Relative Sensitivity of Parkin and Other Cysteine-containing Enzymes to Stress-induced Solubility Alterations*

Loss of parkin function is a predominant cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for sporadic Parkinson disease. We have recently demonstrated that a wide variety of Parkinson disease-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell, a phenomenon that may underlie the progressive susceptibility of the brain to degeneration. The vulnerability of parkin to stress-induced modification is likely due to its abundance of cysteine residues. Here, we performed a comprehensive mutational analysis and demonstrate that Cys residues residing both within and outside of the RING-IBR (in between RING fingers)-RING domain of parkin are important in maintaining its solubility. The majority of these Cys residues are highly conserved in parkin across different species and potentially fulfil important structural roles. Further, we found that both parkin and HHARI (human homologue of Drosophila ariadne), another RING-IBR-RING-type ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes. However, parkin appears to be uniquely sensitive to DA-mediated stress, the specificity of which is likely due to DA modification of 2 Cys residues on parkin (Cys-268 and Cys-323) that are distinct from other RING-IBR-RING members.

Parkinson disease (PD) 4 is the most common neurodegenerative movement disorder characterized pathologically by the rather selective loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal protein inclusions known as Lewy bodies. Although most cases of PD occur in a sporadic manner, a subset of PD cases is inheritable and attributable to mutations in specific genes. These familial PD-linked genes include ␣-synuclein, parkin, DJ-1, PINK1, and LRRK2 (1,2). Of these, mutations in the parkin gene are currently recognized to be a predominant cause of familial, early onset PD (3)(4)(5). Further, emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of PD (6,7).
The importance of functional parkin to dopaminergic neuronal survival is probably related to the multitude of neuroprotective roles it serves (8,9). Parkin functions as a ubiquitin ligase associated with protein homeostasis and apparently confers protection to neurons against a diverse range of cellular insults (8,9). Recently, we have demonstrated that a wide variety of PD-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell (10). Our observations corroborated with a similar study conducted by LaVoie et al. (11), who further showed that DA covalently modifies parkin via its Cys residues, although the number and location of the Cys targeted by DA remain unknown. Since parkin functions as a broad spectrum neuroprotectant, the effects on parkin brought about by these oxidative stressors could deplete the availability of soluble parkin in the brain, and as such, may underlie the progressive susceptibility of the brain to degeneration.
We have previously speculated that enzymes whose structure and function are dependent on catalytic Cys are more susceptible to the consequence of oxidative modification (10). Although the active site Cys-dependent tyrosine phosphatase family of enzymes provides one example (12), the RING finger-containing ubiquitin ligases, all of which are characterized by their Cys-rich catalytic moieties, potentially represent another. In particular, the abundance of Cys residues residing on RING-IBR-RING-containing proteins, such as parkin, conceivably could present a ready source of targets for oxidative modification. As the majority of these highly conserved RING finger Cys residues are thought to fulfil important structural roles (13,14), it is conceivable that their modification by oxidation could disrupt the overall structural integrity of the protein, leading to alterations in their biochemical properties.
In this study, we systematically mutate almost all of parkin's Cys residues and demonstrate that conserved Cys residues residing both within and outside of the RING-IBR-RING domain of parkin are important for maintaining its solubility, although modification of Cys residues within parkin RING-IBR-RING domain also resulted in a significantly higher tendency for the protein to form aggresome-like structures within the cell. Further, we found that both parkin and HHARI, another RING-IBR-RING ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes examined. However, parkin appears to be uniquely sensitive to DA-mediated stress. Further mutational analysis suggests the involvement of 2 nonconserved Cys residues on parkin, Cys-268 and Cys-323, in DAmediated parkin insolubility. As these residues are distinct from other RING-IBR-RING members, including HHARI, they could potentially explain for the enhanced sensitivity of parkin to modification by DA.

