkin gene. However, heterozygous mutations in parkin also exist and are more controversial (17) including some that are likely polymorphisms. Approximately 95 nonstop, missense mutations are associated with ARJP (see http://www.molgen.ua.ac.be/PDmutDB and http://www.hgmd.org) and are located throughout the parkin gene resulting in single residue substitutions in the coded protein (for a summary see refs 18 and 19). This includes at least 20 substitutions at 16 different positions in the Ubl domain. Some disease state mutations localized to the Ubl domain have been shown to decrease the stability of the full-length parkin protein in vivo, although conflicting evidence has been presented for the effects of the mutations on E3 ligase activity. (15, 20, 21) To date, it has been difficult to distinguish whether the observations arise from substitutions causing structural or folding alterations in parkin or are a result of disrupted protein interactions with E2 ubiquitin conjugating enzymes, potential substrates, or the S5a proteasomal subunit. One reason for this uncertainty has been the inability to purify parkin, its constitutive domains, and especially proteins containing disease-causing mutations on a large scale suitable for biophysical studies. To date, only the parkin Ubl (16, 22) and IBR (23) domains have been purified and their three-dimensional structures determined. While some mutant forms of the parkin IBR domain have been isolated and characterized, (23) similar approaches have not been completed for the Ubl domain mutant containing proteins. To date, only two substitutions (K48A, R42P) have been structurally examined in the Ubl domain leaving the majority of the disease-related substitutions uncharacterized. (3, 24) Further, while a large number of mutations in the RING domains have been examined for interactions with different E2 enzymes (25) and ubiquitination, (20, 25-27) similar comprehensive studies have not been completed for the Ubl domain.
In this work we have examined proteins carrying ARJP disease-associated mutations at 13 of the possible 16 positions in the parkin Ubl domain in order to identify the impact of these substitutions on the Ubl domain structure, stability, and interactions with the S5a proteasome. Our work provides evidence that several ARJP disease state mutations cause drastic alterations in the Ubl domain structure and/or stability, while others disrupt binding to the S5a proteasomal subunit. These observations provide two different mechanisms that ARJP-associated missense mutations in the parkin gene may contribute to the dysfunction of the protein in the ubiquitination pathway.

Experimental Procedures

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Cloning

The DNA fragment encoding the Ubl domain (residues 1−77) of human parkin was cloned into the NdeI and BamHI sites of the pET44a vector (Novagen). The substitutions G12R, V15M, D18N, A31D, K32T, R33Q, Q34R, P37L, R42P, A46P, K48A, T55I, and V56E in the Ubl domain were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The DNA fragment encoding the UIMs (residues 196−309) of the human proteasomal S5a subunit was cloned into a pET21a vector (Novagen) modified to include a tobacco etch virus protease cleavage site before the six-residue histidine C-terminal tag. For expression with an in-frame N-terminal GB1 fusion, the DNA fragment encoding the Ubl domain (1−77) was cloned into the NheI and XhoI sites of the GEV1 vector, (28) a generous gift from Dr. M. Clore (NIDDK, Bethesda, MD). The V56E substituted version of the GB1-Ubl domain fusion was created using the QuikChange site-directed mutagenesis kit.

Protein Expression and Purification

The Ubl domain, GB1-Ubl domain, and all substituted proteins were overexpressed in the BL21(DE3) Codon PlusRIL Escherichia coli strain and purified as previously described using the same chromatographic methods. (24) The S5a196−309 fragment containing both UIM regions was expressed with a six-residue histidine tag and purified using a Ni-NTA FPLC affinity column (GE Healthcare) followed by size exclusion chromatography. Ubiquitin from Saccharomyces cerevisiae was expressed from a pET3a vector as previously described. (29) The integrity of all proteins was confirmed by electrospray ionization mass spectrometry (UWO Biological Mass Spectrometry Laboratory).

NMR Spectroscopy

All NMR experiments were performed on a 600 MHz Varian Inova spectrometer equipped with either a 13C-enhanced triple resonance cold probe with z-gradients or an xyz gradient, triple resonance probe (Biomolecular NMR Facility, UWO). 1H chemical shifts were referenced directly to internal DSS at 0 ppm. Sensitivity-enhanced 1H−15N HSQC spectra (30) were recorded at 25 °C on 15N-labeled proteins in 10 mM KH2PO4, 1 mM EDTA, 1 mM DTT, 30 μM DSS, and 10% D2O at pH 7.0. All spectra were processed with NMRPipe (31) software using a 60° shifted, cosine-squared function in both 1H and 15N dimensions and analyzed using NMRView. (32)

Protein Unfolding Experiments

Unfolding experiments were monitored by circular dichroism (CD) spectropolarimetry using a Jasco J-810 instrument (Biomolecular Interactions and Conformations Facility, UWO). Spectra (190−250 nm) were initially measured at 5 °C using ten averaged scans for protein samples ranging in concentration from 20 to 80 μM in a 1 mm cell. Following this, the ellipticity at 222 nm was measured as a function of temperature between 5 and 95 °C using a 1 °C/min temperature gradient. Data were analyzed by plotting the observed ellipticity as a function of temperature and fitting for the melting point transition (Tm) and the enthalpy (ΔHm) according to eq 1. During the fitting process a heat capacity (ΔCp) of 3.1 kJ/(mol K) was used based on calculated accessible surface areas of the folded and unfolded Ubl domain. (33, 34) The data were found to be insensitive to heat capacity as fits done using ΔCp 0 kJ/(mol K) yielded near identical values for Tm and ΔHm.(1)
The differences in stability (ΔΔG) between substituted Ubl domain proteins and the wild-type protein were calculated using ΔHm(wt), Tm(wt), and the differences in melting temperatures (ΔTm) between the wild type and the appropriate substituted proteins (35) according to eq 2.(2)

Ubl Domain−S5a Binding Assays

Purified His-tagged S5a196−309 was mixed with untagged Ubl domain, substituted Ubl domain proteins, or ubiquitin at a 1:2 molar ratio, respectively, in a total volume of 300 μL and placed on a rotating shaker at 4 °C for 1 h. The mixture was then loaded onto a Ni-NTA spin column (Qiagen) preequilibrated in binding buffer (20 mM sodium phosphate, 10 mM imidazole, 300 mM NaCl, pH 8). The column was washed twice with 600 μL of binding buffer and then eluted (20 mM sodium phosphate, 250 mM imidazole, 300 mM NaCl, pH 8). Elution samples were fractionated by electrophoresis on a tricine−polyacrylamide gel and stained with Coomassie brilliant blue dye. Protein concentrations were determined using the extinction coefficient method measured in guanidine hydrochloride. (36)

Results

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The three-dimensional structure of the parkin Ubl domain comprises a β-grasp fold (Figure 1) containing a five-strand β-sheet (β1, H11−V15; β2, I2−F7; β3, R42−F45; β4, K48−L50; and β5, Q64−V70) and two α-helices (α1, S22-Q34; α2, V56-D60). (16) This structure is characteristic of other ubiquitin-like domains such as hPLIC-2 and hHR23a (37, 38) and ubiquitin. (39) Despite these structural similarities, the parkin Ubl domain is nearly 11 kJ/mol less stable than ubiquitin. (24) The lower stability of the parkin Ubl domain may be an inherent property of the domain or might indicate that intramolecular interactions with other domains in the protein are needed to enhance its stability. Therefore, missense mutations in the Ubl domain could contribute to its improper folding, instability that would interfere with the overall structure of parkin or compromise its interactions with other proteins. To determine the effects of ARJP mutations on the structure and stability of the parkin Ubl domain, we used site-directed mutagenesis to create 13 of the possible substituted proteins. NMR spectroscopy was used to assess how each of these single residue substitutions might affect the overall structure of the proteins. Thermal denaturation, as measured by CD spectropolarimetry, was used to identify the change in stability caused by the substitutions. Both of these methods were used to determine whether structural alterations might render the Ubl domain ineffective for interaction with the proteasomal S5a subunit. The substitutions investigated covered most secondary structure regions of the Ubl domain including G12R (UbldG12R) and V15 M (UbldV15M) in β1; R42P (UbldR42P) in β3; K48A (UbldK48A) in β4; A31D (UbldA31D), K32T (UbldK32T), R33Q (UbldR33Q), and Q34R (UbldQ34R) in β1; V56E (UbldV56E) in β2; and D18N (UbldD18N), P37L (UbldP37L), A46P (UbldA46P), and T55I (UbldT55I) in loop regions (Figure 1). The ARJP substitution UbldR42P  (40) and another reported, (41) but not confirmed (UbldK48A), have been previously described (24) and are included here for completeness. The proteins covered 13 of the 16 sites for ARJP mutations. The only substitutions not assessed were at the initiating methionine position (M1L) and S10N and W54R that were reported after this work was started.

