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J. Biol. Chem., Vol. 277, Issue 32, 28572-28577, August 9, 2002

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Effect of Wild-type or Mutant Parkin on Oxidative Damage, Nitric Oxide, Antioxidant Defenses, and the Proteasome^*

Dong-Hoon Hyun, MoonHee Lee, Nobutaka Hattori^§, Shin-Ichiro Kubo^§, Yoshikuni Mizuno^§, Barry Halliwell^¶, and Peter Jenner

From the  Wolfson Centre for Age-related Diseases, GKT School of Biomedical Sciences, King's College London, London SE1 1UL, United Kingdom, the ^§ Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan 113-8421, and the ^¶ Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Singapore

Received for publication, January 22, 2002, and in revised form, May 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutations in Parkin (a ubiquitin protein ligase) are involved in autosomal recessive juvenile parkinsonism, but it is not known how they cause nigral cell death. We examined the effect of Parkin overexpression on cellular levels of oxidative damage, antioxidant defenses, nitric oxide production, and proteasomal enzyme activity. Increasing expression of Parkin by gene transfection in NT-2 and SK-N-MC cells led to increased proteasomal activity, decreased levels of protein carbonyls, 3-nitrotyrosine-containing proteins, and a trend to a reduction in ubiquitinated protein levels. Transfection of these cells with DNA encoding three mutant Parkins associated with autosomal recessive juvenile parkinsonism (Del 3-5, T240R, and Q311X) gave smaller increases in proteasomal activity and led to elevated levels of protein carbonyls and lipid peroxidation. Turnover of the mutant proteins was slower than that of the wild-type protein, and both could be blocked by the proteasome inhibitor, lactacystin. A rise in levels of nitrated proteins and increased levels of NO/NO was also observed in cells transfected with mutant Parkins, apparently because of increased levels of neuronal nitric-oxide synthase. The presence of mutant Parkin in substantia nigra in juvenile parkinsonism may increase oxidative stress and nitric oxide production, sensitizing cells to death induced by other insults.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Parkinson's disease (PD)¹ results from degeneration of dopaminergic neurones in the substantia nigra (1). Although most cases appear sporadic and of unknown cause, oxidative stress and apoptosis are associated with disease progression (1). Consistent with this view, increased levels of oxidative damage to DNA, proteins, and lipids and decreased levels of GSH are found in substantia nigra in PD (1-5).

Autosomal recessive juvenile parkinsonism (AR-JP), an early onset form of PD, is characterized by loss of tyrosine hydroxylase-immunoreactive neurones in substantia nigra pars compacta and locus ceruleus, usually without Lewy body formation (6). Various mutations (including deletion or point mutations) in the Parkin gene located on chromosome 6 (6q25.2-q27) have been found in AR-JP patients, but no clear correlations exist between types of Parkin mutations and clinical or pathologic features (7-14).

Parkin has been identified as a ubiquitin-protein ligase containing 465 amino acids, which consists of a ubiquitin-like (UBL) domain in the N terminus, two ring-finger motifs (termed RING1 and RING2) flanking a Cys-rich domain, named as the in-between RING (IBR) (9, 14). An additional segment is a linker region that connects two regions of UBL and RING1-IBR-RING2 (named as the RING box). Deletional analysis of Parkin revealed that UBL and the linker region are not necessary for association with a specific E2 enzyme. In contrast, the full region of the RING box is necessary for non-covalent association with E2. Therefore, missense mutations in the RING box of Parkin in AR-JP patients have almost completely lost the specific E2-binding activity.² Thus, mutations in the RING finger motifs such as T240R and T415N could cause a loss of ubiquitin-protein isopeptide ligase activity due to no recruiting of E2 (15). In our previous study (16), the N-terminal UBL domain is required for recognition of target protein(s) for ubiquitination and thus may function as a substrate-binding module. Possible Parkin substrates including O-glycosylated -synuclein (16), insoluble Pael receptor (17), CDCrel-1, a synaptic vesicle-associated protein (18), and synphilin-1, an -synuclein-interacting protein (19), have been reported.

