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• W2068806054 abstract "Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated α-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease. Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated α-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease. Parkin, the most commonly mutated gene known to result in familial Parkinson's disease (PD), 1The abbreviations used are: PD, Parkinson's disease; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; UPS, ubiquitin-proteasome system; ERAD, endoplasmic reticulum-associated degradation; ER, endoplasmic reticulum; HD, Huntington's disease; Ubl, ubiquitin-like; poly(Q), polyglutamine; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; HA, hemagglutinin; HEK293, human embryonic kidney-derived 293; htt, Huntingtin.1The abbreviations used are: PD, Parkinson's disease; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; UPS, ubiquitin-proteasome system; ERAD, endoplasmic reticulum-associated degradation; ER, endoplasmic reticulum; HD, Huntington's disease; Ubl, ubiquitin-like; poly(Q), polyglutamine; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; HA, hemagglutinin; HEK293, human embryonic kidney-derived 293; htt, Huntingtin. encodes an E3 ubiquitin ligase (1Shimura 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 (1696) Google Scholar). Several substrates for parkin have been identified including CDCrel-1, an o-glycosylated form of α-synuclein αSp22, Pael-R (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar), and synphilin-1 (3Zhang 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 (833) Google Scholar, 4Shimura 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 (941) Google Scholar, 5Chung 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 (653) Google Scholar). These parkin substrates have little sequence or functional similarities; however, Pael-R and α-synuclein have a propensity to misfold and aggregate (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 4Shimura 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 (941) Google Scholar). This common property of known parkin substrates suggests that parkin may play a general role in the degradation of misfolded proteins, which might otherwise overwhelm the ubiquitin-proteasome system (UPS). Parkin has been demonstrated to function in the endoplasmic reticulum-associated degradation (ERAD) of misfolded ER proteins (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). Parkin is up-regulated during the unfolded protein response (6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). Pael-R overexpression results in ER accumulation of the protein, causing ER stress-induced cell death; parkin overexpression ameliorates these effects (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). Proteasome function is also important for normal ERAD and proteasomal dysfunction can cause ER stress (7Werner E.D. Brodsky J.L. McCracken A.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (388) Google Scholar, 8Chillaron J. Haas I.G. Mol. Biol. Cell. 2000; 11: 217-226Crossref PubMed Scopus (78) Google Scholar). Whereas ERAD is an important pathway for eliminating misfolded proteins in the ER, there are many misfolded aggregation-prone proteins that are translated in the cytosol including most of the polyglutamine (poly(Q)) containing proteins. Accumulation of misfolded cytosolic and ER-translated proteins can ultimately inhibit proteasomal activity (9Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1816) Google Scholar, 10Jana N.R. Zemskov E.A. Wang G. Nukina N. Hum. Mol. Genet. 2001; 10: 1049-1059Crossref PubMed Scopus (384) Google Scholar, 11Waelter S. Boeddrich A. Lurz R. Scherzinger E. Lueder G. Lehrach H. Wanker E.E. Mol. Biol. Cell. 2001; 12: 1393-1407Crossref PubMed Scopus (528) Google Scholar). Whereas the cytotoxicity of expanded poly(Q) proteins may be because of a variety of mechanisms (12Kouroku Y. Fujita E. Jimbo A. Kikuchi T. Yamagata T. Momoi M.Y. Kominami E. Kuida K. Sakamaki K. Yonehara S. Momoi T. Hum. Mol. Genet. 2002; 11: 1505-1515Crossref PubMed Scopus (169) Google Scholar, 13McCampbell A. Taylor J.P. Taye A.A. Robitschek J. Li M. Walcott J. Merry D. Chai Y. Paulson H. Sobue G. Fischbeck K.H. Hum. Mol. Genet. 