Figure 1

Figure 1. Ribbon drawing of the parkin Ubl domain illustrating the location of ARJP disease state substitutions. Side chains are indicated in red and labeled with the ARJP causative substitution. This figure was produced using the program PyMOL (48) using the human parkin Ubl domain structure (Protein Data Bank 1IYF). (16)

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Most Ubl domain substituted proteins with the exception of UbldA31D, UbldR42P, UbldA46P, and UbldV56E expressed well and produced yields comparable to that of the wild-type protein. The UbldA31D protein expressed at very low levels, could not be purified to homogeneity, and was not further investigated in these studies. The UbldA46P and UbldV56E proteins were expressed, but more than 90% of the protein was found in the insoluble fraction of the cell lysate as determined from Coomassie stained gels (data not shown). Attempts to purify these proteins showed wide variations in heterogeneity and susceptibility to proteolysis as determined by mass spectrometry. The UbldT55I protein was expressed and could be purified intact, but preliminary NMR spectra showed a heterogeneous population of peaks with different line widths. The behaviors of UbldA31D, UbldA46P, UbldT55I, and UbldV56E are consistent with poorly folded protein structures that are sensitive to degradation and/or aggregation, similar to that observed for UbldR42P. To circumvent this issue, we selected a candidate protein (UbldV56E) to express as a fusion protein linked to the B1 domain from immunoglobulin G (GB1), similar to that described previously for UbldR42P. (24) The resulting protein GB1-UbldV56E could be expressed to high levels and purified to homogeneity for structural characterization.

Parkin R42P, A46P, and V56E Substitutions Cause Ubl Domain Unfolding

CD spectropolarimetry and NMR spectroscopy were used to determine whether any significant changes to the secondary and tertiary structures of the substituted proteins occurred compared to the wild-type Ubl domain. The CD spectrum of the wild-type Ubl domain at 5 °C had well-defined minima near 208 and 225 nm (Figure 2) consistent with its three-dimensional structure, which contains approximately 21% α-helix and 43% β-sheet. A comparison of the CD spectra for UbldP37L (Figure 2) and several other substituted proteins (UbldG12R, UbldV15M, UbldD18N, UbldK32T, UbldR33Q, UbldQ34R, UbldK48A) with the wild-type Ubl domain showed less than 5% difference between the spectra, indicating that the secondary structure of these Ubl domain proteins was very similar to the normal protein at this temperature. A CD spectrum of UbldA46P (Figure 2) showed very poor signal from α-helix or β-sheet structure indicating this protein was likely unfolded. Analysis of this sample using mass spectrometry subsequent to CD measurement revealed that a portion of the protein had undergone proteolysis.

Figure 2

Figure 2. Folding comparison of substituted Ubl domains using CD spectropolarimetry. The spectrum of the wild-type parkin Ubl domain is shown (−) compared to the UbldP37L protein (- - -) that maintained a similar fold. The spectrum of the UbldA46P protein (···) which had little secondary structure and was shown by mass spectrometry to be sensitive to proteolysis is also shown. All spectra were collected using 20−80 μM protein in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0 at 5 °C.

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Each of the Ubl domain substituted proteins was examined by comparing their 1H−15N HSQC spectrum with the wild-type parkin Ubl domain spectrum (Figure 3). It was expected that a benign single site substitution should have only local effects on the spectrum reflecting the amino acid change whereas a greater disruption in structure would be reflected in a large number of spectral changes, especially for residues remote from the substitution. The 1H−15N HSQC spectra for the UbldG12R, UbldV15M, UbldD18N, UbldK32T, UbldR33Q, UbldQ34R, UbldP37L, and UbldK48A proteins (Figure 3 and Figure S1 of Supporting Information) all had well-dispersed peaks and similar spectral patterns to the wild-type Ubl domain spectrum. In several cases (UbldD18N, UbldK32T, UbldR33Q, UbldP37L, and UbldK48A) the single amino acid substitutions resulted in minimal changes in the NMR spectrum. For example, in the UbldK32T protein (Figure 3A) the appearance of a new peak for T32 and a large shift of the neighboring R33 occurred while the remainder of the spectrum was unaffected. This is consistent with a minor environmental change around position 32 with little alteration in structure. In contrast, the 1H−15N HSQC spectrum for UbldV15M resulted in changes in chemical shift to the adjacent residues E16, V17, and D18 but also to V29 and V30 in the α1 helix and several residues (F4, V5, F7) in β2. In the Ubl domain structure, V15 packs against V29 and V30 and has backbone contacts with M1−V3 near the N-terminus, indicating the V15M substitution likely affected the packing between these residues. A similar observation was noted for the Q34R substitution.

Figure 3

Figure 3. 1H−15N HSQC spectra of parkin Ubl domain and substituted proteins resulting from ARJP mutations. Superposition of spectra for the parkin Ubl domain (black) with (A) UbldK32T (red) and (B) UbldV15M (red). In each panel selected residue assignments are shown that have the largest chemical shift differences from the wild-type Ubl domain. An arrow is used to indicate the positions of peaks for the substituted residue in the wild-type and substituted proteins.

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Since the UbldV56E protein could not be purified in sufficient levels and was susceptible to proteolysis, the protein was linked to the B1 domain from immunoglobulin G (GB1; GB1-UbldV56E) to improve these properties. The resulting 1H−15N HSQC spectrum (Figure 4) displayed a population of well-dispersed peaks that were in excellent agreement with those observed in the 1H−15N HSQC spectrum of the isolated GB1 domain. (28) This indicated that the GB1 domain maintained its native fold having little interaction with the UbldV56E portion of the construct. The remaining peaks arising from the UbldV56E protein had little resemblance to the 1H−15N HSQC spectrum of the wild-type Ubl domain or most of the substituted proteins (Figure 3). The nondisperse nature of these peaks and their tight clustering between 8.0 and 8.5 ppm in the 1H dimension indicated the UbldV56E protein was unfolded. Subsequent to the NMR experiments, size exclusion chromatography showed the elution volume of GB1-UbldV56E was much smaller than expected for a 16 kDa protein, consistent with its unfolded structure. A similar observation has been made previously for UbldR42P. (24) Overall, these results indicate that the Ubl domain proteins containing the ARJP mutations G12R, D18N, K32T, R33Q, P37L, and K48A retain a similar three-dimensional fold as the wild-type domain, while at least two others (UbldV15M, UbldQ34R) have evidence of altered packing. In contrast, three substitutions (R42P, A46P, and V56E) result in gross structural differences due to unfolding of the domain.

Figure 4

Figure 4. 1H−15N HSQC spectra of GB1-UbldV56E showing the unfolded nature of UbldV56E. The spectrum of the GB1-UbldV56E protein is plotted in black contours while the isolated GB1 protein is shown in red contours. The spectra were collected at 600 MHz in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0 at 25 °C.

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Folded ARJP Substituted Proteins Lead to Differential Ubl Domain Stability

The substituted proteins (UbldG12R, UbldV15M, UbldD18N, UbldK32T, UbldR33Q, UbldQ34R, UbldP37L, UbldK48A) that exhibited a folded structure were examined to determine if the substitutions had altered the stabilities of the proteins. This was done using thermal unfolding of each protein and monitoring the change in ellipticity at 222 nm as a function of temperature. Initial spectra were recorded at 5 °C where the CD spectra of the selected substituted proteins were similar to the wild-type protein (Figure 2). Most of the melting curves for native parkin Ubl domain and the ARJP substituted proteins showed smooth sigmoidal transitions between folded and unfolded species indicative of a two-state unfolding process (Figure 5). UbldD18N and UbldK32T had shallower slopes that might indicate some deviation from this model. The data were fit using eq 1 to determine the midpoint of the transition (Tm) and the enthalpy (ΔHm). The data showed that the parkin Ubl domain had a melting temperature (Tm) near 63 °C (Table 1). There were three ARJP-substituted proteins that melted at higher temperatures (UbldG12R, UbldD18N, UbldP37L) and five substituted Ubl domain proteins that unfolded at lower temperatures including UbldR33Q and UbldK48A that melted nearly 10 °C lower than the wild-type protein. The difference in stabilities was calculated using the method of Becktel and Schellman using the ΔHm for the wild type and the Tm for each protein. (35) These results generally followed the order of the melting stabilities indicating that UbldD18N was the most stable protein and UbldK48A had the poorest stability.

Figure 5

Figure 5. Thermal unfolding curves for the parkin Ubl domain and substituted proteins resulting from ARJP disease state mutations. Data were collected using CD spectropolarimetry between 5 and 95 °C using a 1 °C/min temperature gradient. The ellipticity at 222 nm was plotted as fraction unfolded and fit according to equations described in Experimental Procedures. All samples were comprised of 20−80 μM protein in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0.

