Caspase-mediated Parkin Cleavage in Apoptotic Cell Death
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m111534200
ISSN1083-351X
AutoresSøren Kahns, Simon Lykkebo, Lene Diness Jakobsen, Morten S. Nielsen, Poul Henning Jensen,
Tópico(s)Nuclear Receptors and Signaling
ResumoThe parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress. The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress. ubiquitin ligase ubiquitin-activating enzyme ubiquitin-conjugating enzyme Chinese hamster ovary okadaic acid Parkinson's disease is the second most common neurodegenerative disorder, and its symptoms arise primarily from a rather selective loss of dopaminergic neurons in the substantia nigra of the brain stem (1Lang A.E. Lozano A.M. N. Engl. J. Med. 1998; 339: 1044-1053Crossref PubMed Scopus (1749) Google Scholar). Lesions in the parkin gene on chromosome 6 are responsible for a large number of patients affected by early onset Parkinson's disease (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar, 3Lücking C.B. Durr A. Bonifati V. De Vaughan J. Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1237) Google Scholar). The parkin gene encodes the intracellular parkin protein that consists of 465 amino acids with an N-terminal ubiquitin-like domain and a C-terminal domain with two Ring finger motifs (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar). Ring finger domains are present in one class of E31 ubiquitin ligases (4Kornitzer D. Ciechanover A. J. Cell. Physiol. 2000; 182: 1-11Crossref PubMed Scopus (231) Google Scholar), and parkin exhibits ubiquitin ligase activity although it is unclear whether it is a bona fide E3 ubiquitin ligase or exists as part of a multisubunit ubiquitin ligase complex (5Shimura 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 (1681) Google Scholar, 6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar). The C-terminal Ring finger domain mediates the binding to E2 ubiquitin conjugases (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar,7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar).The ubiquitin-proteasome system is responsible for the majority of intracellular protein catabolism and relies on the concerted action of two ATP-dependent enzyme systems, the ubiquitin system and the proteasome (8Hershko A Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6791) Google Scholar). Ubiquitination of substrate proteins is carried out by sequential reactions catalyzed by the ubiquitin-activating enzyme (E1), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). The E3 ubiquitin ligases function either as templates that bring the ubiquitin-bearing E2 molecules in the proximity of the substrate proteins or by directly transferring the ubiquitin moiety to the substrate protein. The target proteins, ranging from misfolded proteins to highly controlled cellular regulators of e.g. the cell cycle, become polyubiquitinated, thereby making them recognizable by the 26 S proteasomal multicatalytic protease complex. The expression of the parkin gene is enhanced during unfolded protein stress as part of the unfolded protein response, and parkin confers cytoprotection toward unfolded protein stress (6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 9Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). Several disease-causing lesions in the parkin gene result in a loss of ubiquitin ligase function (5Shimura 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 (1681) Google Scholar, 7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar, 9Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). The loss of function elicits a preferential degeneration of the dopaminergic neurons in the substantia nigra despite parkin being widely expressed in the central nervous system (10Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (257) Google Scholar, 11Horowitz J.M. Myers J. Stachowiak M.K. Torres G. Neuroreport. 1999; 10: 3393-3397Crossref PubMed Scopus (38) Google Scholar, 12Gu W.J. Abbas N. Lagunes M.Z. Parent A. Pradier L. Bohme G.A. Agid Y. Hirsch E.C. Raisman-Vozari R. Brice A. J. Neurochem. 