MATERIALS AND METHODS
Antibodies and Reagents-The following antibodies were used: monoclonal anti-FLAG-HRP (Sigma), monoclonal anti-Myc-HRP (Roche Applied Science), monoclonal anti-HA-HRP (Roche Applied Science), monoclonal anti-␤ actin (Sigma), rhodamine-conjugated anti-mouse IgG (Molecular Probes), monoclonal anti-UCH-L1 (Dako), polyclonal anti-HHARI (Abcam), polyclonal anti-c-Cbl (Cell Signaling), and polyclonal anti-CHIP (gift from L. Petrucelli). The FLAG-tagged wild-type parkin and Myc-tagged CHIP expression constructs were gifts from R. Takahashi, whereas HA-tagged HHARI and c-Cbl expression constructs were gifts from G. Guy. All other chemicals, unless otherwise stated, were purchased from Sigma. DA stock solution (100 mM in boiled water) was freshly prepared prior to use, whereas NOC-18 stock solution (50 mM in water) was prepared and stored at Ϫ20°C.

Site-directed Mutagenesis and Generation of Parkin Deletion
Constructs-All the Cys 3 Ala (C 3 A) parkin mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions and verified via DNA sequencing. Parkin 1-137 and 1-237 deletion constructs were generated by means of PCR amplification of the designated regions using wild-type FLAG-parkin cDNA as a template and subcloned into pCDNA3 plasmid. For parkin ⌬77-237, the UBL and RING-IBR-RING regions of parkin were amplified separately from wild-type FLAG-parkin cDNA by means of PCR and subsequently cloned in-frame into pCDNA3.
Cell Culture, Treatment with Various Stressors, and Western Blot Analysis-SH-SY5Y neuroblastoma cells were seeded at 2 ϫ 10 5 cell density for all transfections. pCDNA3 plasmid bearing FLAG-tagged wild-type parkin or various C 3 A parkin mutants were transfected using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. At 48 h after transfection, the cells were harvested for sequential fractionation of the cell lysates into Triton-X-soluble and SDSsoluble fractions as described previously (18). An equivalent amount of proteins among different Triton-X-soluble and SDSsoluble fractions was resolved by means of SDS-PAGE, and the levels of various proteins were analyzed by means of Western blotting procedures using ECL detection reagents (Amersham Biosciences). For stress treatments, plasmids containing FLAG-parkin species, HA-HHARI, HA-c-Cbl, Myc-CHIP, or Myc-UCH-L1 were transfected using Lipofectamine 2000 reagent (Invitrogen). At 24 h after transfection, transfected cells were treated with 20 mM H 2 O 2 for 30 min, 0.5-1 mM DA for 12 h, or 0.25-0.5 mM NOC-18 for 24 h.
Immunocytochemistry and Confocal Microscopy-5 ϫ 10 4 SH-SY5Y cells were seeded on coverslips for subsequent transfection with FLAG-tagged wild-type parkin or selected C 3 A parkin mutants using Lipofectamine Plus reagent (Invitrogen). At 48 h after transfection, the cells were fixed with 3% paraformaldehyde (Sigma) for 1 h at 4°C. Cellular distributions of wild-type and mutant parkin were examined by means of immunocytochemistry and confocal microscopy as described previously (4). At least 100 transfected cells were counted to quantify the incidence of inclusions.
Human Tissues and Statistical Analysis-Aliquots of previously described (10) human brain lysates prepared from postmortem brains of control and PD individuals that were stored at Ϫ80°C were used in this study. Statistical significance for all the quantitative data obtained was analyzed using Student's t test (*, p Ͻ 0.05, **, p Ͻ 0.001) unless otherwise stated.

Conservation and Structural Implication of Parkin's Cysteine
Residues-Inspection of the amino acid sequence of human parkin reveals a total of 35 Cys residues, the majority of which (23 out of 35) reside within the RING-IBR-RING domain of the protein (Fig. 1A). Further, comparison of human parkin protein sequence with orthologous sequences from rodent, fish, insect, and worm reveals that most of the parkin's Cys residues are absolutely conserved across these diverse species, a feature that suggests their importance to the protein's structure and/or function ( Fig. 1A and supplemental Fig. S1). Although the invariant Cys residues in parkin across different species are largely found within the catalytically important RING-IBR-RING domain, a number of such highly conserved Cys residues, such as Cys-150, Cys-166, Cys-212, and Cys-457, are notably also found along the length of the protein outside of this domain (Fig. 1A).