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Table 1. Thermodynamic Parameters for Substituted Parkin Ubl Proteins
proteinTm (°C)aΔHm (kJ/mol)bΔΔG (kJ/mol)c
parkin Ubl domain62.7 ± 0.1246.8 ± 9.5 
G12R64.0 ± 0.1271.2 ± 16.2−1.0
V15M60.2 ± 0.1246.2 ± 7.91.8
D18N68.7 ± 0.1336.7 ± 14.9−4.4
K32T61.9 ± 0.2117.1 ± 7.60.6
R33Q54.1 ± 0.1198.9 ± 8.56.3
Q34R58.4 ± 0.1267.3 ± 8.03.2
P37L68.0 ± 0.1223.5 ± 7.1−3.9
K48A53.2 ± 0.2179.2 ± 8.47.0
a

Determined from nonlinear fit using ΔCp = 3.1 kJ mol−1 K−1 according to eq 1.

b

Calculated from the linear fit near the Tm of ln K = ΔS/R − ΔH/R(1/T).

c

Estimated using eq 2 and Tm, ΔHm for parkin Ubl domain wild-type protein.

ARJP-Substituted Proteins Compromise S5a Interaction

The interaction of the parkin Ubl domain with the 19S regulatory subunit S5a was studied using residues 196−309 of the 377 amino acid protein. This fragment (S5a196−309) could be expressed and purified with a six-residue histidine tag and possesses ubiquitin interacting motifs (UIMs) shown previously to interact with the parkin Ubl domain. (16) To assess the impact of different ARJP substitutions on the interaction of the Ubl domain with S5a196−309, affinity pull-down experiments were employed. Figure 6 shows that His-tagged S5a196−309 bound to Ni-NTA beads successfully pulled down untagged parkin Ubl domain. The S5a fragment also bound similarly to UbldG12R, UbldD18N, and UbldQ34R, indicating these substitutions are not involved at the parkin Ubl domain−S5a interface. However, S5a196−309 interactions with the parkin- substituted proteins UbldV15M, UbldK32T, UbldR33Q, and UbldP37L were notably weaker than wild-type Ubl domain, similar to previous observations with the UbldK48A protein. (3) Since NMR experiments show some of these proteins (UbldK32T, UbldR33Q, and UbldP37L) retain a similar three-dimensional fold as the parkin Ubl domain, this indicates the substitutions likely interfere with formation of a proper parkin Ubl domain−S5a interface. As expected, little or no interaction between S5a196−309 and ubiquitin was observed.

Figure 6

Figure 6. ARJP substitutions in the Ubl domain disrupt S5a interactions. His6-S5a196−309 was incubated with 2 molar equiv of the parkin Ubl domain, substituted protein, or ubiquitin for 1 h and then loaded onto a Ni-NTA spin column. Following elution the samples were fractionated on a 16.5% tricine gel and stained with Coomassie dye. The top panel shows the bound proteins eluted with His6-S5a196−309 as follows: UbldG12R (lane 1), UbldV15M (lane 2), UbldD18N (lane 3), UbldR33Q (lane 4), UbldQ34R (lane 5), UbldP37L (lane 6), UbldK48A (lane 7), Ubl domain (lane 8), ubiquitin (lane 9), and His6-S5a196−309 alone (lane 13). Lower quantities of UbldK32T (lane 11) were used in the assay (although at the same molar ratio as the other mutants) so parkin Ubl domain was assayed again at this concentration (lane 12). Molecular weight markers are shown in lane 10. The lower panel illustrates the pure proteins as used in the pull-down assay and correspond to the same labels.

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Discussion

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Of the 13 substituted proteins we examined, five of them (UbldA31D, UbldR42P, UbldA46P, UbldT55I, and UbldV56E) showed significant structural alterations. For the UbldA31D protein, its low level of expression in bacteria compared to wild type is evidence for a poorly folded protein with decreased stability that may be subject to proteolysis. Similarly, UbldA46P showed little secondary or tertiary structure and was easily degraded. The UbldT55I protein showed evidence of heterogeneity and aggregation in NMR spectra. In previous studies we used an N-terminal GB1 tag to stabilize and protect the Ubl domain proteins from degradation. Both UbldV56E and UbldR42P had 1H−15N HSQC spectra consistent with an unfolded state for the Ubl domain portion of the protein. In the parkin Ubl domain, V56 is 95% buried near the N-terminus of the short α2 helix and participates in hydrophobic interactions with the side chains of M1, V17, and L61. Molecular dynamics experiments suggest that substitution of this hydrophobic residue with a charged glutamic acid residue would disrupt these interactions. (42) Similarly, A46 is located in the loop region between β3 and β4, and the substitution of alanine to proline would disrupt its hydrogen bonding to I66 in β5. Further, the addition of proline at this position could restrict the flexibility of the loop. As shown previously for UbldR42P, disruption of hydrogen bonding within the β-sheet regions can lead to unfolding of the entire domain. (24)
The eight proteins that contained substitutions but formed folded domains showed differential interactions with the tandem UIM region from the S5a subunit, proposed to allow for efficient delivery of polyubiquitinated cargo to the proteasome for degradation. The UbldK48A substitution resulted in decreased binding with S5a consistent with previous results showing this residue is involved in the interaction with the UIM region(s) from either S5a or Eps15. (3) In contrast, the UbldV15M, UbldK32T, UbldR33Q, and UbldP37L proteins showed diminished binding compared to wild-type Ubl domain even though none of these residues lies on the binding interface required for S5a interaction. Three of the substitutions (UbldV15M, UbldK32T, UbldR33Q) lead to decreased stability of the Ubl domain. It is noted that V15, K32, and R33 all interact with F13 in sheet β1, a residue affected by S5a binding. The decreased stability of the Ubl domain carrying these substitutions suggests that some modification of the S5a binding site has occurred. It is not clear why decreased S5a binding to the UbldP37L protein occurs as this substitution leads to increased domain stability. The similarity of S5a binding to UbldG12R and UbldD18N compared to wild-type Ubl domain was in agreement with their maintenance of three-dimensional fold and similar or increased stabilities. Although G12 is affected by the interaction with the UIMs from Eps15, neither substitution is at the interaction surface for S5a. (3)
The downstream effects of some interactions of the parkin Ubl domain have been validated in vitro and in vivo. It was recently shown that the interaction of the parkin Ubl domain with the regulatory particle of the proteasome activates the 26S proteasome. (43) This interaction provides a direct link between the cellular ubiquitination machinery and the regulation of the degradation machinery in the cell. The upregulation of proteasome activity was also shown in Drosophila where a K71P substitution (corresponding to R42P here) showed no regulation of proteasome activity, further indicating that a correctly folded Ubl domain is essential to the function of parkin in vivo.
Several ARJP substitutions in the Ubl domain shown to cause unfolding (UbldR42P, UbldV56E) or large decreases in stability (UbldR33Q, UbldK48A) alter the behavior of the full-length protein in vivo. In work done by Henn et al. (15) ARJP substitutions including R33Q, R42P, K48A, and V56E resulted in decreased stability of the full-length protein in N2a cells. This effect was shown to be a result of an increased degradation of parkin by the 26S proteasome. Another interaction where substitutions in the Ubl domain have been shown to affect its function is in the parkin interaction with the SH3 domain of endophilin-A1. (44) It was observed that in vitro parkin ubiquitinated endophilin-A1. The Ubl domain with the reported ARJP causative substitutions R42P and K48A was shown to disrupt this interaction, providing a new link between mutations in parkin and disruption to proteins in synaptic transmission.
These results clearly demonstrate there are multiple outcomes of the different ARJP disease-state mutations in the parkin Ubl domain. Some of these substitutions (A31D, R42P, A46P, T55I, V56E) result in disruption of the domain fold. Others, while maintaining the three-dimensional fold of the Ubl domain, have defects in interactions with the S5a subunit including K48A that has not been clearly linked to ARJP. Further, one must consider the outcomes of three of the substitutions (G12R, D18N, Q34R) that have little effect on the three-dimensional fold or S5a interaction. The corresponding mutations in the parkin gene are observed in the heterozygous state (17, 45-47) and could simply be a result of a polymorphism that has little impact on the protein function. Alternatively, it is possible that some of the disease-state residues in the parkin Ubl domain could be stabilized or are important for other essential, unidentified interactions with the remaining portions of the protein. Further studies will be needed to clarify the roles of these residues in parkin function and in ARJP.

Supporting Information

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1H−15N HSQC spectra of substituted parkin Ubl domain proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Author
    • Gary S. Shaw - Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1;  Email: [email protected]
  • Authors
    • Susan S. Safadi - Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1
    • Kathryn R. Barber - Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1

Acknowledgment

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The authors thank Lee-Ann Briere for maintenance of the Biomolecular Interactions and Conformation Facility, Qin Liu for maintenance of the Biomolecular NMR Facility, and Dr. Helen Walden (Cancer Research, U.K.) for careful reading of the manuscript.