2000; 74: 1773-1776Crossref PubMed Scopus (43) Google Scholar, 13Gu M. Zarate-Lagunes W.J. Blanchard V. Francois C. Muriel M.P. Mouatt-Prigent A. Bonici B. Parent A. Hartmann A. Yelnik J. Boehme G.A. Pradier L. Moussaoui S. Faucheux B. Raisman-Vozari R. Agid Y. Brice A. Hirsch E.C. J. Comp. Neurol. 2001; 432: 184-196Crossref PubMed Scopus (47) Google Scholar). This indicates that parkin performs a vital function for the survival of this particular population of nerve cells. Accordingly, pathological signaling pathways that impinge on parkin function may represent potential contributors to the neurodegeneration in sporadic Parkinson's disease.Caspases comprise a family of cysteine-proteases that regulate the process of apoptosis at several levels and exhibit an ultimate requirement for aspartic residues in the P1 position of their substrates (14Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 15Steinnicke H.R. Salvesen G.S. Biochim. Biophys. Acta. 2000; 1477: 299-306Crossref PubMed Scopus (293) Google Scholar, 16Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (262) Google Scholar). Caspase-mediated cell death is generally considered a swift process, where caspase activation is rapidly followed by cellular fragmentation and phagocytic removal (17Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6132) Google Scholar). However, transient caspase activation is compatible with cellular survival (18Reddien P.W. Cameron S. Horvitz H.R. Nature. 2001; 412: 198-202Crossref PubMed Scopus (282) Google Scholar, 19Hoeppner D.J. Hengartner M.O. Schnabel R. Nature. 2001; 412: 202-206Crossref PubMed Scopus (244) Google Scholar), and nerve cells display a relative resistance toward caspase activity that may allow low-level caspase activation to play a role in long term neurodegenerative processes (20Zhang Y. Goodyer C. LeBlanch A. J. Neurosci. 2000; 20: 8384-8389Crossref PubMed Google Scholar). This is corroborated by the demonstration of activated caspases or their specific proteolytic products in degenerating nerve cells of brain tissue affected by Parkinson's disease (21Hartmann A. Hunot S. Michel P.P. Muriel M.-P. Vyas S. Faucheux B.A. Mouatt-Prigent A. Turmel H. Srinivasan A. Ruberg M. Evan G.I. Agid Y. Hirsch E.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2875-2880Crossref PubMed Scopus (612) Google Scholar) and Alzheimer's disease (22Stadelmann C. Deckwerth T.L. Srinivasan A. Bancher C. Bruck W.K. Lassmann H. Am. J. Pathol. 1999; 155: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 23Rohn T.T. Su E. Head J.H. Anderson A.J. Bahr B.A. Cotman C.W. Cribbs D.H. Am. J. Pathol. 2001; 158: 189-198Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Proteolytic cascades catalyzed by caspases may thus represent signaling pathways involved in the progression of common neurodegenerative diseases.We investigated the presence of apoptosis-associated parkin modifications and demonstrate that parkin is cleaved during apoptosis by caspase-mediated proteolysis. Asp-126 represents the major cellular parkin cleavage site in parkin-expressing Chinese hamster ovary (CHO) and SH-SY5Y dopaminergic neuroblastoma cell lines as determined by site-directed mutagenesis. Proteolysis of parkin after Asp-126 will mimic the disease-causing in-frame truncation of exons 2–3 comprising amino acid residues 3–137 (3Lücking C.B. Durr A. Bonifati V. De Vaughan J. Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1237) Google Scholar) and is thus incompatible with a functional parkin molecule.DISCUSSIONApoptosis-associated proteolysis cleaves parkin and generates a 38-kDa C-terminal fragment as demonstrated by (i) its specific binding of polyclonal antibodies raised against recombinant human parkin and a synthetic peptide corresponding to the 15 C-terminal amino acid residues of human parkin and (ii) the abrogated formation of the 38-kDa fragment in the presence of peptide inhibitors of caspases. Parkin cleavage is induced by as different inducers of apoptosis as the protein phosphatase 2A inhibitor OA, the broad-spectrum kinase inhibitor staurosporine, and the topoisomerase inhibitor camptothecin. This indicates that parkin cleavage is associated to apoptosis in general and not merely reflects the activation of a specific cellular signal transduction pathway. A direct involvement of caspase activity in the cleavage reaction is supported by several