2000; 9: 2197-2202Crossref PubMed Scopus (481) Google Scholar, 14Monoi H. Futaki S. Kugimiya S. Minakata H. Yoshihara K. Biophys. J. 2000; 78: 2892-2899Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Ross C.A. Margolis R.L. Becher M.W. Wood J.D. Engelender S. Cooper J.K. Sharp A.H. Prog. Brain Res. 1998; 117: 397-419Crossref PubMed Google Scholar, 16Luthi-Carter R. Strand A.D. Hanson S.A. Kooperberg C. Schilling G. La Spada A.R. Merry D.E. Young A.B. Ross C.A. Borchelt D.R. Olson J.M. Hum. Mol. Genet. 2002; 11: 1927-1937Crossref PubMed Google Scholar), expanded poly(Q) proteins impair proteasome function (9Bence N.F. Sampat R.M. Kopito R.R. Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1816) Google Scholar, 10Jana N.R. Zemskov E.A. Wang G. Nukina N. Hum. Mol. Genet. 2001; 10: 1049-1059Crossref PubMed Scopus (384) Google Scholar, 11Waelter S. Boeddrich A. Lurz R. Scherzinger E. Lueder G. Lehrach H. Wanker E.E. Mol. Biol. Cell. 2001; 12: 1393-1407Crossref PubMed Scopus (528) Google Scholar). Proteasomal dysfunction has also been demonstrated in PD brain and may play a role in the accumulation of aberrant proteins and neuronal loss that characterize several of the adult neurodegenerative diseases (17McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (551) Google Scholar). Accumulation of aberrant proteins is a hallmark of both PD and the poly(Q) expansion diseases, which include Huntington's disease (HD) and several spino-cerebellar ataxias. Overexpression of aberrant proteins has been very useful for identifying genes and proteins capable of modifying their accumulation or toxicity. Molecular chaperones such as Hsp70 improve cell viability (18Cummings C.J. Mancini M.A. Antalffy B. DeFranco D.B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1998; 19: 148-154Crossref PubMed Scopus (751) Google Scholar, 19Jana N.R. Tanaka M. Wang G. Nukina N. Hum. Mol. Genet. 2000; 9: 2009-2018Crossref PubMed Scopus (363) Google Scholar, 20Kobayashi Y. Kume A. Li M. Doyu M. Hata M. Ohtsuka K. Sobue G. J. Biol. Chem. 2000; 275: 8772-8778Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) and facilitate the elimination of poly(Q) proteins in cellular models and ameliorate disease phenotype in transgenic Drosophila models (18Cummings C.J. Mancini M.A. Antalffy B. DeFranco D.B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1998; 19: 148-154Crossref PubMed Scopus (751) Google Scholar, 21Bailey C.K. Andriola I.F. Kampinga H.H. Merry D.E. Hum. Mol. Genet. 2002; 11: 515-523Crossref PubMed Scopus (209) Google Scholar, 22Fernandez-Funez P. Nino-Rosales M.L. de Gouyon B. She W.C. Luchak J.M. Martinez P. Turiegano E. Benito J. Capovilla M. Skinner P.J. McCall A. Canal I. Orr H.T. Zoghbi H.Y. Botas J. Nature. 2000; 408: 101-106Crossref PubMed Scopus (547) Google Scholar, 23Warrick J.M. Chan H.Y. Gray-Board G.L. Chai Y. Paulson H.L. Bonini N.M. Nat. Genet. 1999; 23: 425-428Crossref PubMed Scopus (728) Google Scholar, 24Marsh J.L. Walker H. Theisen H. Zhu Y.Z. Fielder T. Purcell J. Thompson L.M. Hum. Mol. Genet. 2000; 9: 13-25Crossref PubMed Scopus (208) Google Scholar, 25Kazemi-Esfarjani P. Benzer S. Science. 2000; 287: 1837-1840Crossref PubMed Scopus (496) Google Scholar). Hsp70 also improves the phenotype in a Drosophila PD model in which human α-synuclein is overexpressed (26Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M. Bonini N.M. Science. 2002; 295: 865-868Crossref PubMed Scopus (1053) Google Scholar). Parkin appears to interact with Hsp70 along with the ubiquitinating factor CHIP (27Imai Y. Soda M. Hatakeyama S. Akagi T. Hashikawa T. Nakayama K.I. Takahashi R. Mol. Cell. 2002; 10: 55-67Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The N terminus of parkin contains a domain homologous to ubiquitin called the ubiquitin-like (Ubl) domain. A similar domain in the human homologue of the yeast DNA repair factor (hhRad23) has been shown to interact with expanded poly(Q) proteins (28Wang G. Sawai N. Kotliarova S. Kanazawa I. Nukina N. Hum. Mol. Genet. 