To gain insights into the importance of individual Cys residue on parkin to its overall tertiary architecture, it is essential to elucidate the three-dimensional structure of the protein, information of which is currently lacking. However, the structure of both RING1 and RING2 in related proteins, c-Cbl and HHARI, respectively, have previously been reported (13,14). Accordingly, we used the structure of c-Cbl RING1 and HHARI RING2 as templates to model parkin RING1 and RING2 domains, respectively. Homology models of parkin RING1 and RING2 so obtained reveal the coordination of Cys-238, Cys-241, Cys-260, and Cys-263 to a zinc atom in RING1 and the coordination of Cys-418, Cys-421, Cys-436, and Cys-441 to another zinc atom in RING2 (Fig.  1B). Although our program failed to model a small stretch of parkin sequence containing Cys-289 and Cys-293 residues accurately, these residues, together with Cys-253 and His-257, should coordinate a second zinc atom in RING1 in view of their high sequence homology to c-Cbl RING1 (14) (Fig. 1B). Not surprisingly, all of these structurally important Cys residues are absolutely conserved in parkin across different species ( Fig. 1A and supplemental Fig. S1). On the other hand, Cys-268, which resides on a solvent-exposed surface (supplemental Fig. S2A) and does not appear to have a critical structural role in RING1 (Fig. 1B), is replaced by a leucine (Leu) in C. elegans parkin (supplemental Fig. S1) and by other amino acid residues in related proteins such as RBCK1 (isoleucine) and ariadne-2 (phenylalanine) (19). Similarly, the presumably nonstructural Cys-451 residue proximal to RING 2 ( Fig. 1B) is poorly conserved among parkin from different species (supplemental Fig. S1). Since the structure of parkin's UBL domain is known (20), we also inspected the structural position of Cys-59 and found that this Cys residue, like Cys-268, is located at a solvent-exposed loop on the surface of the UBL domain and not within its core (Fig. 1C), thereby offering some structural flexibility. Notably, in C. elegans parkin, Cys-59 is substituted with a Leu (supplemental Fig. S1). Taken together, the degree of Cys conservation in parkin across different species appears to correlate with their structural importance. Conceivably, modification of any of the numerous highly conserved Cys residues on parkin is likely to influence its structural topology, and thereby, its biochemical properties.
Conserved Cysteine Residues on Parkin Residing Both Within and Outside the RING-IBR-RING Domain Are Important in Maintaining Its Solubility-To examine whether the modification of parkin's Cys residues would influence its solubility, we generated a large series of parkin Cys 3 Ala (C 3 A) point mutants that cover the length of the protein via site-directed mutagenesis and expressed each of these mutants in SH-SY5Y neuroblastoma cells ( Fig. 2A). When cells transfected with these mutants were subjected to sequential detergent extraction, we found that all the C 3 A mutations occurring on Cys residues that are invariant in parkin across different species, except C431A, show preferential localization to the detergent-insoluble (P) fraction relative to control, wild-type parkin ( Fig. 2A). Conversely, Cys residues on parkin such as Cys-59, Cys-95, Cys-268, Cys-323, Cys-431, and Cys-451 that are either not absolutely conserved among different species or otherwise appear structurally unimportant, or both, do not significantly alter parkin solubility when mutated ( Fig. 2A). Consistent with our in silico sequence and structural prediction, our results suggest the importance of conserved parkin's Cys residues in maintaining the structure and thus solubility of the protein and that alteration of parkin solubility via the modification of its Cys residues is not limited to those residing within the RING-IBR-RING motif.