The ubiquitin/proteasome system plays an important role in cellular function, e.g. it degrades oxidatively damaged, nitrated, and ubiquitinated proteins (20-23). Indeed, decreases in proteasomal enzyme activity as measured by trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolase (PGPH) activities are found in substantia nigra in PD (24). Although the mechanisms by which mutations in Parkin induce cell death in AR-JP are far from clear, a rise in levels of abnormal proteins due to proteasomal dysfunction may induce oxidative stress, apoptosis, and formation of protein aggregates that might be cytotoxic (25-28).

In the present study, we investigated how overexpression of wild-type and mutant Parkin proteins (Del 3-5, T240R, and Q311X) modulates proteasomal activity, the accumulation of ubiquitinated proteins, indices of oxidative stress, nitric oxide production, and antioxidant defenses. We chose two very different cell types, a human neuroblastoma cell line, SK-N-MC cells (29), and a human teratocarcinoma cell line, NT-2 cells, with cholinergic characteristics (30), to examine whether the effects could be reproduced in different cell lines.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- NT-2, SK-N-MC and their Parkin transfectants were maintained in 100-mm tissue culture plates (Greiner, Frickenhausen, Germany) containing high glucose-Dulbecco's modified Eagle's medium (Invitrogen), 1 mM sodium pyruvate (Sigma), 10% fetal bovine serum (Invitrogen), 100 µg/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) under humidified 5% CO₂ and 95% air.

DNA Transfection-- pcDNA3.1(+)-myc-neo, pcDNA3.1(+)-myc-wild-type parkin, pcDNA3.1(+)-myc-Del 3-4 mutant parkin, pcDNA3.1(+)-myc-Del 3-5 mutant parkin, pcDNA3.1(+)-myc-Del 5 mutant parkin, pcDNA3.1(+)-myc-R42P mutant parkin, pcDNA3.1(+)-myc-K161N mutant parkin, pcDNA3.1(+)-myc-K211N mutant parkin, pcDNA3.1(+)-myc-T240R mutant parkin, pcDNA3.1(+)-myc-R275W mutant parkin, pcDNA3.1(+)-myc-C289G mutant parkin, pcDNA3.1(+)-myc-Q311X mutant parkin, pcDNA3.1(+)-myc-R334C mutant parkin, pcDNA3.1 (+)-myc-T415N mutant parkin, pcDNA3.1(+)-myc-G430D mutant parkin and pcDNA3.1(+)-myc-W453X mutant parkin were used. Transfection of these cDNAs into NT-2 and SK-N-MC cell lines was performed as described by Lee et al. (31, 32). Transfectants were selected with 400 µg/ml G418 (a neomycin analogue, Invitrogen).

Twenty clones from each transfectant were selected as stably expressing wild-type or mutant Parkin (Del 3-5, T240R, and Q311X) in both cell lines. After control experiments measuring growth rate and protein expression, 10 clones were chosen, which showed consistent traits as follows: 1) stable protein expression and 2) similar growth rates. For viability tests, four clones were used. For parameters of oxidative stress (levels of GSH and oxidative damage), levels of ubiquitinated proteins, proteasome activity, and enzyme activity, six clones were used.

Western Blotting-- Cells were lysed with a buffer containing 5 µg/ml aprotinin (Sigma), 5 µg/ml leupeptin (Sigma), 5 µg/ml pepstastin A (Sigma), and 10% SDS (Sigma) and placed on ice for 5 min. The lysates were centrifuged (12,000 × g, 10 min), and the supernatants were transferred into new Eppendorf tubes. Protein levels were measured (33), and a total of 50 µg of protein was then electrophoresed on 8% SDS-polyacrylamide gels for 1.5 h (100 V). Separated proteins were transferred to nitrocellulose membranes (Bio-Rad) at 20 V overnight. The membranes were incubated with anti-Myc monoclonal antibody (1:500 dilution, Invitrogen, Groningen, the Netherlands) for 2 h and then washed with phosphate-buffered saline (pH 7.4). The membranes were incubated with alkaline phosphatase-conjugated anti-IgG antibody (1:500 dilution, Vector Laboratories, Burlingame, CA) for 1 h. A color reaction was performed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma). The blotting membranes were washed with phosphate-buffered saline to stop the color reaction and then were dried for determining the amount of expressed protein in an image analyzer (Imaging System, St. Catherine, Ontario, Canada).

