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• W2169363048 abstract "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 NO2−/NO3− 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. 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 NO2−/NO3− 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. Parkinson's disease autosomal recessive juvenile parkinsonism Cu,Zn-superoxide dismutase Mn-superoxide dismutase trypsin-like chymotrypsin-like peptidylglutamyl peptide hydrolase high pressure liquid chromatography analysis of variance inducible nitric-oxide synthase neuronal nitric-oxide synthase ubiquitin carrier protein ubiquitin-like Parkinson's disease (PD)1 results from degeneration of dopaminergic neurones in the substantia nigra (1Jenner P. Olanow C.W. Ann. Neurol. 1998; 44: S72-S84Crossref PubMed Scopus (633) Google Scholar). Although most cases appear sporadic and of unknown cause, oxidative stress and apoptosis are associated with disease progression (1Jenner P. Olanow C.W. Ann. Neurol. 1998; 44: S72-S84Crossref PubMed Scopus (633) Google Scholar). 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 (1Jenner P. Olanow C.W. Ann. Neurol. 1998; 44: S72-S84Crossref PubMed Scopus (633) Google Scholar, 2Dexter D.T. Carter C.J. Wells F.R. Javoy-Agid F. Agid Y. Lees A. Jenner P. Marsden C.D. J. Neurochem. 1989; 52: 381-389Crossref PubMed Scopus (1236) Google Scholar, 3Sian J. Dexter D.T. Lees A.J. Daniel S. Agid Y. Javoy-Agid F. Jenner P. Marsden C.D. Ann. Neurol. 1994; 36: 56-66Google Scholar, 4Alam Z.I. Daniel S.E. Lees A.J. Marsden D.C. Jenner P. Halliwell B. J. Neurochem. 1997; 69: 1326-1329Crossref PubMed Scopus (484) Google Scholar, 5Alam Z.I. Jenner A. Daniel S.E. Lees A.J. Cairns N. Marsden C.D. Jenner P. Halliwell B. J. Neurochem. 1997; 69: 1196-1203Crossref PubMed Scopus (713) Google Scholar). 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 (6Ishikawa A. Takahashi H. J. Neurol. 1998; 245 Suppl. 3: P4-P9Crossref Google Scholar). 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 (7Hattori N. Kitada T. Matsumine H. Asakawa S. Yamamura Y. Yoshino H. Kobayashi T. Yokochi M. Wang M. Yoritaka A. Kondo T. Kuzuhara S. Nakamura S. Shimizu N. Mizuno Y. Ann. Neurol. 1998; 44: 935-941Crossref PubMed Scopus (290) Google Scholar, 8Hattori N. Matsumine H. Asakawa S. Kitada T. Yoshino H. Elibol B. Brookes A.J. Yamamura Y. Kobayashi T. Wang M. Yoritaka A. Minoshima S. Shimizu N. Mizuno Y. Biochem. Biophys. Res. Commun. 1998; 249: 754-758Crossref PubMed Scopus (182) Google Scholar, 9Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4226) Google Scholar, 10Nisipeanu P. Inzelberg R. Blumen S.C. Carasso R.L. Hattori N. Matsumine H. Mizuno Y. Neurology. 1999; 53: 1602-1604Crossref PubMed Google Scholar, 11Kruger R. Vieira-Sacker A.M. Kuhn W. Muller T. Woitalla D. Schols L. Przuntek H. Epplen J.T. Riess O. J. Neural Transm. 1999; 106: 159-163Crossref PubMed Scopus (15) Google Scholar, 12Klein C. Pramstaller P.P. Kis B. Page C.C. Kann M. Leung J. Woodward H. Castellan C.C. Scherer M. Vieregge P. Breakefield X.O. Kramer P.L. Ozelius L.J. Ann. Neurol. 2000; 48: 65-71Crossref PubMed Scopus (196) Google Scholar, 13Klein C. Schumacher K. Jacobs H. Hagenah J. Kis B. Garrels J. Schwinger E. Ozelius L. Pramstaller P. Vieregge P. Kramer P.L. Ann. Neurol. 2000; 48: 126-127Crossref PubMed Google Scholar, 14Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1710) Google Scholar). 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) (9Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4226) Google Scholar, 14Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1710) Google Scholar). 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. 2N. Hattori, S.-I. Kubo, and Y. Mizuno, unpublished data. 