We have previously demonstrated an association between altered parkin solubility and its propensity to form intracellular aggregates (18). Since mutations of conserved and non-con-served Cys residues on parkin produce different effects on the protein's solubility, we were interested to know their respective influence in promoting parkin aggregation within the cell. For this purpose, representative pairs of mutants containing C 3 A substitutions of either an invariant or a non-invariant Cys at different regions of parkin were examined (Fig. 2B). Between the paired mutants, we found that aggresome-like structures occur more frequently in cells expressing the one substituting for the absolutely conserved Cys and vice versa (Fig. 2B). This is consistent with their respective solubility profile as described above ( Fig. 2A). Quantitatively, C 3 A mutations occurring on invariant Cys residues located on the RING-IBR-RING domain or the C-terminal tail of the protein show the highest propensity to generate intracellular inclusions when compared with wildtype parkin as well as mutants bearing similar mutations at the N-terminal region of parkin (Fig. 2C). It thus appears that Cys modification occurring on the RING-IBR-RING domain or C-terminal end of parkin result in more significant alterations of the protein (i.e. solubility changes and higher tendency to aggregate) when compared with analogous modification occurring at other regions of the protein.

Susceptibility of Parkin and Other Cysteine-containing
Enzymes to Stress-induced Solubility Alterations-Given the demonstrated importance of parkin's Cys residues in maintaining its structural and biochemical properties, one could appreciate the reported alteration of parkin function via modification of its Cys residues (11). Since parkin and HHARI contain a comparable number of Cys residues (35 and 32, respectively), and the majority of these Cys are found within their RING-IBR-RING domains, it is likely that they share comparable sensitivities to modification by various stress-inducing agents. Thus, we were intrigued by recent findings showing that parkin, but not HHARI, is selectively vulnerable to DA-mediated modification (11). To examine the relative susceptibility of parkin and other cysteine-containing enzymes to stress-induced solubility alterations, we subjected SH-SY5Y cells ectopically expressing parkin, HHARI, c-Cbl (RING domain ubiquitin-protein isopeptide ligase (E3) with 23 Cys residues), CHIP (U box protein with 7 Cys residues), or UCH-L1 (ubiquitin hydrolase with 6 Cys residues) to a variety of stresses, including hydrogen peroxide (H 2 O 2 ), NOC-18 (a nitrogen oxide donor), and DA, all of which have been previously reported to induce parkin insolu- bility (10,11). Consistent with their similar structure and abundance of Cys, we found that both parkin and HHARI are more or less equally susceptible to solubility alterations promoted by the treatment of cells with H 2 O 2 and NOC-18 (Fig. 3A). On the other hand, H 2 O 2 -mediated stress has no apparent effects on the solubility of c-Cbl, CHIP, and UCH-L1 (Fig. 3A). Although NOC-18 treatment of transfected cells at 0.25 mM markedly altered the solubility of parkin and HHARI, its effects on c-Cbl, CHIP, and UCH-L1 are more apparent only at a higher dose of 0.5 mM (Fig. 3A). For DA-mediated stress, we used a similar treatment paradigm to that reported by LaVoie et al. (11). Interestingly, we found that DA treatment of cells ectopically expressing parkin promotes a significant increase in the amount of monomeric and high molecular weight (HMW) parkin species in the detergent-insoluble fraction without a corresponding decrease in the levels of soluble parkin (Fig. 3B). Surprisingly, this phenomenon is specific to parkin as all the other Cys-containing enzymes examined, including HHARI, remain unaffected, suggesting that parkin is uniquely sensitive to DA-mediated modification (Fig. 3B). Although our findings on the effect of DA on parkin are consistent with that reported earlier (11), HHARI, despite sharing similar RING-IBR-RING structure and comparable sensitivity to parkin toward the other stress paradigms examined, is spared from DA-mediated effects. Nonetheless, when taken together, our results suggest that RING-IBR-RING proteins are more sensitive than other cysteine-containing enzymes to stress-induced modification but parkin is selectively vulnerable to DA-induced alterations.