lines of evidence. Firstly, two peptide-based caspase inhibitors inhibit the cleavage, thereby demonstrating a caspase as being upstream of or as responsible for the cleavage. Secondly, the inhibition of cleavage by site-directed mutagenesis of Asp-126 to Glu and Ala indicates that the protease requires an aspartate in the P1 position. Thirdly, the requirement for an aspartate in the P1 position is corroborated by the efficient inhibition of the cleavage by the tetrapeptide aldehyde LHTD-CHO based on the amino acid sequence N-terminal to the putative scissile peptide bond Asp-126–Ser-127. Caspases may cleave other sites in parkin since some caspase inhibitor-sensitive proteolysis of parkin-(D126E) was observed as a smear below the uncleaved 52-kDa parkin. Asp-86–Asp-87 has been reported as a cleavage site in parkin (40Tsai Y. Fishmann P.S. Oyler G.A. Abstract of the Society for Neuroscience's 30th Annual Meeting. 2000; 2000: 4-9Google Scholar), but this site has no quantitative significance in our cellular models based on site-directed mutagenesis. The identity of the parkin cleaving caspase cannot be inferred from the potency of the inhibitors employed as they have a low cell permeability. The LHTD sequence N-terminal to the cleavage site does not conform to any of the optimal sequences for caspase cleavage sites, but may be cleaved with some efficiency by caspases 1, 2, and 9 as judged on the combinatorial approach employed by Thornberry et al. (27Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzage R.V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar). The identification of the responsible caspase will have to await the development of a cell-free assay, specific cell permeable caspase inhibitors, or the use of cell lines established from caspase gene knock-out mice.Subcellular fractionation of parkin-expressing cells showed that parkin predominantly is a vesicle-associated protein as previously demonstrated (28Kubo S.I. Kitami T. Noda S. Shimura H. Uchiyama Y. Asakawa S. Minoshima S. Shimizu N. Mizuno Y. Hattori N. J. Neurochem. 2001; 78: 42-54Crossref PubMed Scopus (115) Google Scholar). This localization is maintained in the apoptotic cells where the 38-kDa fragment remains vesicle-associated. This indicates that the caspase-mediated parkin cleavage occurs at or near the membranes where caspases previously have been localized (29Krebs J.S. Srinivasan A. Wong A.M. Tomaselli K.J. Fritz L.C. Wu J.C. Biochemistry. 2000; 39: 16056-16063Crossref PubMed Scopus (8) Google Scholar,30Nakagawa T. Zhu H. Li N. Xu E. Morishima J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2926) Google Scholar).Proteolysis of parkin after Asp-126 will liberate the ubiquitin-like domain from the remaining large polypeptide containing the Ring box domain (Fig. 5). This is incompatible with a functional parkin enzyme based on several data. Clinical data demonstrate that a single missense mutation in the ubiquitin-like domain R42P or in-frame deletion of exons 2 and 3, resulting in the loss of amino acid residues 3–137, causes early onset autosomal-recessive juvenile parkinsonism based on parkin dysfunction (3Lücking C.B. Durr A. Bonifati V. De Vaughan J. Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1237) Google Scholar, 25Abbas N. Lucking C.B. Ricard S. Durr A. De Bonifati V. Michele G. Bouley S. Vaughan J.R. Gasser T. Marconi R. Broussolle E. Brefel-Courbon C. Harhangi B.S. Oostra B.A. Fabrizio E. Bohme G.A. 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Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 224-225Crossref PubMed Scopus (933) Google Scholar). Subcellular fractionation of the P1–126-CHO1 cells expressing the N-terminal 126-amino acid parkin peptide demonstrates its localization in the cytosol. This suggests that it may function independently in this environment e.g.as suggested by inhibiting the proteasome (40Tsai Y. Fishmann P.S. Oyler G.A. Abstract of the Society for Neuroscience's 30th Annual Meeting. 