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 (15Zhang Y. Gao J. Chung K.K. Huang H. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13359Crossref PubMed Scopus (841) Google Scholar). In our previous study (16Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 263-269Crossref PubMed Scopus (953) Google Scholar), 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 α-synuclein (16Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 263-269Crossref PubMed Scopus (953) Google Scholar), insoluble Pael receptor (17Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar), CDCrel-1, a synaptic vesicle-associated protein (18Mizuno Y. Hattori N. Mori H. Suzuki T. Tanaka K. Curr. Opin. Neurol. 2001; 14: 477-482Crossref PubMed Scopus (93) Google Scholar), and synphilin-1, an α-synuclein-interacting protein (19Chung K.K.K. Zhang Y. Lim K.L. Tanaka Y. Huang H. Gao J. Ross C.A. Dawson V.L. Dawson T.M. Nat. Med. 2001; 7: 1144-1150Crossref PubMed Scopus (666) Google Scholar), have been reported. The ubiquitin/proteasome system plays an important role in cellular function, e.g. it degrades oxidatively damaged, nitrated, and ubiquitinated proteins (20Grune T. Blasig I.E. Sitte N. Roloff B. Haseloff R. Davies K.J. J. Biol. Chem. 1998; 273: 10857-10862Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 21Stadtman E.R. Berlett B.S. Drug Metab. Rev. 1998; 30: 225-243Crossref PubMed Scopus (477) Google Scholar, 22Halliwell B. Drugs Aging. 2001; 18: 685-716Crossref PubMed Scopus (1248) Google Scholar, 23Sitte N. Merker K. Von Zglinicki T. Grune T. Free Radic. Biol. Med. 2000; 28: 701-708Crossref PubMed Scopus (134) Google Scholar). 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 (24McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (555) Google Scholar). 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 (25Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1831) Google Scholar, 26Mezey E. Dehejia A. Harta G. Papp M.I. Polymeropoulos M.H. Brownstein M.J. Nat. Med. 1998; 4: 755-759Crossref PubMed Scopus (178) Google Scholar, 27Jesenberger V. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 112-121Crossref PubMed Scopus (310) Google Scholar, 28McNaught K.S. Olanow C.W. Halliwell B. Isacson O. Jenner P. Nat. Rev. Neurosci. 2001; 2: 589-594Crossref PubMed Scopus (455) Google Scholar). 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 (29Biedler J.L. Roffler-Tarlov S. Schner M. Freeman L.S. Cancer Res. 1978; 38: 3751-3757PubMed Google Scholar), and a human teratocarcinoma cell line, NT-2 cells, with cholinergic characteristics (30Angulo A. Suto C. Ghazal P. J. Virol. 1995; 69: 3831-3837Crossref PubMed Google Scholar), to examine whether the effects could be reproduced in different cell lines. 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% CO2and 95% air. pcDNA3.1(+)-myc-neo, pcDNA3.1(+)-myc-wild-type parkin, pcDNA3.1(+)-myc-Del 3–4 mutant parkin, pcDNA3.1(+)-myc-Del 3–5 mutantparkin, pcDNA3.1(+)-myc-Del 5 mutant parkin, pcDNA3.1(+)-myc-R42P mutant parkin, pcDNA3.1(+)-myc-K161N mutant parkin, pcDNA3.1(+)-myc-K211N mutantparkin, pcDNA3.1(+)-myc-T240R mutant parkin, pcDNA3.1(+)-myc-R275W mutant parkin, pcDNA3.1(+)-myc-C289G mutant parkin, pcDNA3.1(+)-myc-Q311X mutantparkin, 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. (31Lee M. Hyun D.-H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (72) Google Scholar, 32Lee M. Hyun D.-H. Jenner P. Halliwell B. J. Neurochem. 2001; 76: 957-965Crossref PubMed Scopus (70) Google Scholar). 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. 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 (33Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 269-275Abstract Full Text PDF Google Scholar), 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). 