DA Modifies Residues Residing on the RING-IBR-RING Motif as well as the Linker Region of Parkin-To
map the region on parkin that confers its unique susceptibility to DAinduced modification, we generated deletion mutants of FLAG-tagged parkin devoid either of its linker region (parkin ⌬77-237) or its RING-IBR-RING domain (parkin 1-237 and parkin 1-137) (Fig. 4A). We then treated cells expressing these various parkin deletion mutants with the same concentrations of DA as described above. Although DA treatment of cells expressing parkin 1-237 and parkin ⌬77-237 promotes an accumulation of detergent-insoluble parkin species in a manner similar to that observed with full-length parkin, parkin 1-137 is apparently spared from DA-mediated modification, even at the higher concentration of DA dose used (Fig. 4B). Thus, the region on parkin stretching from amino acids 138 -237 appears to be as susceptible to modification by DA as the RING-IBR-RING domain. Interestingly, this stretch of parkin sequence contains several Cys residues that are not found in HHARI. Given the apparent selective sensitivity of parkin to DA-mediated modification, it is tempting to suggest from our results that DA preferentially modifies Cys residues that are located either at the unique linker region and/or at the C-terminal portion of protein involving residues such as Cys-268, Cys-323, and Cys-451 that are also unique to parkin.

DA Preferentially Modifies Cys-268 and Cys-323 on Parkin-
Since DA modification of parkin promotes the formation of insoluble monomeric and HMW parkin species without causing a corresponding decrease in the levels of soluble parkin (Fig.  3B), it is reasonable to assume that DA targets either non-conserved Cys residues or those that are structurally less important. Although four of such Cys residues (Cys-268, Cys-323, Cys-431, and Cys-451) are present at the C-terminal region of parkin, only 1 (Cys-182) is found between amino acids 138 and 237. Accordingly, we repeated the above experiment with a compendium of parkin mutants containing C 3 A substitution at position Cys-182, Cys-268, Cys-323, Cys-431, or Cys-451. We also included an insoluble mutant, C441A, as a control. Surprisingly, we found that DA modifies the parkin C182A mutant in a similar fashion to that observed with the wild-type protein (Fig. 5A). Parkin C441A, found predominantly in the detergent-insoluble fraction, also appears to be modified by DA (Fig. 5A). On the other hand, two parkin mutants, C268A and C323A, residing on RING1 and IBR domain of parkin, respectively, are apparently significantly more resistant to DA-medi-ated modification when compared with their counterparts (Fig.  5A). Although DA treatment of cells expressing wild-type, C431A, or C451A parkin produce robust amounts of insoluble monomeric and HMW parkin species, the amounts of both these insoluble parkin species are dramatically reduced in the case of the C268A and C323A mutants (Fig. 5A). Notably, the levels of insoluble parkin C323A species generated by DA treatment are so modest that they compare well with untreated wild-type parkin control (Fig. 5A). We next examined the effects of DA on the cellular localization of wild-type, Cys-268, and Cys-323 parkin. Consistent with its susceptibility to DAmediated modification, we found that cells expressing wildtype parkin exhibit a high tendency to form parkin-positive inclusions following DA treatment. In contrast, such inclusions occur rarely in DA-treated cells expressing C268A and C323A parkin mutant (Fig. 5B). Taken together, our results demonstrate that mutation of Cys-268 and Cys-323, respectively, to alanine render parkin less susceptible to insolubility induced by DA treatment, suggesting that DA modification of parkin's Cys residues takes place predominantly at these Cys residues.