Abbreviations

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ARJP

autosomal recessive juvenile parkinsonism

Ubl

ubiquitin-like

Ub

ubiquitin

UIM

ubiquitin-interacting motif

Ni-NTA

nickel nitrilotriacetic acid

HSQC

heteronuclear single-quantum coherence.

References

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This article references 48 other publications.

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    Nuytemans, Karen; Theuns, Jessie; Cruts, Marc; Van Broeckhoven, Christine
    Human Mutation (2010), 31 (7), 763-780CODEN: HUMUE3; ISSN:1059-7794. (Wiley-Liss, Inc.)
    A review. To date, mol. genetic analyses have identified over 500 distinct DNA variants in five disease genes assocd. with familial Parkinson disease; α-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include ∼82% simple mutations and ∼18% copy no. variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database (http://www.molgen.ua.ac.be/PDmutDB). In addn., we address the biol. relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of obsd. genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiol. of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups.
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  19. 19
    Stenson, P. D., Mort, M., Ball, E. V., Howells, K., Phillips, A. D., Thomas, N. S., and Cooper, D. N. (2009) The Human Gene Mutation Database: 2008 Update Genome Med. 1, 13
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    The Human Gene Mutation Database: 2008 update
    Stenson Peter D; Mort Matthew; Ball Edward V; Howells Katy; Phillips Andrew D; Thomas Nick St; Cooper David N
    Genome medicine (2009), 1 (1), 13 ISSN:.
    The Human Gene Mutation Database (HGMD((R))) is a comprehensive core collection of germline mutations in nuclear genes that underlie or are associated with human inherited disease. Here, we summarize the history of the database and its current resources. By December 2008, the database contained over 85,000 different lesions detected in 3,253 different genes, with new entries currently accumulating at a rate exceeding 9,000 per annum. Although originally established for the scientific study of mutational mechanisms in human genes, HGMD has since acquired a much broader utility for researchers, physicians, clinicians and genetic counselors as well as for companies specializing in biopharmaceuticals, bioinformatics and personalized genomics. HGMD was first made publicly available in April 1996, and a collaboration was initiated in 2006 between HGMD and BIOBASE GmbH. This cooperative agreement covers the exclusive worldwide marketing of the most up-to-date (subscription) version of HGMD, HGMD Professional, to academic, clinical and commercial users.
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  20. 20
    Hampe, C., Ardila-Osorio, H., Fournier, M., Brice, A., and Corti, O. (2006) Biochemical Analysis of Parkinson’s Disease-Causing Variants of Parkin, an E3 Ubiquitin-Protein Ligase with Monoubiquitylation Capacity Hum. Mol. Genet. 15, 2059– 2075
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    20
    Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity
    Hampe, Cornelia; Ardila-Osorio, Hector; Fournier, Margot; Brice, Alexis; Corti, Olga
    Human Molecular Genetics (2006), 15 (13), 2059-2075CODEN: HMGEE5; ISSN:0964-6906. (Oxford University Press)
    Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degrdn. of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochem. properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochem. characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the soly. of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin mols. only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.
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  21. 21
    Wang, C., Tan, J. M., Ho, M. W., Zaiden, N., Wong, S. H., Chew, C. L., Eng, P. W., Lim, T. M., Dawson, T. M., and Lim, K. L. (2005) Alterations in the Solubility and Intracellular Localization of Parkin by Several Familial Parkinson’s Disease-Linked Point Mutations J. Neurochem. 93, 422– 431
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    21
    Alterations in the solubility and intracellular localization of parkin by several familial Parkinson's disease-linked point mutations
    Wang, Cheng; Tan, Jeanne M. M.; Ho, Michelle W. L.; Zaiden, Norazean; Wong, Siew Heng; Chew, Constance L. C.; Eng, Pei Woon; Lim, Tit Meng; Dawson, Ted M.; Lim, Kah Leong
    Journal of Neurochemistry (2005), 93 (2), 422-431CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
    Mutations in the parkin gene, which encodes a ubiquitin ligase, are currently recognized as the main contributor to familial forms of Parkinson's disease (PD). A simple assumption about the effects of PD-linked mutations in parkin is that they impair or ablate the enzyme activity. However, a no. of recent studies, including ours, have indicated that many disease-linked point mutants of parkin retain substantial catalytic activity. To understand how the plethora of mutations on parkin contribute to its dysfunction, the authors have conducted a systematic anal. of a significant no. of parkin point mutants (22 in total), which represent the majority of parkin missense/nonsense mutations reported to date. The authors found that more than half of these mutations, including many located outside of the parkin RING fingers, produce alteration in the soly. of parkin which influences its detergent extn. property. This mutation-mediated alteration in parkin soly. is also assocd. with its propensity to form intracellular, aggresome-like, protein aggregates. However, they do not represent sites where parkin substrates become sequestered. As protein aggregation sequesters the functional forms away from their normal sites of action, the authors' results suggest that alterations in parkin soly. and intracellular localization may underlie the mol. basis of the loss of function caused by several of its mutations.
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    Tashiro, M., Okubo, S., Shimotakahara, S., Hatanaka, H., Yasuda, H., Kainosho, M., Yokoyama, S., and Shindo, H. (2003) NMR Structure of Ubiquitin-Like Domain in PARKIN: Gene Product of Familial Parkinson’s Disease J. Biomol. NMR 25, 153– 156
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    Beasley, S. A., Hristova, V. A., and Shaw, G. S. (2007) Structure of the Parkin in-between-Ring Domain Provides Insights for E3-Ligase Dysfunction in Autosomal Recessive Parkinson’s Disease Proc. Natl. Acad. Sci. U.S.A. 104, 3095– 3100
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    Safadi, S. S. and Shaw, G. S. (2007) A Disease State Mutation Unfolds the Parkin Ubiquitin-Like Domain Biochemistry 46, 14162– 14169
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    Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S., Shimizu, N., Iwai, K., Chiba, T., Tanaka, K., and Suzuki, T. (2000) Familial Parkinson Disease Gene Product, Parkin, is a Ubiquitin-Protein Ligase Nat. Genet. 25, 302– 305
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    25
    Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase
    Shimura H; Hattori N; Kubo S i; Mizuno Y; Asakawa S; Minoshima S; Shimizu N; Iwai K; Chiba T; Tanaka K; Suzuki T
    Nature genetics (2000), 25 (3), 302-5 ISSN:1061-4036.
    Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.
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    Lee, J. Y., Nagano, Y., Taylor, J. P., Lim, K. L., and Yao, T. P. (2010) Disease-Causing Mutations in Parkin Impair Mitochondrial Ubiquitination, Aggregation, and HDAC6-Dependent Mitophagy J. Cell Biol. 189, 671– 679
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    Sriram, S. R., Li, X., Ko, H. S., Chung, K. K., Wong, E., Lim, K. L., Dawson, V. L., and Dawson, T. M. (2005) Familial-Associated Mutations Differentially Disrupt the Solubility, Localization, Binding and Ubiquitination Properties of Parkin Hum. Mol. Genet. 14, 2571– 2586
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    Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin
    Sriram, Sathya R.; Li, Xiaojie; Ko, Han Seok; Chung, Kenny K. K.; Wong, Esther; Lim, Kah Leong; Dawson, Valina L.; Dawson, Ted M.
    Human Molecular Genetics (2005), 14 (17), 2571-2586CODEN: HMGEE5; ISSN:0964-6906. (Oxford University Press)
    Mutations in parkin are largely assocd. with autosomal recessive juvenile parkinsonism. The underlying mechanism of pathogenesis in parkin-assocd. Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity. A subset of missense and nonsense point mutations in parkin that span the entire gene and represent the numerous inheritance patterns that are assocd. with parkin-linked PD were investigated for their E3 ligase activity, localization and their ability to bind, ubiquitinate and effect the degrdn. of two substrates, synphilin-1 and aminoacyl-tRNA synthetase complex cofactor, p38. Parkin mutants vary in their intracellular localization, binding to substrates and enzymic activity, yet they are ultimately deficient in their ability to degrade substrate. These results suggest that not all parkin mutations result in loss of parkin's E3 ligase activity, but they all appear to manifest as loss-of-function mutants due to defects in soly., aggregation, enzymic activity or targeting proteins to the proteasome for degrdn.
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    Huth, J. R., Bewley, C. A., Jackson, B. M., Hinnebusch, A. G., Clore, G. M., and Gronenborn, A. M. (1997) Design of an Expression System for Detecting Folded Protein Domains and Mapping Macromolecular Interactions by NMR Protein Sci. 6, 2359– 2364
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    Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR
    Huth, Jeffrey R.; Bewley, Carole A.; Jackson, Belinda M.; Hinnebusch, Alan G.; Clore, G. Marius; Gronenborn, Angela M.
    Protein Science (1997), 6 (11), 2359-2364CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
    Two protein expression vectors have been designed for the prepn. of NMR samples. The vectors encode the Ig-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic anal. of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the soly. and stability of a protein can be detd. In addn., 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.
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    Hodgins, R., Gwozd, C., Arnason, T., Cummings, M., and Ellison, M. J. (1996) The Tail of a Ubiquitin-Conjugating Enzyme Redirects Multi-Ubiquitin Chain Synthesis from the Lysine 48-Linked Configuration to a Novel Nonlysine-Linked Form J. Biol. Chem. 271, 28766– 28771