2000; 9: 1795-1803Crossref PubMed Scopus (134) Google Scholar) and bind the proteasome (29Hiyama H. Yokoi M. Masutani C. Sugasawa K. Maekawa T. Tanaka K. Hoeijmakers J.H. Hanaoka F. J. Biol. Chem. 1999; 274: 28019-28025Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 30Elsasser S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (378) Google Scholar). Other Ubl domain containing proteins such as Dsk2 (31Kaye F.J. Modi S. Ivanovska I. Koonin E.V. Thress K. Kubo A. Kornbluth S. Rose M.D. FEBS Lett. 2000; 467: 348-355Crossref PubMed Scopus (90) Google Scholar, 32Saeki Y. Sone T. Toh-e A. Yokosawa H. Biochem. Biophys. Res. Commun. 2002; 296: 813-819Crossref PubMed Scopus (119) Google Scholar) and Ubp6 (33Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Mol Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar) also bind the proteasome. These findings suggest that parkin may also bind expanded poly(Q) proteins and proteasomes via its Ubl domain. In the current investigations we have chosen to use an expanded poly(Q) protein as a model for cellular pathology mediated by misfolded proteins more generally. The relationship between genetically determined neurodegeneration and abnormal misfolding of a disease causing protein is well established in poly(Q) diseases (34Sherman M.Y. Goldberg A.L. Neuron. 2001; 29: 15-32Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar). Poly(Q)-mediated neurodegeneration is likely to serve as a model for a number of neurodegenerative disorders in which genetic mutations of the disease-related proteins causes misfolding and aggregation such as in α-synuclein, SOD1, and Tau mutations (35Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1088) Google Scholar, 36Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Crossref PubMed Scopus (511) Google Scholar, 37DeTure M. Ko L.W. Easson C. Yen S.H. Am. J. Pathol. 2002; 161: 1711-1722Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Investigations of the role of parkin in facilitating the degradation of misfolded proteins may also be relevant to sporadic PD and several of the neurodegenerative conditions (38Souza J.M. Giasson B.I. Chen Q. Lee V.M. Ischiropoulos H. J. Biol. Chem. 2000; 275: 18344-18349Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 39Giasson B.I. Duda J.E. Murray I.V. Chen Q. Souza J.M. Hurtig H.I. Ischiropoulos H. Trojanowski J.Q. Lee V.M. Science. 2000; 290: 985-989Crossref PubMed Scopus (1380) Google Scholar). In this study, we address whether parkin promotes the ubiquitination and degradation of expanded poly(Q) proteins, thus reducing impairment of the UPS. We also examine the interaction of parkin with expanded poly(Q) proteins, Hsp70, and the proteasome. Our goal was to further understand the role of Hsp70 binding in parkin function and the role of parkin in preventing cell death induced by misfolded proteins. Proteasomal dysfunction can result in ER stress-associated cell death because retrotranslocation of misfolded proteins from the ER requires ongoing ubiquitination and proteasome function in the cytosol (8Chillaron J. Haas I.G. Mol. Biol. Cell. 2000; 11: 217-226Crossref PubMed Scopus (78) Google Scholar, 40Mancini R. Fagioli C. Fra A.M. Maggioni C. Sitia R. FASEB J. 2000; 14: 769-778Crossref PubMed Scopus (89) Google Scholar). For this reason, we assess the effects of parkin overexpression not only on UPS function but also on the activation of pathways involved in ER stress-induced cell death. Total RNA was extracted from cultures of human embryonic kidney-derived 293 (HEK293) cells using TRIzol reagent (Invitrogen). Oligo(dT)-primed first strand cDNAs were generated using Thermo-Script™ reverse transcriptase-PCR system (Invitrogen). Polymerase chain reaction (PCR) amplification of parkin cDNA was performed using parkin-specific primers. Parkin mutants were generated by PCR with wild-type parkin cDNA as template and cloned into the mammalian expression vectors pcDNA3.1(+) (Invitrogen) and CMV-FLAG 7.1 (Sigma). Wild type and mutant parkin were cloned into pGex-2T (Amersham Biosciences) and pRSETA vector (Invitrogen). GST-E6AP, GST-parkin, and mutants were produced in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B (Amersham Biosciences). His6-parkin was expressed in BL21(DE3) bacteria and purified with TALON resin (Clontech). Poly(Q) proteins (Gln26 and Gln79) fused to green fluorescent protein (GFP) were generated using a fragment of ataxin-3 with 26 and 79 glutamine repeats, respectively. The ataxin-3 fragments were generated by PCR and subcloned into EGFP-C1 vector (Clontech) leaving 44 amino acids of ataxin-3 