Distribution of Various Cysteine-containing Enzymes in Normal and PD Human Brains-We and others have previously demonstrated a significant increase in the amount of detergentinsoluble parkin in the caudate region of idiopathic PD brains when compared with those in control brains (10,11). To examine whether a similar phenomenon occurs with other cysteinecontaining enzymes, particularly HHARI, in PD and control brains, we performed anti-HHARI, anti-UCH-L1, anti-c-Cbl, and anti-CHIP immunoblotting on fractionated detergent-insoluble lysates prepared from post-mortem normal and PD brains (Fig. 6A). In contrast to parkin, as reported previously (10, 11), we did not observe an accumulation of any of the enzymes examined in the detergent-insoluble fraction of PD over control brain samples (Fig. 6B). Quantitatively, we only recorded a significant difference in the levels of CHIP between PD and control brains (Fig. 6B). However, the amount of CHIP decreased, instead of increased, in PD brains when compared with controls (Fig. 6B). Taken together, our results demonstrate that unlike parkin, HHARI, UCH-L1, c-Cbl, and CHIP do not accumulate in the detergent-insoluble fractions of PD brains, suggesting that parkin is uniquely sensitive to stress-induced modification in the caudate region of the brain.

DISCUSSION
In this report, we demonstrate that the modification of any of the numerous highly conserved Cys residues on parkin (except Cys-431), both within and outside its RING-IBR-RING motif, leads to alterations of the protein biochemical and cellular properties, and thereby, its function. As several of these structurally important Cys on parkin are not found in other related proteins, our findings suggest an increased vulnerability of parkin to stress-induced modification. Indeed, parkin appears to be more susceptible to oxidative and nitrosative stress and uniquely sensitive to DA-mediated stress. Given parkin's neuroprotective roles and the oxidative environment of dopaminergic neurons, the enhanced sensitivity of parkin to intracellular stress may underlie the progressive susceptibility of an individual to PD. Of the 20 naturally occurring amino acids found in proteins, cysteines are recognized to be exceptionally susceptible to oxidative modification due to the presence of sulfhydryl groups (21). Sulfhydryl groups are the strongest nucleophile in the cell at physiological pH and thus represent ideal targets for nucleophilic attack by oxidants or nitrosative agents. Accordingly, the abundance of Cys residues on a protein should, in part, contribute to the tendency for the protein to be modified by cellular oxidants. We found that this appears to be the case when we subjected enzymes with different cysteine content to the effects of oxidative/nitrosative stress agents. Although parkin and HHARI, both containing over 30 Cys residues, are comparably susceptible to H 2 O 2 -or NOC-18-mediated effects, c-Cbl, CHIP, and UCH-L1, containing 23, 7, and 6 Cys residues, respectively, either remain inert to these stress agents or otherwise require higher concentrations of these agents to produce a similar effect observed with parkin and HHARI.
The extent of a protein's alteration via its Cys modification is obviously also related to the importance of the targeted Cys residue to the overall tertiary structure of the protein. We have demonstrated that the large majority of Cys residues residing on parkin (23 out of 28 examined), both within and outside the RING-IBR-RING domain, are important in maintaining its solubility. With the notable exception of Cys-431, all the Cys residues of parkin found to be invariant across diverse species resulted in parkin insolubility when they are mutated to alanine, suggesting their importance in fulfilling critical structural roles. Although the Zn 2ϩ -coordinating Cys residues in RING1 and RING2 are obviously structurally important, it is interesting that almost all the Cys residues located at the IBR, a domain whose function remains unclear, appears to be critically important for the native folding of parkin. On the other hand, despite being an absolutely conserved residue, Cys-431 does not appear to play an important role in maintaining parkin's RING2 structure, which probably explains its lack of effects on parkin solubility when mutated. Nonetheless, our result with the C431A mutant in this study appears to contradict our previous finding that the C431F mutant promotes parkin insolubilty and intracellular aggregation (18). In the latter case, it is conceivable that the substitution of Cys with the bulky Phe could result in marked steric hindrance that might indirectly affect parkin's RING2 structure. Accordingly, we generated a homology model of parkin's RING2 structure with Phe substituting for Cys at position 431 and found that the benzene ring of Phe may interfere sterically with the RING structure (supplemental Fig. S2B). We speculated that substitution of Cys-431 with the equally bulky Tyr residue would bring about similar effects to that observed with the C431F mutant. Using both homology modeling and in vitro experiments similar to the ones described above, we were able to confirm our speculation with the C431Y mutant (supplemental Fig. S2, B and C). Thus, parkin solubility alteration mediated by C431F mutation is likely to arise from steric rather than overt structural aberrations.