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    Kay, L., Keifer, P., and Saarinen, T. (1992) Pure Absorption Gradient Enhanced Heteronuclear Single Quantum Correlation Spectroscopy with Improved Sensitivity J. Am. Chem. Soc. 114, 10663– 10665
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    Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity
    Kay, Lewis, E.; Keifer, Paul; Saarinen, Tim
    Journal of the American Chemical Society (1992), 114 (26), 10663-5CODEN: JACSAT; ISSN:0002-7863.
    A pulse sequence for recording gradient enhanced pure absorption 1H-15N heteronuclear single quantum correlation spectra is presented which offers improvements in sensitivity over existing gradient enhanced pulse schemes for moderately sized proteins (100-150 amino acids). Selection of protons directly bound to 15N and suppression of the intense water resonance is achieved solely through the application of gradient pulses. The method is demonstrated on a 100 amino acid fragment comprising the cellulose binding domain of a cellulase from Cellulomonas fimi.
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    Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: A Multidimensional Spectral Processing System Based on UNIX Pipes J. Biomol. NMR 6, 277– 293
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    NMRPipe: a multidimensional spectral processing system based on UNIX pipes
    Delaglio, Frank; Grzesiek, Stephan; Vuister, Geerten W.; Zhu, Guang; Pfeifer, John; Bax, Ad
    Journal of Biomolecular NMR (1995), 6 (3), 277-93CODEN: JBNME9; ISSN:0925-2738. (ESCOM)
    The NMRPipe system is a UNIX software environment of processing, graphics, and anal. tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
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    Johnson, B. A. (2004) Using NMRView to Visualize and Analyze the NMR Spectra of Macromolecules Methods Mol. Biol. 278, 313– 352
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    Using NMRView to visualize and analyze the NMR spectra of macromolecules
    Johnson, Bruce A.
    Methods in Molecular Biology (Totowa, NJ, United States) (2004), 278 (Protein NMR Techniques), 313-352CODEN: MMBIED; ISSN:1064-3745. (Humana Press Inc.)
    NMR expts. on macromols. can generate a tremendous amt. of data that must be analyzed and correlated to generate conclusions about the structure and dynamics of the mol. system. NMRView is a computer program that is designed to be useful in visualizing and analyzing these data. NMRView works with various types of NMR datasets and can have multiple datasets and display windows opened simultaneously. Virtually all actions of the program can be controlled through the Tcl scripting language, and new graphical user interface components can be added with the Tk toolkit. NMR spectral peaks can be analyzed and assigned. A suite of tools exists within NMRView for assigning the data from triple-resonance expts. The std. method for obtaining the most up-to-date version of NMRView is by downloading it from the website www.onemoonscientific.com.
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    Myers, J. K., Pace, C. N., and Scholtz, J. M. (1995) Denaturant m Values and Heat Capacity Changes: Relation to Changes in Accessible Surface Areas of Protein Unfolding Protein Sci. 4, 2138– 2148
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    Denaturant m values and heat capacity changes: relation to changes in accessible surface area of protein unfolding
    Myers, Jeffrey K.; Pace, C. Nick; Scholtz, J. Martin
    Protein Science (1995), 4 (10), 2138-48CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
    Denaturant m values, the dependence of the free energy of unfolding on denaturant concn., have been collected for a large set of proteins. The m value correlates very strongly with the amt. of protein surface exposed to solvent upon unfolding, with linear correlation coeffs. of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded proteins is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (ΔCp), which is confirmed here for the set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amt. of surface exposed to solvent upon unfolding is a main structural determinant for both m values and ΔCp.
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    Protein Structure and the Energetics of Protein Stability
    Robertson, Andrew D.; Murphy, Kenneth P.
    Chemical Reviews (Washington, D. C.) (1997), 97 (5), 1251-1267CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review with 102 refs. The focus of this review is on the relationships between protein stability and protein structure that can be established with the primary observables, the thermodn. parameters derived from calorimetric and spectroscopic studies and the structural models derived from X-ray crystallog. and NMR spectroscopy. This purely empirical approach will rely on coarse but regular features of structure such as solvent-exposed surface areas, secondary structure content, and nos. of disulfide bonds. The questions at hand are (1) how much information regarding the mol. origins of protein stability can be gleaned from the protein data alone and (2) can these data be used to resolve some of the controversies now in the literature.
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    Protein stability curves
    Becktel, Wayne J.; Schellman, John A.
    Biopolymers (1987), 26 (11), 1859-77CODEN: BIPMAA; ISSN:0006-3525.
    The stability curve of a protein is defined as the plot of the free energy of unfolding as a function of temp. For most proteins, the change in heat capacity on denaturation, or unfolding, is large but approx. const. When unfolding is a 2-state process, most of the salient features of the stability curves of proteins can be derived from this fact. A no. of relations are obtained, including the special features of low-temp. denaturation, the properties of the max. in stability, and the interrelations of the characteristic temps. of the protein. A formula that permits one to calc. small changes in stabilization free energy from changes in the melting temp. of the protein is given.
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    Quantitation of protein
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    Methods in Enzymology (1990), 182 (Guide Protein Purif.), 50-68CODEN: MENZAU; ISSN:0076-6879.
    A review with 48 refs. Assays that are easy to perform, require simple instrumentation, and are highly sensitive including methods to conc. samples or to eliminate interfering reagents are discussed.
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    Walters, K. J., Kleijnen, M. F., Goh, A. M., Wagner, G., and Howley, P. M. (2002) Structural Studies of the Interaction between Ubiquitin Family Proteins and Proteasome Subunit S5a Biochemistry 41, 1767– 1777
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    Mueller, T. D. and Feigon, J. (2003) Structural Determinants for the Binding of Ubiquitin-Like Domains to the Proteasome EMBO J. 22, 4634– 4645
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    Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987) Structure of Ubiquitin Refined at 1.8 Å Resolution J. Mol. Biol. 194, 531– 544
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    Structure of ubiquitin refined at 1.8 Å resolution
    Vijay-Kumar, Senadhi; Bugg, Charles E.; Cook, William J.
    Journal of Molecular Biology (1987), 194 (3), 531-44CODEN: JMOBAK; ISSN:0022-2836.
    The crystal structure of human erythrocyte ubiquitin has been refined at 1.8-Å resoln. using a restrained least-squares procedure. The crystallog. R-factor for the final model is 0.176. Bond lengths and bond angles in the mol. have root-mean-square deviations from ideal values of 0.016 Å and 1.5°, resp. A total of 58 water mols./mol. of ubiquitin are included in the final model. The last 4 residues in the mol. appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly H-bonded; ∼87% of the polypeptide chain is involved in H-bonded secondary structure. Prominent secondary structural features include 3 and one-half turns of α-helix, a short piece of 310-helix, a mixed β-sheet that contains 5 strands, and 7 reverse turns. There is a marked hydrophobic core formed between the β-sheet and α-helix. The mol. features a no. of unusual secondary structural features, including a parallel G1 β-bulge, 2 reverse aspartate (asparagine) turns, and a sym. H-bonding region that involves the 2 helixes and 2 of the reverse turns.
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    Terreni, L., Calabrese, E., Calella, A. M., Forloni, G., and Mariani, C. (2001) New Mutation (R42P) of the Parkin Gene in the Ubiquitinlike Domain Associated with Parkinsonism Neurology 56, 463– 466
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    Tomoo, K., Mukai, Y., In, Y., Miyagawa, H., Kitamura, K., Yamano, A., Shindo, H., and Ishida, T. (2008) Crystal Structure and Molecular Dynamics Simulation of Ubiquitin-Like Domain of Murine Parkin Biochim. Biophys. Acta 1784, 1059– 1067
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    Um, J. W., Im, E., Lee, H. J., Min, B., Yoo, L., Yoo, J., Lubbert, H., Stichel-Gunkel, C., Cho, H. S., Yoon, J. B., and Chung, K. C. (2010) Parkin Directly Modulates 26S Proteasome Activity J. Neurosci. 30, 11805– 11814
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  • Abstract

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    Figure 1

    Figure 1. Ribbon drawing of the parkin Ubl domain illustrating the location of ARJP disease state substitutions. Side chains are indicated in red and labeled with the ARJP causative substitution. This figure was produced using the program PyMOL (48) using the human parkin Ubl domain structure (Protein Data Bank 1IYF). (16)

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    Figure 2

    Figure 2. Folding comparison of substituted Ubl domains using CD spectropolarimetry. The spectrum of the wild-type parkin Ubl domain is shown (−) compared to the UbldP37L protein (- - -) that maintained a similar fold. The spectrum of the UbldA46P protein (···) which had little secondary structure and was shown by mass spectrometry to be sensitive to proteolysis is also shown. All spectra were collected using 20−80 μM protein in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0 at 5 °C.