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. 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. (34Kelner G.S. Lee M. Clark M.E. Maciejewski D. McGrath D. Rabizadeh S. Lyons T. Bredesen D. Jenner P. Maki R.A. J. Biol. Chem. 2000; 275: 580-584Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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. 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. (31Lee M. Hyun D.-H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (72) Google Scholar). GSH level was assessed as described by Hissin and Hilf (35Hissin P.J. Hilf R. Anal. Biochem. 1976; 74: 214-226Crossref PubMed Scopus (3720) Google Scholar) 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). DNA extraction and assessment of purity were carried out as described by Lyraset al. (36Lyras L. Cairns N.J. Jenner A. Jenner P. Halliwell B. J. Neurochem. 1997; 68: 2061-2069Crossref PubMed Scopus (466) Google Scholar). DNA hydrolysis with formic acid and separation of modified 8-hydroxyguanine by HPLC were performed as described by Kaur and Halliwell (37Kaur H. Halliwell B. Biochem. J. 1996; 318: 21-23Crossref PubMed Scopus (143) Google Scholar). Protein carbonyl content was determined by method A of Lyras et al. (38Lyras L. Evans P.J. Shaw P.J. Ince P.G. Halliwell B. Free Radic. Res. 1996; 24: 397-406Crossref PubMed Scopus (66) Google Scholar), except that the final protein pellets were dissolved in 1 ml of 6 m guanidinium hydrochloride. Carbonyl content was calculated as nmol/mg protein (39Reznick A.Z. Packer L. Methods Enzymol. 1994; 223: 357-363Crossref Scopus (2009) Google Scholar). Measurement of protein-bound 3-nitrotyrosine content was carried out as described by Khan et al. (40Khan J. Brennand D.M. Bradley N. Gao B. Bruckdorfer R. Jacobs M. Biochem. J. 1998; 332: 807-808Crossref PubMed Google Scholar). Peroxynitrite (ONOO−) was prepared in a quenched flow reaction system (41Yermilov V. Rubio J. Ohshima H. FEBS Lett. 1995; 376: 207-210Crossref PubMed Scopus (285) Google Scholar). Nitrated bovine serum albumin was prepared as described by Whiteman and Halliwell (42Whiteman M. Halliwell B. Biochem. Biophys. Res. Commun. 1999; 258: 168-172Crossref PubMed Scopus (37) Google Scholar). The conjugation of horseradish peroxidase with anti-3-nitrotyrosine antibody was performed with the periodate method as described by Harlow and Lane (43Harlow E. Lane D. Antibodies: A Laboratory Manual. 2nd Ed. Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY1988: 471-612Google Scholar). For measuring lipid peroxidation, thiobarbituric acid-reactive material was measured by HPLC as described by Lyras et al. (44Lyras L. Perry R.H. Perry E.K. Ince P.G. Jenner A. Jenner P. Halliwell B. J. Neurochem. 1998; 71: 302-312Crossref PubMed Scopus (110) Google Scholar), employing 3 × 106 cells. Levels of NO2−/NO3− and expression of nitric-oxide synthase were measured as described by Lee et al. (31Lee M. Hyun D.-H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (72) Google Scholar). The levels of NO2−/NO3− in medium alone (including fetal bovine serum) were subtracted from the levels of NO2−/NO3− in cells plus medium. The levels of NO2−/NO3− in cells plus medium were 20–25 μm and in medium (plus fetal bovine serum) alone 10–12 μm. Three different proteasome activities were measured as described by Canu et al. (45Canu N. Barbato C. Ciotti M.T. Serafino A. Dus L. Calissano P. J. Neurosci. 2000; 20: 589-599Crossref PubMed Google Scholar), using fluorogenic substrates, Suc-LLVY-MCA (50 μm, Sigma), Boc-LRR-MCA (100 μm, Bachem, Merseyside, UK). or Z-LLE-βNap (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). 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.(31Lee M. Hyun D.