Interestingly, all the invariant Cys residues residing within the parkin RING-IBR-RING motif are also conserved among members of the RING-IBR-RING family (19). This feature probably explains the comparable susceptibility of HHARI and parkin to H 2 O 2 or NOC-18-mediated solubility alterations, and at the same time, suggests the vulnerability of other RING-IBR-RING members to stress-induced modifications. The implication of this is that oxidative stress occurring in the brain, particularly in dopaminergic neurons, could promote the dysfunction of not just parkin but potentially all RING-IBR-RING family members as well. It is well known that dopaminergic neurons in the brain are particularly exposed to oxidative stress because the metabolism of DA produces various reactive oxygen species (peroxide, superoxide, and hydroxyl radicals) (21,22). If not handled properly, the reactive oxygen species generated could create a considerable damaging environment. Further, DA could auto-oxidize to DA-quinone, a reactive species that has been demonstrated to covalently modify cellular macromolecules, including parkin, and contribute to DA-induced neurotoxicity (11,21,22). However, unlike our previous observation with parkin (10), we did not observe an accumulation of HHARI in the detergent-insoluble fraction from the caudate region of PD brains relative to controls. It would appear that parkin is uniquely vulnerable in this region of the brain, at least when compared with HHARI, to PD-linked stress. Conceivably, the modification of parkin in this case must involve regions or residues of the protein that are distinct from HHARI. Interestingly, DA modification of parkin appears to fit this requirement nicely, as suggested by our following findings in this study. 1) DA-mediated promotion of insoluble parkin species is apparently a specific phenomenon as neither HHARI nor the other cysteine-containing enzymes examined are modified by DA in such a manner, even at the higher concentration of DA used. 2) DA could modify a truncated form of parkin bearing its unique linker region but devoid of the RING-IBR-RING motif in a similar manner to that observed with the wild-type protein. However, we failed to pinpoint the Cys residue involved in this DA-mediated effect and therefore cannot exclude alternative possibilities. 3) Importantly, DA appears to predominantly tar-get 2 Cys residues, Cys-268 and Cys-323, on RING1 and IBR domain, respectively, which are unique to parkin and thus not found in HHARI. It is noteworthy that C 3 A substitution of these 2 non-conserved Cys residues does not produce markedly insoluble proteins. This might explain why DA-mediated accumulation of detergent-insoluble parkin species occurs without apparent depletion of corresponding detergent-soluble parkin species, a milder effect when compared with the solubility alterations of parkin produced by H 2 O 2 or NOC-18 as described above or by various other stressors that we have previously reported (10). Consistent with this, both C268A and C323A fail to protect parkin against H 2 O 2 -induced solubility alterations (data not shown). Given the selective vulnerability of brain parkin to stress-induced modifications, as shown in this study, it is tempting to suggest that the principal culprit promoting brain parkin insolubility is likely DA, DA-related, or otherwise one that influences parkin in a similar manner to that brought about by DA. Whether and how detergent-insoluble monomeric and HMW parkin species influence soluble parkin function and cellular survivability remain to be characterized. However, their association with PD brains (10, 11) would suggest pathogenic roles.
In conclusion, our results here offer significant insights into the components important for parkin solubility, and at the same time, provide some structural basis for the solubility alterations of the protein produced by cellular stress. Further, we have mapped 2 Cys residues on parkin that render the protein less susceptible to insolubility induced by DA treatment, thus potentially representing the major target sites of DA-mediated modification. Notably, both these residues are unique to parkin and might therefore explain the selective vulnerability of parkin to DA-induced changes. Future experiments should clarify the potential pathogenecity of the insoluble parkin species promoted by DA modification.