Parkin Enzyme Activity-- The ubiquitin-protein ligase activity was assessed by measuring formation of ubiquitinated protein. Cell extracts (after lysis using 0.5% Nonidet P-40) were incubated in 50 mM potassium phosphate buffer (pH 7.4) containing Enzyme E1 (Affiniti Research Products, Exeter, UK), UbcH7 (3 µg, Affiniti Research Products), and free ubiquitin (1 µg, Sigma). The reaction mixtures were incubated at 37 °C for 1 h. The levels of ubiquitinated proteins were measured as described below.

Cell Viability Assays-- Viability assays (trypan blue exclusion test and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide test) were performed as described by Kelner et al. (34).

Turnover Rate of Overexpressed Parkins-- Cells were cultured in the absence or presence of 1 µM lactacystin for an appropriate time. Cycloheximide (0.5 µM) was also added to prevent further protein synthesis. Then cells were lysed for Western blotting.

Measurement of Antioxidant Enzyme Activity-- Activities of Cu/Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase, glutathione peroxidase, and glutathione reductase were measured as described by Lee et al. (31).

Measurement of GSH Level-- GSH level was assessed as described by Hissin and Hilf (35) with 10⁵ cells. This assay detects GSH by its reaction with o-phthalaldehyde at pH 8. A standard curve was made using commercial GSH (Sigma).

Measurement of Oxidative Damage-- DNA extraction and assessment of purity were carried out as described by Lyras et al. (36). DNA hydrolysis with formic acid and separation of modified 8-hydroxyguanine by HPLC were performed as described by Kaur and Halliwell (37).

Protein carbonyl content was determined by method A of Lyras et al. (38), except that the final protein pellets were dissolved in 1 ml of 6 M guanidinium hydrochloride. Carbonyl content was calculated as nmol/mg protein (39).

Measurement of protein-bound 3-nitrotyrosine content was carried out as described by Khan et al. (40). Peroxynitrite (ONOO) was prepared in a quenched flow reaction system (41). Nitrated bovine serum albumin was prepared as described by Whiteman and Halliwell (42). The conjugation of horseradish peroxidase with anti-3-nitrotyrosine antibody was performed with the periodate method as described by Harlow and Lane (43).

For measuring lipid peroxidation, thiobarbituric acid-reactive material was measured by HPLC as described by Lyras et al. (44), employing 3 × 10⁶ cells.

Levels of NO/NO and Expression of Nitric-oxide Synthase-- Levels of NO/NO and expression of nitric-oxide synthase were measured as described by Lee et al. (31). The levels of NO/NO in medium alone (including fetal bovine serum) were subtracted from the levels of NO/NO in cells plus medium. The levels of NO/NO in cells plus medium were 20-25 µM and in medium (plus fetal bovine serum) alone 10-12 µM.

Assessment of Proteasomal Activity-- Three different proteasome activities were measured as described by Canu et al. (45), using fluorogenic substrates, Suc-LLVY-MCA (50 µM, Sigma), Boc-LRR-MCA (100 µM, Bachem, Merseyside, UK). or Z-LLE-Nap (400 µM, Sigma). Hydrolysis of these substrates was independent of the ubiquitin system. Standard curves were made with 20 S proteasome (Calbiochem) and trypsin (Sigma).

Ubiquitinated Proteins-- Protein ubiquitination was assessed by dot blotting with alkaline phosphatase-conjugated anti-ubiquitin antibody (Santa Cruz Biotechnology), which recognize free ubiquitin and mono-ubiquitinated proteins, as described by Lee et al. (31). After cell lysis, the lysates were filtered to remove free ubiquitin (Centricon, molecular weight cut-off 10,000, Millipore, Bedford, MA). The conjugation of alkaline phosphatase with anti-ubiquitin antibody was performed with the periodate method as described by Harlow and Lane (43). Data were analyzed by densitometry (Imaging System, St. Catherine, Ontario, Canada).

Data Analysis-- Statistical differences were analyzed by one- and two-way ANOVA tests. Trypan blue exclusion and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide data were analyzed by a two-way ANOVA test. Multiple comparisons were performed by the post hoc Bonferroni t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Overexpression of wild-type or mutant Parkin did not significantly affect the viability of either NT-2 or SK-N-MC cell lines compared with nontransfected cells and vector-only transfectants under normal culture conditions over the time period of our experiments.