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    Figure 3

    Figure 3. 1H−15N HSQC spectra of parkin Ubl domain and substituted proteins resulting from ARJP mutations. Superposition of spectra for the parkin Ubl domain (black) with (A) UbldK32T (red) and (B) UbldV15M (red). In each panel selected residue assignments are shown that have the largest chemical shift differences from the wild-type Ubl domain. An arrow is used to indicate the positions of peaks for the substituted residue in the wild-type and substituted proteins.

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    Figure 4

    Figure 4. 1H−15N HSQC spectra of GB1-UbldV56E showing the unfolded nature of UbldV56E. The spectrum of the GB1-UbldV56E protein is plotted in black contours while the isolated GB1 protein is shown in red contours. The spectra were collected at 600 MHz in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0 at 25 °C.

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    Figure 5

    Figure 5. Thermal unfolding curves for the parkin Ubl domain and substituted proteins resulting from ARJP disease state mutations. Data were collected using CD spectropolarimetry between 5 and 95 °C using a 1 °C/min temperature gradient. The ellipticity at 222 nm was plotted as fraction unfolded and fit according to equations described in Experimental Procedures. All samples were comprised of 20−80 μM protein in 10 mM KH2PO4, 1 mM EDTA, and 1 mM DTT, pH 7.0.

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    Figure 6

    Figure 6. ARJP substitutions in the Ubl domain disrupt S5a interactions. His6-S5a196−309 was incubated with 2 molar equiv of the parkin Ubl domain, substituted protein, or ubiquitin for 1 h and then loaded onto a Ni-NTA spin column. Following elution the samples were fractionated on a 16.5% tricine gel and stained with Coomassie dye. The top panel shows the bound proteins eluted with His6-S5a196−309 as follows: UbldG12R (lane 1), UbldV15M (lane 2), UbldD18N (lane 3), UbldR33Q (lane 4), UbldQ34R (lane 5), UbldP37L (lane 6), UbldK48A (lane 7), Ubl domain (lane 8), ubiquitin (lane 9), and His6-S5a196−309 alone (lane 13). Lower quantities of UbldK32T (lane 11) were used in the assay (although at the same molar ratio as the other mutants) so parkin Ubl domain was assayed again at this concentration (lane 12). Molecular weight markers are shown in lane 10. The lower panel illustrates the pure proteins as used in the pull-down assay and correspond to the same labels.

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  • References

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    This article references 48 other publications.