N-terminal to the poly(Q) tract and 26 amino acids at the C terminus. GST-E6AP (originally from A. Weissman) and His6-ubiquitin (originally from D. Bohman (41Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar)) cDNAs were generous gifts from Cecile Pickart (Johns Hopkins). GFPu plasmid was kindly provided by Ron Kopito (Stanford); myc-E6AP cDNA was kindly provided by Allan Weissman (National Institutes of Health, NCI); mouse procaspase-12 cDNA was kindly provided by Junying Yuan (Harvard); cDNA for Hsp70 and ataxin-3 Gln28 and Gln84 were kindly provided by Henry Paulson (University of Iowa). N18 and HEK293 cells were transfected with various expression vectors using FuGENE 6 (Roche Diagnostics) according to the manufacturer's recommendations. Total amounts of plasmid DNA in individual transfections were adjusted to be equivalent in all transfections with empty vector. Transfected cells were cultured for 48–72 h and harvested for immunoprecipitation, Western blotting, or cell death assay. HEK293 cells were transfected with GFPu plasmid and cultured in medium containing G418. Isolated foci were selected for expansion and the cell lines were screened for an increase in GFPu fluorescence upon treatment with the proteasome inhibitor MG132. The YAC72 (42Hodgson J.G. Agopyan N. Gutekunst C.A. Leavitt B.R. LePiane F. Singaraja R. Smith D.J. Bissada N. McCutcheon K. Nasir J. Jamot L. Li X.J. Stevens M.E. Rosemond E. Roder J.C. Phillips A.G. Rubin E.M. Hersch S.M. Hayden M.R. Neuron. 1999; 23: 181-192Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar) transgenic mice, which express human Huntingtin with 72 glutamine repeats as a model for Huntington's disease, were used in this study. Brains from 12–15-month-old YAC72 transgenic mice were removed after perfusion with 4% formaldehyde and post-fixed overnight in 2% paraformaldehyde and 30% sucrose in phosphate-buffered saline (PBS). Brains were sectioned in series of 10 20-μm coronal sections on a cryostat and collected in PBS (pH 7.5). Immunofluorescence detection in cultured cells was performed as described in Ref. 43Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar. The following antibodies were used: anti-FLAG M2 (Sigma), anti-HA (Clontech), anti-parkin (Cell Signaling Technologies), anti-Huntingtin (Chemicon), and anti-ubiquitin (generous gift from Cecile Pickart). Cy5-conjugated donkey anti-rabbit and fluorescein isothiocyanate or Cy3-conjugated donkey anti-mouse were used as secondary antibodies (Chemicon). Controls included omission of primary antibody or primary antibody alone. As additional controls of parkin immunofluorescent colocalization with poly(Q)-containing proteins, pre-adsorption of the parkin antibody was performed using purified parkin expressed in bacteria (5 mg of parkin protein/ml of parkin antibody). All tissue was collected in accordance with the institutional review board-approved guidelines. Human HD brains were obtained anonymously from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland. Frontal cortex and caudate tissue samples were taken from two different patients. The brains were from male patients with genetically confirmed HD. The patients died at ages 47 and 51 years old, respectively. The brains were preserved in 10% formalin for 5 to 6 years prior to immunofluorescence observations. Tissue from the selected regions were dissected into 1-cm cubes and immersed in 30% sucrose in PBS for 72 h at 4 °C. The tissue samples were sectioned on a cryostat in 10-μm sections and collected in PBS. Immunofluorescence was performed using anti-Huntingtin (Chemicon) and anti-parkin antibodies HP2A recognizing amino acids 342–353 of parkin (a generous gift of Michael Schlossmacher). The HP2A antibody has been successfully used on human brain autopsy samples to demonstrate localization of parkin to Lewy bodies (44Schlossmacher M.G. Frosch M.P. Gai W.P. Medina M. Sharma N. Forno L. Ochiishi T. Shimura H. Sharon R. Hattori N. Langston J.W. Mizuno Y. Hyman B.T. Selkoe D.J. Kosik K.S. Am. J. Pathol. 