Overexpression and Activity of Wild-Type and Mutant Parkins-- Expression of wild-type and mutant Parkins in both NT-2 and SK-N-MC cell lines was examined using Western blotting (Fig. 1A). Wild-type and the three mutant Parkin proteins were stably expressed. The ubiquitin-protein ligase activity of Parkin proteins was also assessed. Both NT-2 and SK-N-MC cell lines contained basal levels of the ubiquitin-protein ligase activity (Fig. 1B). Overexpression of wild-type and Del 3-5 mutant Parkins elevated the enzymatic activity 2.5-fold compared with non- or vector-only transfectants. However, the enzyme activity in T240R or Q311X mutant Parkin transfectants was not significantly increased despite increased protein expression (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Overexpression, enzyme activity, and turnover of both wild-type and mutant Parkin proteins in NT-2 and SK-N-MC cell lines. A, expression. When cells reached 80% confluence in the presence of 400 µg/ml G418, proteins were extracted for Western blotting. Lane 1, NT-2/vector-only; lane 2, NT-2/wild-type parkin; lane 3, NT-2/Del 3-5 mutant parkin; lane 4, NT-2/T240R mutant parkin; lane 5, NT-2/Q311X mutant parkin; lane 6, SK-N-MC/vector-only; lane 7, SK-N-MC/wild-type parkin; lane 8, SK-N-MC/Del 3-5 mutant parkin; lane 9, SK-N-MC/T240R mutant parkin; and lane 10, SK-N-MC/Q311X mutant parkin. B, enzyme activity. When cells reached 80% confluence in the presence of 400 µg/ml G418, they were extracted to measure enzyme activities. A, NT-2 (nontransfectant); B, NT-2/vector-only; C, NT-2/wild-type parkin; D, NT-2/Del 3-5 mutant parkin; E, NT-2/T240R mutant parkin; F, NT-2/Q311X mutant parkin; G, SK-N-MC (nontransfectant); H, SK-N-MC/vector-only; I, SK-N-MC/wild-type parkin; J, SK-N-MC/Del 3-5 mutant parkin; K, SK-N-MC/T240R mutant parkin; and L, SK-N-MC/Q311X mutant parkin. Values are the means ± S.E., n = 6. One-way ANOVA was carried out to test significance. *, p < 0.01, significant difference compared with non- or vector-only transfectants under normal conditions. C and D, turnover. Cells were cultured using culture medium containing 0.5 µM cycloheximide and 1 µM lactacystin for an appropriate time and lysed for Western blot analysis. , WT (no lactacystin); , WT (lactacystin-treated);, Del 3-5 (no lactacystin); , Del 3-5 (lactacystin-treated); , T240R (no lactacystin); , T240R (lactacystin-treated); , Q311X (no lactacystin); and , Q311X (lactacystin-treated).

Turnover of Overexpressed Wild-type and Mutant Parkins-- The turnover rate of Parkins was examined. The half-life of wild-type Parkin was 7.31 ± 0.54 h, and the turnover could be almost completely blocked by 1 µM lactacystin (Fig. 1C). The mutant proteins turned over less rapidly (half-life 13.78 ± 1.17 h), and this was again blocked by lactacystin.

Antioxidant Defenses and Oxidative Damage to DNA, Proteins, and Lipids-- Expression of wild-type or mutant Parkins did not affect activities of antioxidant enzymes, namely Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase, glutathione peroxidase, and glutathione reductase (Table I). However, levels of GSH in cells expressing mutant Parkins measured at 5 days were lowered (p < 0.01). Levels of 8-hydroxyguanine as a biomarker of oxidative damage to DNA (46) were not significantly different in nontransfectants and Parkin transfectants, whether with the mutant or wild-type enzyme (Table II), although there was a trend for a rise with mutant Parkins in both cell types. Protein carbonyl levels were significantly lowered in both cell lines expressing wild-type Parkin (p < 0.01). However, transfection with mutant Parkins produced a significant rise in carbonyls in both cell types (p < 0.01). Lipid peroxidation was measured as the formation of thiobarbituric acid-reactive material by using an HPLC-based assay to remove interfering chromogens (47). Levels of lipid peroxidation were higher in cells expressing mutant Parkins (p < 0.01), but there was no significant difference between control cells and wild-type Parkin transfectants. The possible role of peroxynitrite (ONOO) and/or other reactive nitrogen species was assessed by measuring the level of protein-bound 3-nitrotyrosine, a biomarker of attack upon proteins by such species (48, 49). Expression of mutant Parkins in both cell lines elevated the levels of protein-bound 3-nitrotyrosine (p < 0.01), but levels were lower in cells expressing wild-type Parkin proteins (p < 0.01).