-H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (72) Google Scholar). 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 (43Harlow E. Lane D. Antibodies: A Laboratory Manual. 2nd Ed. Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY1988: 471-612Google Scholar). Data were analyzed by densitometry (Imaging System, St. Catherine, Ontario, Canada). 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. 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. Expression of wild-type and mutant Parkins in both NT-2 and SK-N-MC cell lines was examined using Western blotting (Fig.1 A). 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. 1 B). 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.1 A). 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.1 C). The mutant proteins turned over less rapidly (half-life 13.78 ± 1.17 h), and this was again blocked by lactacystin. 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 (TableI). 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 (46Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, Oxford, UK1999: 246-350Google Scholar) 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 (47Chirico S. Methods Enzymol. 1994; 233: 314-318Crossref PubMed Scopus (158) Google Scholar). 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 (48Halliwell B. FEBS Lett. 1997; 411: 157-160Crossref PubMed Scopus (439) Google Scholar, 49Greenacre S.A. Ischiropoulos H. Free Radic. Res. 2001; 34: 541-581Crossref PubMed Scopus (478) Google Scholar). 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).Table IAntioxidant defensesCell lineSOD1SOD2CatalaseGPxGRGSHunit/mg proteinmmol/mg proteinNT-2Nontransfectant11.4 ± 0.92.9 ± 0.218.2 ± 1.520.3 ± 2.134.2 ± 2.465.2 ± 6.1Vector-only12.1 ± 1.13.1 ± 0.118.0 ± 1.321.5 ± 2.335.2 ± 2.569.4 ± 5.0WT13.4 ± 1.03.2 ± 0.220.1 ± 1.322.1 ± 2.236.2 ± 2.362.6 ± 3.4Del 3–512.3 ± 1.02.9 ± 0.218.4 ± 1.119.7 ± 2.734.2 ± 2.640.9 ± 2.51-ap < 0.01, significant difference compared with none or vector-only transfectants.T240R11.7 ± 1.23.0 ± 0.119.4 ± 1.519.7 ± 2.433.3 ± 2.342.2 ± 1.61-ap < 0.01, significant difference compared with none or vector-only transfectants.Q331X12.3 ± 1.22.8 ± 0.117.7 ± 1.920.2 ± 2.134.9 ± 2.641.0 ± 2.51-ap < 0.01, significant difference compared with none or vector-only transfectants.SK-N-MCNontransfectant10.5 ± 1.12.8 ± 0.217.2 ± 1.529.2 ± 2.131.5 ± 2.582.6 ± 6.0Vector-only11.2 ± 0.93.1 ± 0.217.0 ± 1.328.6 ± 2.433.2 ± 2.785.4 ± 5.7WT12.1 ± 0.93.2 ± 0.218.3 ± 1.230.1 ± 2.234.1 ± 2.677.4 ± 5.3Del 3–511.3 ± 1.03.0 ± 0.217.7 ± 1.528.9 ± 2.232.2 ± 2.758.1 ± 3.21-ap < 0.01, significant difference compared with none or vector-only transfectants.T240R11.0 ± 1.03.0 ± 0.218.6 ± 1.729.7 ± 2.333.2 ± 2.559.5 ± 3.41-ap < 0.01, significant difference compared with none or vector-only transfectants.Q331X11.2 ± 1.13.1 ± 0.217.0 ± 1.228.9 ± 2.232.9 ± 2.751.5 ± 2.41-ap < 0.01, significant difference compared with none or vector-only transfectants.1 unit defines SOD1 and SOD2, the amount of enzyme inhibiting the rate of NBT reduction by 50%; catalase, the amount of enzyme consuming 1 μmol of H2O2 per min; glutathione peroxidase (GPx), the amount of enzyme leading to oxidation of 1 nmol of NADPH per min; glutathione reductase (GR), the amount of enzyme oxidizing 1 nmol of NADPH per min. Values are the means ± S.E., n=6. One-way ANOVA was carried out to test significance.1-a p < 0.01, significant difference compared with none or vector-only transfectants. Open table in a new tab Table IILevels of oxidative damage to DNA, proteins, and lipidsCell line8-OHGProtein carbonylsLipid peroxidation3-Nitrotyrosinenmol/mg DNAnmol/mg proteinμmpmol/mg proteinNT-2Nontransfectant2.3 ± 0.16.1 ± 0.44.6 ± 0.321.4 ± 1.5Vector-only2.4 ± 0.15.9 ± 0.34.6 ± 0.220.9 ± 1.2WT2.4 ± 0.23.2 ± 0.22-ap < 0.01, significant difference compared with none or vector-only transfectants.4.9 ± 0.213.2 ± 0.82-ap < 0.01, significant difference compared with none or vector-only transfectants.Del 3–52.7 ± 0.29.2 ± 0.62-bp < 0.01, significant difference compared with none or vector-only transfectants.6.9 ± 0.32-bp < 0.01, significant difference compared with none or vector-only transfectants.30.5 ± 2.52-bp < 0.01, significant difference compared with none or vector-only transfectants.T240R2.7 ± 0.29.0 ± 0.52-bp < 0.01, significant difference compared with none or vector-only transfectants.6.9 ± 0.22-bp < 0.01, significant difference compared with none or vector-only transfectants.32.2 ± 2.52-bp < 0.01, significant difference compared with none or vector-only transfectants.Q331X2.7 ± 0.19.5 ± 0.62-bp < 0.01, significant difference compared with none or vector-only transfectants.7.0 ± 0.32-bp < 0.01, significant difference compared with none or vector-only transfectants.33.3 ± 2.52-bp < 0.01, significant difference compared with none or vector-only transfectants.SK-N-MCNontransfectant2.5 ± 0.16.6 ± 0.34.2 ± 0.229.2 ± 2.0Vector-only2.3 ± 0.16.7 ± 0.44.3 ± 0.230.3 ± 2.2WT2.5 ± 0.14.0 ± 0.32-ap < 0.01, significant difference compared with none or vector-only transfectants.4.3 ± 0.219.8 ± 1.32-ap < 0.01, significant difference compared with none or vector-only transfectants.Del 3–52.9 ± 0.29.3 ± 0.52-bp < 0.01, significant difference compared with none or vector-only transfectants.7.1 ± 0.32-bp < 0.01, significant difference compared with none or vector-only transfectants.42.3 ± 2.52-bp < 0.01, significant difference compared with none or vector-only transfectants.T240R2.9 ± 0.29.3 ± 0.52-bp < 0.01, significant difference compared with none or vector-only transfectants.6.9 ± 0.32-bp < 0.01, significant difference compared with none or vector-only transfectants.41.4 ± 2.12-bp < 0.01, significant difference compared with none or vector-only transfectants.Q331X2.9 ± 0.29.6 ± 0.72-bp < 0.01, significant difference compared with none or vector-only transfectants.7.3 ± 0.32-bp < 0.01, significant difference compared with none or vector-only transfectants.44.5 ± 2.92-bp < 0.01, significant difference compared with none or vector-only transfectants.When cells were grown to 80% confluence in 100-mm tissue culture plates, they were extracted. Values are the means ± S.E.,n = 6. One-way ANOVA was carried out to test significance.2-a p < 0.01, significant difference compared with none or vector-only transfectants.2-b p < 0.01, significant difference compared with none or vector-only transfectants. Open table in a new tab 1 unit defines SOD1 and SOD2, the amount of enzyme inhibiting the rate of NBT reduction by 50%; catalase, the amount of enzyme consuming 1 μmol of H2O2 per min; glutathione peroxidase (GPx), the amount of enzyme leading to oxidation of 1 nmol of NADPH per min; glutathione reductase (GR), the amount of enzyme oxidizing 1 nmol of NADPH per min. Values are the means ± S.E., n=6. One-way ANOVA was carried out to test significance. When cells were grown to 80% confluence in 100-mm tissue culture plates, they were extracted. Values are the means ± S.E.,n = 6. One-way ANOVA was carried out to test significance. Levels of NO2−/NO3− and expression of nitric-oxide synthase (iNOS and nNOS) were also assessed. Levels of NO2−/NO3− were increased in cells transfected with mutant" @default.
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• W2169363048 date "2002-08-01" @default.
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• W2169363048 title "Effect of Wild-type or Mutant Parkin on Oxidative Damage, Nitric Oxide, Antioxidant Defenses, and the Proteasome" @default.
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