2002; 160: 1655-1667Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Fluorescein isothiocyanate-conjugated donkey anti-mouse and Cy5-conjugated donkey anti-rabbit were used as secondary antibodies (both from Chemicon). Transfected cells were harvested, washed in PBS, and lysed in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.5–1.0% Triton X-100). Lysates were centrifuged at 15,000 × g for 10 min, and the supernatant was precleared before immunoprecipitation. Samples (300 μg) were incubated with 40 μl of anti-FLAG M2 affinity gel (Sigma) at 4 °C for 2 h with constant mixing. The immobilized immunocomplexes were collected by centrifugation, washed 3× with lysis buffer, and boiled in SDS sample buffer for SDS-PAGE. For brain homogenates, mouse brains were removed and homogenized in lysis buffer containing 2 mm ATP and 1% Nonidet P-40 with a Dounce homogenizer. Brain homogenates were centrifuged at 15,000 × g for 10 min at 4 °C and used for immunoprecipitation (1 mg/ml) as described above using anti-Huntingtin (Chemicon) or anti-parkin antibodies (Cell Signaling Technologies or HP2A kindly provided by Michael Schlossmacher). Rabbit anti-mouse was used as control IgG for nonspecific co-immunoprecipitation. The antigen complex was eluted and processed for SDS-PAGE. For immunoblots, cells were lysed in lysis buffer and briefly sonicated. Equal amounts (50 μg) of cell lysates were separated by SDS-PAGE, transferred to membranes, probed with the appropriate antibody, and visualized using chemiluminescence. GFP-Gln79, GFP-Gln26, and GFP were detected with rabbit polyclonal anti-GFP (Clontech). FLAG-tagged wild-type and mutant parkin were detected using anti-FLAG M2 (Sigma). HA-tagged poly(Q) proteins were detected with anti-HA (Clontech). Caspase-12 was detected by rabbit polyclonal anti-caspase-12 (Cell Signaling Technologies). The Rpt6/S8 and HC3 subunits of the proteasome were detected with rabbit polyclonal antibodies against p45 and HC3, respectively (Affiniti). All images were acquired on a Zeiss LSM 510 confocal microscope. Images were minimally processed for presentation. In all experiments in which aggregates and cell viability were quantified, the same observer, blinded to the transfection, scored the cells. For GFP-Gln79 aggregates, cells with large visible inclusions were counted (see Fig. 4A). Cell viability was assayed by propidium iodide exclusion under a fluorescence microscope (Zeiss Axiovert). Only cells expressing GFP-poly(Q) proteins were scored for propidium iodide exclusion. Transfection efficiency is about 50%. For each experiment, 200-500 cells were counted for each treatment. Auto-ubiquitination—FLAG-tagged wild-type and mutant parkin expressed in HEK293 cells were immunoprecipitated using anti-FLAG M2 antibody (300 μg, described above). The ubiquitination reaction contained the immunocomplexes, mammalian ubiquitin-activating enzyme E1 (70 nm), the E2 ubiquitin-conjugating enzyme UbcH7 (100 nm), and 125I-labeled ubiquitin (5 μm) in a reaction buffer of 50 mm Tris (pH 7.6), 5 mm MgCl2, 2 mm ATP with an ATP regenerating system. The reaction (50 μl) was incubated at 37 °C with gentle agitation for the indicated period of time and quenched with 2× SDS sample buffer (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). In Vitro Ubiquitination of Expanded Poly(Q) Proteins—35S-Labeled ataxin-3 Gln79 was translated in vitro using either a rabbit reticulocyte lysate or S30 T7 bacteria lysate system (Promega). Ubiquitination reactions contained 1 μl of translation mixture, mammalian E1 (100 nm), UbcH7 (300 nm), bovine ubiquitin (5 μm, Sigma), E3 (GST-E6AP, GST-parkin or mutants, 500 nm) in 50 μl of reaction buffer (above). Hsp70 (1 μg, Sigma) was added where indicated. The reaction was incubated for 2 h at 37 °C and quenched with 2× SDS sample buffer. The reaction mixtures were separated by SDS-PAGE and processed for visualization on a Storm PhosphorImager (Amersham Biosciences). HEK293 cells were transfected with the indicated expression plasmids and cultured for 30 h. Cells were washed and starved in Met/Cys-free medium for 1 h before labeling with 50 μCi/ml [35S]Met and 35S-Cys for 1 h (Promix, Amersham Biosciences). After labeling, cells were washed three times and chased in normal medium supplemented with unlabeled Met and Cys. Where indicated, 50 μm MG132 was added to the labeling and chase medium to inhibit proteasome activity. At the indicated times, cells were washed twice with PBS, lysed in RIPA buffer, and briefly sonicated before immunoprecipitation with anti-GFP