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      Parkinson's disease is a common neurodegenerative disease with complex clin. features. Autosomal recessive juvenile parkinsonism (AR-JP) maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253; the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, we have now isolated a complementary DNA clone of 2,960 base paris with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3-7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product 'Parkin'.
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      Mutations in the parkin gene are responsible for a common familial form of Parkinson's disease. As parkin encodes an E3 ubiquitin ligase, defects in proteasome-mediated protein degrdn. are believed to have a central role in the pathogenesis of Parkinson's disease. Here, we report a novel role for parkin in a proteasome-independent ubiquitination pathway. We have identified a regulated interaction between parkin and Eps15, an adaptor protein that is involved in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking. Treatment of cells with EGF stimulates parkin binding to both Eps15 and the EGFR and promotes parkin-mediated ubiquitination of Eps15. Binding of the parkin ubiquitin-like (Ubl) domain to the Eps15 ubiquitin-interacting motifs (UIMs) is required for parkin-mediated Eps15 ubiquitination. Furthermore, EGFR endocytosis and degrdn. are accelerated in parkin-deficient cells, and EGFR signaling via the phosphoinositide 3-kinase (PI(3)K)-Akt pathway is reduced in parkin knockout mouse brain. We propose that by ubiquitinating Eps15, parkin interferes with the ability of the Eps15 UIMs to bind ubiquitinated EGFR, thereby delaying EGFR internalization and degrdn., and promoting PI(3)K-Akt signaling. Considering the role of Akt in neuronal survival, our results have broad new implications for understanding the pathogenesis of Parkinson's disease.
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      Parkin is a multidomain E3 ligase assocd. with autosomal recessive Parkinson disease. The N-terminal ubiquitin-like domain (Ubld) of parkin functions with the S5a proteasomal subunit, positioning substrate proteins for degrdn. In addn., the parkin Ubld recruits the endocytotic protein Eps15, allowing the E3 ligase to ubiquinate Eps15 distal from its parkin-interacting site. The recognition sequences in the S5a subunit and Eps15 for the parkin Ubld are known as ubiquitin-interacting motifs (UIM). Each protein has two UIM sequences sepd. by a 50-residue spacer in S5a, but only ∼5 residues in Eps15. In this work we used NMR spectroscopy to det. how the parkin Ubld recognizes the proteasomal subunit S5a compared with Eps15, a substrate for ubiquitination. We show that Eps15 contains two flexible α-helixes each encompassing a UIM sequence. The α-helix surrounding UIM II is longer than that for UIM I, a situation that is reversed from S5a. Furthermore, we show the parkin Ubld preferentially binds to UIM I in the S5a subunit. This interaction is strongly diminished in a K48A substitution, found near the center of the S5a-interacting surface on the parkin Ubld. In contrast to S5a, parkin recruits Eps15 using both its UIM sequences, resulting in a larger interaction surface that includes residues from β1 and β2, not typically known to interact with UIM sequences. These results show that the parkin Ubld uses differential surfaces to recruit UIM regions from the S5a proteasomal subunit compared with Eps15 involved in cell signaling.
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      Imam, Syed Z.; Zhou, Qing; Yamamoto, Ayako; Valente, Anthony J.; Ali, Syed F.; Bains, Mona; Roberts, James L.; Kahle, Philipp J.; Clark, Robert A.; Li, Senlin
      Journal of Neuroscience (2011), 31 (1), 157-163CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Mutations in parkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function both in vitro and in vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.
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    9. 9
      Goldberg, M. S., Fleming, S. M., Palacino, J. J., Cepeda, C., Lam, H. A., Bhatnagar, A., Meloni, E. G., Wu, N., Ackerson, L. C., Klapstein, G. J., Gajendiran, M., Roth, B. L., Chesselet, M. F., Maidment, N. T., Levine, M. S., and Shen, J. (2003) Parkin-Deficient Mice Exhibit Nigrostriatal Deficits but Not Loss of Dopaminergic Neurons J. Biol. Chem. 278, 43628– 43635
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    10. 10
      Dawson, T. M. and Dawson, V. L. (2010) The Role of Parkin in Familial and Sporadic Parkinson’s Disease Mov. Disord. 25 (Suppl. 1) S32– S39
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    11. 11
      Lee, J. Y., Nagano, Y., Taylor, J. P., Lim, K. L., and Yao, T. P. (2010) Disease-Causing Mutations in Parkin Impair Mitochondrial Ubiquitination, Aggregation, and HDAC6-Dependent Mitophagy J. Cell Biol. 189, 671– 679
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    12. 12
      Keeney, P. M., Xie, J., Capaldi, R. A., and Bennett, J. P., Jr. (2006) Parkinson’s Disease Brain Mitochondrial Complex I has Oxidatively Damaged Subunits and is Functionally Impaired and Misassembled J. Neurosci. 26, 5256– 5264
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      12
      Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled
      Keeney, Paula M.; Xie, Jing; Capaldi, Roderick A.; Bennett, James P., Jr.
      Journal of Neuroscience (2006), 26 (19), 5256-5264CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidn. of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured NAD (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidn. levels and redns. of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes. We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidn. status and pos. correlate with redn. in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidn. of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidn. may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.
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    13. 13
      Parker, W. D., Jr., Parks, J. K., and Swerdlow, R. H. (2008) Complex I Deficiency in Parkinson’s Disease Frontal Cortex Brain Res. 1189, 215– 218
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      13
      Complex I deficiency in Parkinson's disease frontal cortex
      Parker, W. Davis; Parks, Janice K.; Swerdlow, Russell H.
      Brain Research (2008), 1189 (), 215-218CODEN: BRREAP; ISSN:0006-8993. (Elsevier Ltd.)
      A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a no. of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue prepns. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodol. issues are crit. in demonstrating this loss of activity.
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    14. 14
      Hristova, V. A., Beasley, S. A., Rylett, R. J., and Shaw, G. S. (2009) Identification of a Novel Zn2+-Binding Domain in the Autosomal Recessive Juvenile Parkinson-Related E3 Ligase Parkin J. Biol. Chem. 284, 14978– 14986
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    15. 15
      Henn, I. H., Gostner, J. M., Lackner, P., Tatzelt, J., and Winklhofer, K. F. (2005) Pathogenic Mutations Inactivate Parkin by Distinct Mechanisms J. Neurochem. 92, 114– 122
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      15
      Pathogenic mutations inactivate parkin by distinct mechanisms
      Henn, Iris H.; Gostner, Johanna M.; Lackner, Peter; Tatzelt, Joerg; Winklhofer, Konstanze F.
      Journal of Neurochemistry (2005), 92 (1), 114-122CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
      Loss of parkin function is the major cause of autosomal recessive Parkinson's disease (ARPD). A wide variety of parkin mutations have been identified in patients; however, the pathophysiol. mechanisms leading to the inactivation of mutant parkin are poorly understood. In this study we characterized pathogenic C- and N-terminal parkin mutants and found distinct pathways of parkin inactivation. Deletion of the C terminus abrogated the assocn. of parkin with cellular membranes and induced rapid misfolding and aggregation. Four N-terminal missense mutations, located within the ubiquitin-like domain (UBL), decrease the stability of parkin; as a consequence, these mutants are rapidly degraded by the proteasome. Furthermore, we present evidence that a smaller parkin species of 42 kDa, which is present in exts. prepd. from human brain and cultured cells, originates from an internal start site and lacks the N-terminal UBL domain.
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    16. 16
      Sakata, E., Yamaguchi, Y., Kurimoto, E., Kikuchi, J., Yokoyama, S., Yamada, S., Kawahara, H., Yokosawa, H., Hattori, N., Mizuno, Y., Tanaka, K., and Kato, K. (2003) Parkin Binds the Rpn10 Subunit of 26S Proteasomes through its Ubiquitin-Like Domain EMBO Rep. 4, 301– 306
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      16
      Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain
      Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi
      EMBO Reports (2003), 4 (3), 301-306CODEN: ERMEAX; ISSN:1469-221X. (Nature Publishing Group)
      Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we detd. the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chem.-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP.
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    17. 17
      Chaudhary, S., Behari, M., Dihana, M., Swaminath, P. V., Govindappa, S. T., Jayaram, S., Goyal, V., Maitra, A., Muthane, U. B., Juyal, R. C., and Thelma, B. K. (2006) Parkin Mutations in Familial and Sporadic Parkinson’s Disease among Indians Parkinsonism Relat. Disord. 12, 239– 245
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      17
      Parkin mutations in familial and sporadic Parkinson's disease among Indians
      Chaudhary Shashi; Behari Madhuri; Dihana Maninder; Swaminath Pazhayannur V; Govindappa Shyla T; Jayaram Sachi; Goyal Vinay; Maitra Arindam; Muthane Uday B; Juyal R C; Thelma B K
      Parkinsonism & related disorders (2006), 12 (4), 239-45 ISSN:1353-8020.
      We observed a mutation frequency of 8.5% in Parkin gene among Indian PD patients based on sequencing and gene dosage analysis of its exons. We identified nine point mutations of which seven are novel and hitherto unreported. These mutations accounted for 14.3% familial PD, 6.9% young onset and 5.9% late onset sporadic PD. Of the 20 PD patients with mutations only two had homozygous mutations and one was a compound heterozygote. Homozygous exonic deletions were absent but heterozygous exon rearrangements were observed in 9.2% of patients (19% familial PD and 4.5% young onset sporadic PD).
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    18. 18
      Nuytemans, K., Theuns, J., Cruts, M., and Van Broeckhoven, C. (2010) Genetic Etiology of Parkinson Disease Associated with Mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 Genes: A Mutation Update Hum. Mutat. 31, 763– 780
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      18
      Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update
      Nuytemans, Karen; Theuns, Jessie; Cruts, Marc; Van Broeckhoven, Christine
      Human Mutation (2010), 31 (7), 763-780CODEN: HUMUE3; ISSN:1059-7794. (Wiley-Liss, Inc.)
      A review. To date, mol. genetic analyses have identified over 500 distinct DNA variants in five disease genes assocd. with familial Parkinson disease; α-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include ∼82% simple mutations and ∼18% copy no. variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database (http://www.molgen.ua.ac.be/PDmutDB). In addn., we address the biol. relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of obsd. genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiol. of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups.
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    19. 19
      Stenson, P. D., Mort, M., Ball, E. V., Howells, K., Phillips, A. D., Thomas, N. S., and Cooper, D. N. (2009) The Human Gene Mutation Database: 2008 Update Genome Med. 1, 13
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      19
      The Human Gene Mutation Database: 2008 update
      Stenson Peter D; Mort Matthew; Ball Edward V; Howells Katy; Phillips Andrew D; Thomas Nick St; Cooper David N
      Genome medicine (2009), 1 (1), 13 ISSN:.
      The Human Gene Mutation Database (HGMD((R))) is a comprehensive core collection of germline mutations in nuclear genes that underlie or are associated with human inherited disease. Here, we summarize the history of the database and its current resources. By December 2008, the database contained over 85,000 different lesions detected in 3,253 different genes, with new entries currently accumulating at a rate exceeding 9,000 per annum. Although originally established for the scientific study of mutational mechanisms in human genes, HGMD has since acquired a much broader utility for researchers, physicians, clinicians and genetic counselors as well as for companies specializing in biopharmaceuticals, bioinformatics and personalized genomics. HGMD was first made publicly available in April 1996, and a collaboration was initiated in 2006 between HGMD and BIOBASE GmbH. This cooperative agreement covers the exclusive worldwide marketing of the most up-to-date (subscription) version of HGMD, HGMD Professional, to academic, clinical and commercial users.
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    20. 20