2000; 2000: 4-9Google Scholar).Caspase activation has for several years been associated to the swift apoptotic process observed in cell culture models and certain animal models. However, increasing evidence demonstrate that activated caspases and their specific proteolytic products are present in viable neurons in brain tissue from normal-aged control brains (32Su J.H. Zhao M. Anderson A.J. Srinivasan A. Cotman C.W. Brain Res. 2001; 898: 350-357Crossref PubMed Scopus (195) Google Scholar, 33Su J.H. Nichol K.E. Sitch T. Sheu P. Chubb C. Miller B.L. Tomaselli K.J. Kim R.C. Cotman C.W. Exp. Neurol. 2000; 163: 9-19Crossref PubMed Scopus (94) Google Scholar) and brains affected by Parkinson's disease (17Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6132) Google Scholar, 34Tatton N.A. Exp. Neurol. 2000; 166: 29-43Crossref PubMed Scopus (341) Google Scholar) and other neurodegenerative diseases (18Reddien P.W. Cameron S. Horvitz H.R. Nature. 2001; 412: 198-202Crossref PubMed Scopus (282) Google Scholar, 19Hoeppner D.J. Hengartner M.O. Schnabel R. Nature. 2001; 412: 202-206Crossref PubMed Scopus (244) Google Scholar, 31Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 224-225Crossref PubMed Scopus (933) Google Scholar, 32Su J.H. Zhao M. Anderson A.J. Srinivasan A. Cotman C.W. Brain Res. 2001; 898: 350-357Crossref PubMed Scopus (195) Google Scholar, 35Xu F.G. Gervais D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 36Selznick L.A. Holtzman D.M. Han B.H. Gokden M. Srinivasan A.N. Roth K.A. J. Neuropathol. Exp. Neurol. 1999; 58: 1020-1026Crossref PubMed Scopus (145) Google Scholar) where they are likely to persist for months. The caspase-positive neurons display degenerative characteristics but are viable, thus demonstrating that low-level proapoptotic caspase activity is compatible with continued survival of functionally compromised nerve cells (17Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6132) Google Scholar, 18Reddien P.W. Cameron S. Horvitz H.R. Nature. 2001; 412: 198-202Crossref PubMed Scopus (282) Google Scholar, 19Hoeppner D.J. Hengartner M.O. Schnabel R. Nature. 2001; 412: 202-206Crossref PubMed Scopus (244) Google Scholar, 36Selznick L.A. Holtzman D.M. Han B.H. Gokden M. Srinivasan A.N. Roth K.A. J. Neuropathol. Exp. Neurol. 1999; 58: 1020-1026Crossref PubMed Scopus (145) Google Scholar) probably due to antiapoptotic mechanisms (for a review, see Ref. 37Robertson G.S. Crocker S.J. Nicholson D.W. Schulz J.B. Brain Pathol. 2000; 10: 283-292Crossref PubMed Scopus (213) Google Scholar). This idea is corroborated by cell culture experiments where cultured neurons display strong resistance toward microinjection of active caspases as compared with cell lines that die rapidly (16Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (262) Google Scholar). Shimura et al. (10Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (257) Google Scholar) have previously reported the existence of an anti-parkin-immunoreactive band of ∼41 kDa in brain extracts from patients with Parkinson's disease, which is absent in control substantia nigra. However, with our current antibodies we are unable to unambiguously demonstrate a parkin cleavage product compatible with a Asp-126–Ser-127 proteolysis in extracts of substantia nigra (data not shown). The demonstration of such putative in vivo cleavage of parkin must await the generation of antibodies that specifically recognize Asp-126-cleaved parkin as previously demonstrated,e.g. amyloid precursor protein (35Xu F.G. Gervais D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar).Parkin cleavage may thus contribute to a vicious circle in neurons where caspase activation, initiated by e.g. hitherto unknown causes in sporadic Parkinson's disease, compromises parkin function that subsequently lowers the cellular stress threshold and thus leads to further caspase activation. Whether the degenerating caspase-positive neurons represent a reversible state is unknown but a reversible dysfunctional degenerative state has been demonstrated in a transgenic mouse model of Huntington's disease, where inhibition of the expression of the transgene polyglutamine-containing protein was followed by improvement of the motor symptoms of the mice (38Yamamoto A. Lucas J.J. Hen R. Cell. 2000; 101: 57-66Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar).We did not observe any cytoprotective effect of the D126E mutation as compared with wild type parkin when challenging the cells with OA, staurosporine, and