Levels of NO/NO and Expression of Nitric-oxide Synthase-- Levels of NO/NO and expression of nitric-oxide synthase (iNOS and nNOS) were also assessed. Levels of NO/NO were increased in cells transfected with mutant Parkins at day 5 (p < 0.01, Fig. 2A) but not wild-type Parkin. There was also an increase in nNOS expression (1.4 ± 0.2-fold, n = 4, a representative blot is shown in Fig. 2, C and D). iNOS was not detected before or after transfection of wild-type or mutant Parkins (data not shown), although it could be detected in a positive control for iNOS (LPS-treated rat astrocytes). Antisense DNA against the 5'-end of nNOS mRNA (5'-ATT GGG CTG GAT TTG CTG AAC ACC GAA CAT GTG ATC CTC CAT-3') decreased nNOS expression, as expected (Fig. 2C).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Levels of reactive nitrogen species and expression of nNOS. When cells were grown to 80% confluence in 96-well plates, they were extracted. A, NO and B, NO. A, NT-2 (nontransfectant); B, NT-2/vector-only; C, NT-2/wild-type parkin; D, NT-2/Del 3-5 mutant parkin; E, NT-2/T240R mutant parkin: F, NT-2/Q311X mutant parkin; G, SK-N-MC (nontransfectant); H, SK-N-MC/vector-only; I, SK-N-MC/wild-type parkin; J, SK-N-MC/Del 3-5 mutant parkin; K, SK-N-MC/T240R mutant parkin; L, SK-N-MC/Q311X mutant parkin. Values are the means ± S.E., n = 6. Significance was examined by one-way ANOVA. *, p < 0.01 compared with the same cells in normal condition. C, expression of nNOS. 50 µg of protein was loaded in each lane. Lane 1, control NT-2 cell line; lane 2, antisense DNA-treated NT-2 cell line; lane 3, NT-2/vector-only; lane 4, NT-2/wild-type parkin; lane 5, NT-2/Del 3-5 mutant parkin; lane 6, NT-2/T240R mutant parkin; lane 7, NT-2/Q311X mutant parkin; lane 8, SK-N-MC/vector-only; lane 9, SK-N-MC/wild-type parkin; lane 10, SK-N-MC/Del 3-5 mutant parkin; lane 11, SK-N-MC/T240R mutant parkin; lane 12, SK-N-MC/Q311X mutant parkin. Three independent experiments were carried out. Significance was examined by one-way ANOVA. *, p < 0.01 compared with the same cells in normal condition.

Proteasomal Activity and Levels of Ubiquitinated Proteins-- Proteasomal activities, namely trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolase (PGPH) were measured by using fluorogenic substrates, whose hydrolysis is independent of ubiquitin. The activities were higher in both wild-type and (to a lesser extent) in mutant Parkin transfectants at day 5 (p < 0.01, Fig. 3, A-C). Levels of ubiquitinated proteins tended to be higher in mutant Parkin transfectants and lower in cells transfected with wild-type Parkin, but neither change was significant (Fig. 3D).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Proteasome activity and levels of ubiquitinated proteins. When cells were grown to 80% confluence in 100-mm tissue culture plates, they were extracted. A, NT-2 (nontransfectant); B, NT-2/vector-only; C, NT-2/wild-type parkin; D, NT-2/Del 3-5 mutant parkin; E, NT-2/T240R mutant parkin; F, NT-2/Q311X mutant parkin; G, SK-N-MC (nontransfectant); H, SK-N-MC/vector-only; I, SK-N-MC/wild-type parkin; J, SK-N-MC/Del 3-5 mutant parkin; K, SK-N-MC/T240R mutant parkin; L, SK-N-MC/Q311X mutant parkin. A, trypsin-like activity; B, chymotrypsin-like activity; C, PGPH activity. Values are the means ± S.E., n = 6. One-way ANOVA was carried out to test significance. *, p < 0.01 and **, p < 0.05; significant difference compared with non- or vector-only transfectants. D, levels of ubiquitinated proteins. Values are the means ± S.E., n = 6.