      Hampe, C., Ardila-Osorio, H., Fournier, M., Brice, A., and Corti, O. (2006) Biochemical Analysis of Parkinson’s Disease-Causing Variants of Parkin, an E3 Ubiquitin-Protein Ligase with Monoubiquitylation Capacity Hum. Mol. Genet. 15, 2059– 2075
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      20
      Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity
      Hampe, Cornelia; Ardila-Osorio, Hector; Fournier, Margot; Brice, Alexis; Corti, Olga
      Human Molecular Genetics (2006), 15 (13), 2059-2075CODEN: HMGEE5; ISSN:0964-6906. (Oxford University Press)
      Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degrdn. of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochem. properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochem. characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the soly. of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin mols. only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.
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    21. 21
      Wang, C., Tan, J. M., Ho, M. W., Zaiden, N., Wong, S. H., Chew, C. L., Eng, P. W., Lim, T. M., Dawson, T. M., and Lim, K. L. (2005) Alterations in the Solubility and Intracellular Localization of Parkin by Several Familial Parkinson’s Disease-Linked Point Mutations J. Neurochem. 93, 422– 431
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      21
      Alterations in the solubility and intracellular localization of parkin by several familial Parkinson's disease-linked point mutations
      Wang, Cheng; Tan, Jeanne M. M.; Ho, Michelle W. L.; Zaiden, Norazean; Wong, Siew Heng; Chew, Constance L. C.; Eng, Pei Woon; Lim, Tit Meng; Dawson, Ted M.; Lim, Kah Leong
      Journal of Neurochemistry (2005), 93 (2), 422-431CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
      Mutations in the parkin gene, which encodes a ubiquitin ligase, are currently recognized as the main contributor to familial forms of Parkinson's disease (PD). A simple assumption about the effects of PD-linked mutations in parkin is that they impair or ablate the enzyme activity. However, a no. of recent studies, including ours, have indicated that many disease-linked point mutants of parkin retain substantial catalytic activity. To understand how the plethora of mutations on parkin contribute to its dysfunction, the authors have conducted a systematic anal. of a significant no. of parkin point mutants (22 in total), which represent the majority of parkin missense/nonsense mutations reported to date. The authors found that more than half of these mutations, including many located outside of the parkin RING fingers, produce alteration in the soly. of parkin which influences its detergent extn. property. This mutation-mediated alteration in parkin soly. is also assocd. with its propensity to form intracellular, aggresome-like, protein aggregates. However, they do not represent sites where parkin substrates become sequestered. As protein aggregation sequesters the functional forms away from their normal sites of action, the authors' results suggest that alterations in parkin soly. and intracellular localization may underlie the mol. basis of the loss of function caused by several of its mutations.
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    22. 22
      Tashiro, M., Okubo, S., Shimotakahara, S., Hatanaka, H., Yasuda, H., Kainosho, M., Yokoyama, S., and Shindo, H. (2003) NMR Structure of Ubiquitin-Like Domain in PARKIN: Gene Product of Familial Parkinson’s Disease J. Biomol. NMR 25, 153– 156
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    23. 23
      Beasley, S. A., Hristova, V. A., and Shaw, G. S. (2007) Structure of the Parkin in-between-Ring Domain Provides Insights for E3-Ligase Dysfunction in Autosomal Recessive Parkinson’s Disease Proc. Natl. Acad. Sci. U.S.A. 104, 3095– 3100
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    24. 24
      Safadi, S. S. and Shaw, G. S. (2007) A Disease State Mutation Unfolds the Parkin Ubiquitin-Like Domain Biochemistry 46, 14162– 14169
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    25. 25
      Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S., Shimizu, N., Iwai, K., Chiba, T., Tanaka, K., and Suzuki, T. (2000) Familial Parkinson Disease Gene Product, Parkin, is a Ubiquitin-Protein Ligase Nat. Genet. 25, 302– 305
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      25
      Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase
      Shimura H; Hattori N; Kubo S i; Mizuno Y; Asakawa S; Minoshima S; Shimizu N; Iwai K; Chiba T; Tanaka K; Suzuki T
      Nature genetics (2000), 25 (3), 302-5 ISSN:1061-4036.
      Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.
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    26. 26
      Lee, J. Y., Nagano, Y., Taylor, J. P., Lim, K. L., and Yao, T. P. (2010) Disease-Causing Mutations in Parkin Impair Mitochondrial Ubiquitination, Aggregation, and HDAC6-Dependent Mitophagy J. Cell Biol. 189, 671– 679
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      Sriram, S. R., Li, X., Ko, H. S., Chung, K. K., Wong, E., Lim, K. L., Dawson, V. L., and Dawson, T. M. (2005) Familial-Associated Mutations Differentially Disrupt the Solubility, Localization, Binding and Ubiquitination Properties of Parkin Hum. Mol. Genet. 14, 2571– 2586
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      27
      Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin
      Sriram, Sathya R.; Li, Xiaojie; Ko, Han Seok; Chung, Kenny K. K.; Wong, Esther; Lim, Kah Leong; Dawson, Valina L.; Dawson, Ted M.
      Human Molecular Genetics (2005), 14 (17), 2571-2586CODEN: HMGEE5; ISSN:0964-6906. (Oxford University Press)
      Mutations in parkin are largely assocd. with autosomal recessive juvenile parkinsonism. The underlying mechanism of pathogenesis in parkin-assocd. Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity. A subset of missense and nonsense point mutations in parkin that span the entire gene and represent the numerous inheritance patterns that are assocd. with parkin-linked PD were investigated for their E3 ligase activity, localization and their ability to bind, ubiquitinate and effect the degrdn. of two substrates, synphilin-1 and aminoacyl-tRNA synthetase complex cofactor, p38. Parkin mutants vary in their intracellular localization, binding to substrates and enzymic activity, yet they are ultimately deficient in their ability to degrade substrate. These results suggest that not all parkin mutations result in loss of parkin's E3 ligase activity, but they all appear to manifest as loss-of-function mutants due to defects in soly., aggregation, enzymic activity or targeting proteins to the proteasome for degrdn.
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    28. 28
      Huth, J. R., Bewley, C. A., Jackson, B. M., Hinnebusch, A. G., Clore, G. M., and Gronenborn, A. M. (1997) Design of an Expression System for Detecting Folded Protein Domains and Mapping Macromolecular Interactions by NMR Protein Sci. 6, 2359– 2364
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      28
      Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR
      Huth, Jeffrey R.; Bewley, Carole A.; Jackson, Belinda M.; Hinnebusch, Alan G.; Clore, G. Marius; Gronenborn, Angela M.
      Protein Science (1997), 6 (11), 2359-2364CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
      Two protein expression vectors have been designed for the prepn. of NMR samples. The vectors encode the Ig-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic anal. of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the soly. and stability of a protein can be detd. In addn., 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.
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    29. 29
      Hodgins, R., Gwozd, C., Arnason, T., Cummings, M., and Ellison, M. J. (1996) The Tail of a Ubiquitin-Conjugating Enzyme Redirects Multi-Ubiquitin Chain Synthesis from the Lysine 48-Linked Configuration to a Novel Nonlysine-Linked Form J. Biol. Chem. 271, 28766– 28771
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    30. 30
      Kay, L., Keifer, P., and Saarinen, T. (1992) Pure Absorption Gradient Enhanced Heteronuclear Single Quantum Correlation Spectroscopy with Improved Sensitivity J. Am. Chem. Soc. 114, 10663– 10665
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      30
      Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity
      Kay, Lewis, E.; Keifer, Paul; Saarinen, Tim
      Journal of the American Chemical Society (1992), 114 (26), 10663-5CODEN: JACSAT; ISSN:0002-7863.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXitValsLw%253D&md5=78d0376e0fdafecd901d5e0c9139dc14
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      Journal of Neuroscience (2010), 30 (35), 11805-11814CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Parkinson's disease (PD) is a common neurodegenerative disease that involves the deterioration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiol. of PD remains poorly understood, recent genetic, postmortem, and exptl. evidence shows that abnormal protein accumulation and subsequent aggregate formation are prominent features of both sporadic and familial PD. While proteasome dysfunction is obsd. in PD, diverse mutations in the parkin gene are linked to early-onset autosomal-recessive forms of familial PD. We demonstrate that parkin, an E3 ubiquitin ligase, activates the 26S proteasome in an E3 ligase activity-independent manner. Furthermore, an N-terminal ubiquitin-like domain within parkin is crit. for the activation of the 26S proteasome through enhancing the interaction between 19S proteasomal subunits, whereas the PD-linked R42P mutant abolishes this action. The current findings point to a novel role for parkin for 26S proteasome assembly and suggest that parkin mutations contribute to the proteasomal dysfunction in PD.
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      Parkinsonism & related disorders (2009), 15 (2), 105-9 ISSN:1353-8020.
      A family history of Parkinson's disease (PD) is the most commonly reported risk factor after age, suggesting a genetic component to the disease in a sub-group of patients. Mutations in at least six genes have been identified that can lead to monogenic forms of PD. We screened a sample of 74 early-onset PD cases out of a cohort of 950 patients (onset <50 years) for genetic abnormalities in known familial Parkinsonism genes. A self-reported family history of PD existed for 30 patients (40.5%). Of these, 13 each had a first- or a second-degree relative with PD and four reported a more distant relative with PD. The entire coding region of the PRKN (MIM 602544), DJ-1 (MIM 602533) and PINK1 (MIM 698309) genes, and exon 41 of the LRRK2 gene (MIM 609007) were screened by direct sequencing. All exons of PRKN were examined for gene-dosage abnormalities. Screening identified five patients with putative genetic disease: two patients carried PRKN mutations (p.G12R heterozygous and p.G430D homozygous), one patient carried a p.G411S heterozygous amino acid change in the PINK1 gene and two individuals were heterozygous for the common p.G2019S mutation in LRRK2. No alpha-synuclein or DJ-1 variants were observed. Our data suggest that approximately 7% of early-onset PD cases seen in Queensland movement disorders clinics have mutations involving known PARK genes.
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      Human genetics (2007), 122 (3-4), 415 ISSN:0340-6717.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c7ptFKmsw%253D%253D&md5=90d94687e24d6531a26db04a1212e56e
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      Biswas, A., Gupta, A., Naiya, T., Das, G., Neogi, R., Datta, S., Mukherjee, S., Das, S. K., Ray, K., and Ray, J. (2006) Molecular Pathogenesis of Parkinson’s Disease: Identification of Mutations in the Parkin Gene in Indian Patients Parkinsonism Relat. Disord. 12, 420– 426
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      Parkinsonism & related disorders (2006), 12 (7), 420-6 ISSN:1353-8020.
      Parkinson's disease (PD), the second most common neurodegenerative disorder, affects at least 1% of the population over the age of 50. However, very little information is available regarding the molecular basis of PD among Indians. Since the largest number of mutations have been detected in the Parkin gene among all known PD loci, we aim to use Parkin as the candidate gene to assess its role in PD-related pathogenesis in Indian patients. A total of 138 PD patients, with the mean age of onset being 47+/-14 (age range, 5-77 years), and 100 controls were recruited for the study from eastern India. Parkin mutations were detected by amplification of exons of the gene along with the flanking splice junctions by polymerase chain reaction, single-stranded conformation polymorphism and DNA sequencing. A total of 18 nucleotide variants including six novel changes were detected. These include five missense mutations (Gln34Arg, Arg42Cys, Arg42His, Tyr143Cys and Arg334Cys) detected in eight patients in heterozygous condition and a homozygous deletion encompassing exons 3 and 4 in two sibs affected with PD. Clinical features of the Parkin mutants were compared. Among eastern Indian PD patients, mutation in Parkin was identified in 7.24% cases.
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    1H−15N HSQC spectra of substituted parkin Ubl domain proteins. This material is available free of charge via the Internet at http://pubs.acs.org.


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