camptothecin. However, this does not exclude the existence of such an effect when using another apoptotic paradigm as the protective effect of parkin previously have been demonstrated to be rather selective. Accordingly, parkin cleavage may represent a novel drug target in neuroprotective strategies against Parkinson's disease aiming at slowing the functional decline of those dopaminergic neurons that remain at the time of diagnosis. Transplantation of dopamine-producing cells represents another treatment strategy used in Parkinson's disease. Such cells are subject to a considerable stress as evidenced by the large caspase-dependent cell loss upon transplantation (39Schierle G.S. Hansson O. Leist M. Nicotera P. Widner H. Brundin P. Nat. Med. 1999; 5: 97-100Crossref PubMed Scopus (250) Google Scholar). Transgenic expression of parkin mutagenized at Asp-126 in these cells might represent an attractive method for improving the survival provided the mutation does not affect the enzymatic activity of parkin. Parkinson's disease is the second most common neurodegenerative disorder, and its symptoms arise primarily from a rather selective loss of dopaminergic neurons in the substantia nigra of the brain stem (1Lang A.E. Lozano A.M. N. Engl. J. Med. 1998; 339: 1044-1053Crossref PubMed Scopus (1749) Google Scholar). Lesions in the parkin gene on chromosome 6 are responsible for a large number of patients affected by early onset Parkinson's disease (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar, 3Lücking C.B. Durr A. Bonifati V. De Vaughan J. Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1237) Google Scholar). The parkin gene encodes the intracellular parkin protein that consists of 465 amino acids with an N-terminal ubiquitin-like domain and a C-terminal domain with two Ring finger motifs (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar). Ring finger domains are present in one class of E31 ubiquitin ligases (4Kornitzer D. Ciechanover A. J. Cell. Physiol. 2000; 182: 1-11Crossref PubMed Scopus (231) Google Scholar), and parkin exhibits ubiquitin ligase activity although it is unclear whether it is a bona fide E3 ubiquitin ligase or exists as part of a multisubunit ubiquitin ligase complex (5Shimura 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 (1681) Google Scholar, 6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar). The C-terminal Ring finger domain mediates the binding to E2 ubiquitin conjugases (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar,7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar). The ubiquitin-proteasome system is responsible for the majority of intracellular protein catabolism and relies on the concerted action of two ATP-dependent enzyme systems, the ubiquitin system and the proteasome (8Hershko A Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6791) Google Scholar). Ubiquitination of substrate proteins is carried out by sequential reactions catalyzed by the ubiquitin-activating enzyme (E1), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). The E3 ubiquitin ligases function either as templates that bring the ubiquitin-bearing E2 molecules in the proximity of the substrate proteins or by directly transferring the ubiquitin moiety to the substrate protein. The target proteins, ranging from misfolded proteins to highly controlled cellular regulators of e.g. the cell cycle, become polyubiquitinated, thereby making them recognizable by the 26 S proteasomal multicatalytic protease complex. The expression of the parkin gene is enhanced during unfolded protein stress as part of the unfolded protein response, and parkin confers cytoprotection toward unfolded protein stress (6Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 9Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). Several disease-causing lesions in the parkin gene result in a loss of ubiquitin ligase function (5Shimura 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 (1681) Google Scholar, 7Zhang Y Gao J. Chung K.K.K. Huang H. Dawson V. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13559Crossref PubMed Scopus (830) Google Scholar, 9Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. 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