antibody. Immunocomplexes were washed, boiled in SDS sample buffer, and separated by SDS-PAGE. Radiolabeled proteins were visualized by exposure to phosphorimage screens and analyzed with a Storm PhosphorImager (Amersham Biosciences). HEK293 cells stably expressing GFPu were transiently transfected with HA-Gln79 and FLAG-tagged wild-type or mutant parkin. After 72 h, cells were imaged for GFPu fluorescence and HA-Gln79 and FLAG-parkin expression (immunofluorescence). For cells transfected with HA-Gln79, GFPu fluorescence of individual cells expressing HA-Gln79 was measured. To evaluate the effect of overexpressing parkin on GFPu fluorescence, GFPu fluorescence of cells expressing FLAG-parkin were measured. An aliquot containing 10 μg of GST, GST-parkin, or GST-parkin mutants immobilized on glutathione-Sepharose 4B was incubated with 0.1 μm purified bovine 26 S proteasomes (gift of Y. Lam, Johns Hopkins (45Lam Y.A. Pickart C.M. Alban A. Landon M. Jamieson C. Ramage R. Mayer R.J. Layfield R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9902-9906Crossref PubMed Scopus (300) Google Scholar)) in 20 μl of binding buffer (50 mm Tris, pH 7.6, 1 mm dithiothreitol, 1 mm ATP) for 2 h at 4 °C with constant mixing. The immobilized proteins were collected by centrifugation, washed 3× with binding buffer, and boiled in SDS sample buffer for SDS-PAGE (2Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). HA-Gln84 was translated in vitro using S30 T7 bacteria lysate. An aliquot of the translation mixture was incubated with His6-parkin (1 μg) in 25 μl of buffer containing 50 mm Tris (pH 7.6), 1 mg/ml ovalbumin for 2 h at room temperature with constant mixing. HA-Gln84 was immunoprecipitated with anti-HA antibody and His6-parkin immunoprecipitated with anti-His6 antibody (Clontech). Non-parametric statistics were used in this study to avoid assumptions about the underlying distributions of the data. The Kruskal-Wallis test was used for multiple comparisons and p < 0.05 considered statistically significant. For two-sample comparison, treatment was compared with control (transfection with empty vector) using two-tailed Wilcoxon signed-rank test. Data were analyzed with S-Plus and presented as mean ± S.D. Parkin Interacts with Expanded Poly(Q) Proteins in Vivo and in Cells—Immunofluorescence revealed that parkin was localized in the Huntingtin (htt)-containing aggregates in brains of YAC72 transgenic mice (42Hodgson J.G. Agopyan N. Gutekunst C.A. Leavitt B.R. LePiane F. Singaraja R. Smith D.J. Bissada N. McCutcheon K. Nasir J. Jamot L. Li X.J. Stevens M.E. Rosemond E. Roder J.C. Phillips A.G. Rubin E.M. Hersch S.M. Hayden M.R. Neuron. 1999; 23: 181-192Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), which express human htt with 72 glutamines under the control of the htt promoter (Fig. 1A). The aggregates also contained ubiquitin (Fig. 1A). We next determined if parkin colocalized to htt-containing inclusion bodies in human brain tissue from HD patients (Fig. 2). Immunofluorescence showed that parkin was localized to both cytoplasmic and nuclear inclusions of htt found in the caudate and frontal cortex of HD brains. Pre-adsorption of the parkin antibody eliminated parkin immunofluorescence showing that parkin colocalized with inclusions containing poly(Q)-expanded htt in HD brain and in transgenic mouse models of HD.Fig. 2Colocalization of parkin with poly(Q)-expanded Huntingtin in human HD brains. Fixed sections of caudate (upper panel) or frontal cortex (lower panel) from two separate HD brains were prepared for immunofluorescence as described under ”Materials and Methods.“ Immunofluorescence of H" @default.
• W2068806054 created "2016-06-24" @default.
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• W2068806054 date "2003-06-01" @default.
• W2068806054 modified "2023-10-17" @default.
• W2068806054 title "Parkin Facilitates the Elimination of Expanded Polyglutamine Proteins and Leads to Preservation of Proteasome Function" @default.
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• W2068806054 doi "https://doi.org/10.1074/jbc.m212235200" @default.
• W2068806054 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12676955" @default.
• W2068806054 hasPublicationYear "2003" @default.
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