Effect of Expression of Other Parkin Mutations-- Overexpression of other Parkin mutations such as Del 3-4, Del 5, R42P, K161N, K211N, R275W, C289G, G328E, R334C, T415N, G430D, and W453X was also carried out in both cell lines. The effects of these mutations on oxidative and nitrative damage and antioxidant defenses were similar to those for Del 3-5, T240R, and Q311X (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There is considerable evidence that oxidative stress is involved in the progression of sporadic PD (1, 4, 5, 50). The formation of reactive nitrogen species such as ONOO has also been implicated (51). Growing evidence indicates that the malfunction of the ubiquitin/proteasome system is closely associated with development of a range of neurodegenerative diseases including PD (26, 28). Recently, deletion or point mutations in a member of the ubiquitin protein ligase family (Parkin) were found to be associated with young onset PD, where nigral pathology is not usually associated with Lewy body formation, in several families in Japan, Germany, Turkey, and Yemen (7-14). Although the mechanism by which mutations in Parkin cause nigral degeneration is not clear, accumulation of abnormal proteins (ubiquitinated or non-ubiquitinated) may directly cause cell death (25, 27) and/or may render cells sensitive to death caused by mechanisms such as oxidative stress (18, 52). Thus, it is important to determine to what extent Parkin mutations affect antioxidant defenses and oxidative damage. We examined in detail these different mutations, one that has ubiquitin protein ligase activity and two of that do not. The enzyme activity of Del 3-5 mutant Parkin was as high as wild-type Parkin, but T240R or Q311X mutant Parkin had no ligase activity. Shimura et al. (14) suggested that the RING box is responsible for the enzyme activity due to interaction with the specific E2 enzymes, and the latter two mutations are located in this motif. On the other hand, Del 3-5 mutant Parkin has normal enzymatic activity. However, the patients with Del 3-5 have the same clinical phenotypes as other mutations. Although the UBL could be essential for the trapping the specific substrate(s), Del 3-5 includes a part of UBL that consists of exons 1-2 and a part of exon 3. Thus, this mutant has the potential to trap the substrate(s) but may not bind the specific substrate(s) needed to prevent development of AR-JP. Alternatively, the localization of Del 3-5 may not be changed compared with the wild-type Parkin. Indeed, all the fractionation of Del 3-5 revealed the soluble fraction in contrast to the wild-type Parkin (54). It should be noted that none of the mutants significantly increased the levels of ubiquitinated proteins in either cell type studied by us.

Overexpression of mutant Parkins (Del 3-5, T240R, and Q311X) did not alter antioxidant enzyme activities, namely SOD1, SOD2, catalase, glutathione peroxidase, and glutathione reductase. However, Parkin mutations increased oxidative stress as indicated by decreased levels of GSH and elevated levels of oxidative damage to proteins and lipids. This was independent of whether or not the mutant had enzyme activity; mutants with or without enzyme activity imposed oxidative stress to comparable extents. Levels of 3-nitrotyrosine, a biomarker of attack by ONOO and other reactive nitrogen species upon protein-bound tyrosine residues (47, 48), were also significantly increased in the mutant Parkin transfectants, again independent of Parkin enzyme activity. We hypothesized that overexpression of these Parkin mutants might affect proteasomal function, since we recently found that proteasomal inhibition increases oxidative damage and protein nitration in these cell types (55). Carbonylated and nitrated proteins are degraded by the proteasome (55-57). The mutant proteins could elevate levels of NO/NO, for which increased expression of nNOS (but not iNOS) was presumably responsible. Again, such effects have been observed in cells treated with proteasomal inhibitors (55). However, the mutant proteins did not decrease proteasomal hydrolytic activities; rather they were increased although the increase with wild-type Parkin was greater than for the mutants (as measured using substrates whose hydrolysis is not ubiquitin-dependent). This increased activity of proteasomal enzymes could explain why levels of protein carbonyls fell in cells transfected with wild-type Parkin, as carbonylated proteins are degraded by this system (21, 23). It may that the mutant proteins competitively inhibit or reversibly overload the proteasome, an effect that would not be seen in assays on cell extracts. This would explain the rise in protein carbonyls and nitrated proteins and the trend to a rise in ubiquitinated protein levels. Indeed, the effect of lactacystin (Fig. 1C) shows that the proteasome is responsible for turnover of both wild-type and mutant proteins. The slower turnover of all these mutants is consistent with the idea that they might be poor substrates for the proteasome, perhaps causing reversible inhibition. Increased levels of protein carbonyls in the mutant transfectants could be related to impaired proteasomal function as well as to the observed rise in lipid peroxidation, because protein carbonyls can arise from direct oxidative damage to certain amino acid residues and/or by the binding of certain aldehyde end products of lipid peroxidation to proteins (22, 58). Indeed, we reported marked increase in iron accumulation in the substantia nigra of AR-JP brains, indicating that oxidative stress appears to play an important role on nigral cell death in AR-JP (59).

Overall, our data support the growing view that proteasomal dysfunction may be a significant contributor to neuronal cell death in the major neurodegenerative diseases (18, 28, 52, 53, 55). Our data shown that abnormal proteins such as mutated Parkins impact on the ubiquitin-proteasome system, leading to oxidative stress and excess NO production. This itself is not sufficient to kill the cells, at least in short term culture, but it may render them very sensitive to other insults. The mechanism of up-regulation of NO production and proteasomal activity is the subject of investigation in our laboratory and may involve mitogen-activated protein kinase signaling.

	FOOTNOTES

^* This work was supported by the Parkinson's Disease Society (UK), the National Parkinson Foundation (United States), and the National Medical Research Council, Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Wolfson Centre for Age-related Diseases, GKT School of Biomedical Sciences, King's College, London SE1 1UL, UK. Tel.: 44-20-7848-6011; Fax: 44-20-7848-6034; E-mail: div.pharm@kcl.ac.uk.

Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M200666200

² N. Hattori, S.-I. Kubo, and Y. Mizuno, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PD, Parkinson's disease; AR-JP, autosomal recessive juvenile parkinsonism; SOD1, Cu,Zn-superoxide dismutase; SOD2, Mn-superoxide dismutase; T-L, trypsin-like; ChT-L, chymotrypsin-like; PGPH, peptidylglutamyl peptide hydrolase; HPLC, high pressure liquid chromatography; ANOVA, analysis of variance; iNOS, inducible nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; E2, ubiquitin carrier protein; UBL, ubiquitin-like.

	REFERENCES

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				D. Versari, J. Herrmann, M. Gossl, D. Mannheim, K. Sattler, F. B. Meyer, L. O. Lerman, and A. Lerman Dysregulation of the Ubiquitin-Proteasome System in Human Carotid Atherosclerosis Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2132 - 2139. [Abstract] [Full Text] [PDF]

				Y. Yang, S. Gehrke, Y. Imai, Z. Huang, Y. Ouyang, J.-W. Wang, L. Yang, M. F. Beal, H. Vogel, and B. Lu Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin PNAS, July 11, 2006; 103(28): 10793 - 10798. [Abstract] [Full Text] [PDF]

				W. W. Smith, H. Jiang, Z. Pei, Y. Tanaka, H. Morita, A. Sawa, V. L. Dawson, T. M. Dawson, and C. A. Ross Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity Hum. Mol. Genet., December 15, 2005; 14(24): 3801 - 3811. [Abstract] [Full Text] [PDF]

				W. Springer, T. Hoppe, E. Schmidt, and R. Baumeister A Caenorhabditis elegans Parkin mutant with altered solubility couples {alpha}-synuclein aggregation to proteotoxic stress Hum. Mol. Genet., November 15, 2005; 14(22): 3407 - 3423. [Abstract] [Full Text] [PDF]

				C. Cecchi, S. Baglioni, C. Fiorillo, A. Pensalfini, G. Liguri, D. Nosi, S. Rigacci, M. Bucciantini, and M. Stefani Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates J. Cell Sci., August 1, 2005; 118(15): 3459 - 3470. [Abstract] [Full Text] [PDF]

				A. J. Whitworth, D. A. Theodore, J. C. Greene, H. Benes, P. D. Wes, and L. J. Pallanck Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease PNAS, May 31, 2005; 102(22): 8024 - 8029. [Abstract] [Full Text] [PDF]

				A.C. Williams, L.S. Cartwright, and D.B. Ramsden Parkinson's disease: the first common neurological disease due to auto-intoxication? QJM, March 1, 2005; 98(3): 215 - 226. [Abstract] [Full Text] [PDF]