FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080806128> ?p ?o ?g. }

• W2080806128 endingPage "14986" @default.
• W2080806128 startingPage "14978" @default.
• W2080806128 abstract "Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys150–Cys169 and Cys196–His215 consisting of CX2-3CX11CX2C and CX4–6CX10–16-CX2(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn2+ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease. Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys150–Cys169 and Cys196–His215 consisting of CX2-3CX11CX2C and CX4–6CX10–16-CX2(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn2+ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease. Autosomal recessive juvenile Parkinson disease (ARJP) 2The abbreviations used are: ARJP, autosomal recessive juvenile Parkinson; E1, ubiquitin-activating enzyme; E3, ubiquitin-protein isopeptide ligase; UPD, unique parkin domain; ESI-MS, electrospray ionization mass spectrometry; ICP-AES, inductively coupled plasma-atomic emission spectrometry; GST, glutathione S-transferase; LC-MS, liquid chromatography-mass spectrometry. is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4231) Google Scholar, 2Valente E.M. Abou-Sleiman P.M. Caputo V. Muqit M.M. Harvey K. Gispert S. Ali Z. Del Turco D. Bentivoglio A.R. Healy D.G. Albanese A. Nussbaum R. Gonzalez-Maldonado R. Deller T. Salvi S. Cortelli P. Gilks W.P. Latchman D.S. Harvey R.J. Dallapiccola B. Auburger G. Wood N.W. Science. 2004; 304: 1158-1160Crossref PubMed Scopus (2725) Google Scholar, 3Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2287) Google Scholar, 4Zimprich A. Biskup S. Leitner P. Lichtner P. Farrer M. Lincoln S. Kachergus J. Hulihan M. Uitti R.J. Calne D.B. Stoessl A.J. Pfeiffer R.F. Patenge N. Carbajal I.C. Vieregge P. Asmus F. Muller-Myhsok B. Dickson D.W. Meitinger T. Strom T.M. Wszolek Z.K. Gasser T. Neuron. 2004; 44: 601-607Abstract Full Text Full Text PDF PubMed Scopus (2318) Google Scholar). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4231) Google Scholar, 5Hattori 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, 6Foroud T. Uniacke S.K. Liu L. Pankratz N. Rudolph A. Halter C. Shults C. Marder K. Conneally P.M. Nichols W.C. Parkinsons Study GroupNeurology. 2003; 60: 796-801Crossref PubMed Scopus (195) Google Scholar, 7Gu W.J. Corti O. Araujo F. Hampe C. Jacquier S. Lucking C.B. Abbas N. Duyckaerts C. Rooney T. Pradier L. Ruberg M. Brice A. Neurobiol. Dis. 2003; 14: 357-364Crossref PubMed Scopus (67) Google Scholar, 8Lucking C.B. Durr A. Bonifati V. Vaughan J. De 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 (1264) Google Scholar, 9West A. Periquet M. Lincoln S. Lucking C.B. Nicholl D. Bonifati V. Rawal N. Gasser T. Lohmann E. Deleuze J.F. Maraganore D. Levey A. Wood N. Durr A. Hardy J. Brice A. Farrer M. Disease Genetics Study Group and the European Consortium on Genetic Susceptibility on Parkinson's Disease.Am. J. Med. Genet. 2002; 114: 584-591Crossref PubMed Scopus (191) Google Scholar, 10Periquet M. Latouche M. Lohmann E. Rawal N. De Michele G. Ricard S. Teive H. Fraix V. Vidailhet M. Nicholl D. Barone P. Wood N.W. Raskin S. Deleuze J.F. Agid Y. Durr A. Brice A. Brain. 2003; 126: 1271-1278Crossref PubMed Scopus (244) Google Scholar, 11Lohmann E. Periquet M. Bonifati V. Wood N.W. De Michele G. Bonnet A.M. Fraix V. Broussolle E. Horstink M.W. Vidailhet M. Verpillat P. Gasser T. Nicholl D. Teive H. Raskin S. Rascol O. Destee A. Ruberg M. Gasparini F. Meco G. Agid Y. Durr A. Brice A. French Parkinson's Disease Genetics Study Group, and the European Consortium on Genetic Susceptibility in Parkinson's Disease.Ann. Neurol. 2003; 54: 176-185Crossref PubMed Scopus (265) Google Scholar, 12Henn I.H. Gostner J.M. Lackner P. Tatzelt J. Winklhofer K.F. J. Neurochem. 2005; 92: 114-122Crossref PubMed Scopus (90) Google Scholar, 13Hampe C. Ardila-Osorio H. Fournier M. Brice A. Corti O. Hum. Mol. Genet. 2006; 15: 2059-2075Crossref PubMed Scopus (192) Google Scholar, 14von Coelln R. Dawson V.L. Dawson T.M. Cell Tissue Res. 2004; 318: 175-184Crossref PubMed Scopus (117) Google Scholar, 15Shyu W.C. Lin S.Z. Chiang M.F. Pang C.Y. Chen S.Y. Hsin Y.L. Thajeb P. Lee Y.J. Li H. Parkinsonism Relat. Disord. 2005; 11: 173-180Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 16Satoh J. Kuroda Y. Neuroreport. 1999; 10: 2735-2739Crossref PubMed Scopus (53) Google Scholar, 17Abbas N. Lucking C.B. Ricard S. Durr A. Bonifati V. De 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. Pradier L. Wood N.W. Filla A. Meco G. Denefle P. Agid Y. Brice A. Hum. Mol. Genet. 1999; 8: 567-574Crossref PubMed Scopus (491) Google Scholar, 18Pineda-Trujillo N. Carvajal-Carmona L.G. Buritica O. Moreno S. Uribe C. Pineda D. Toro M. Garcia F. Arias W. Bedoya G. Lopera F. Ruiz-Linares A. Neurosci. Lett. 2001; 298: 87-90Crossref PubMed Scopus (46) Google Scholar). Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin ligase enzymes involved in proteosome-mediated protein degradation (19Marin I. Ferrus A. Mol. Biol. Evol. 2002; 19: 2039-2050Crossref PubMed Scopus (84) Google Scholar, 20Marin I. Lucas J.I. Gradilla A.C. Ferrus A. Physiol. Genomics. 2004; 17: 253-263Crossref PubMed Scopus (95) Google Scholar, 21Lucas J.I. Arnau V. Marin I. J. Mol. Biol. 2006; 357: 9-17Crossref PubMed Scopus (31) Google Scholar). The currently accepted domain architecture of parkin, deduced from multiple sequence alignment, shows that the C terminus of the protein is characterized by two ∼50-residue RING (really interesting new gene) domains separated by a 51-residue IBR (In-Between-RING) domain (22Morett E. Bork P. Trends Biochem. Sci. 1999; 24: 229-231Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23Beasley S.A. Hristova V.A. Shaw G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3095-3100Crossref PubMed Scopus (79) Google Scholar). The RING domains of parkin are proposed to interact with the ubiquitin-conjugating enzymes UbcH7, UbcH8, Ubc7, and Ubc13 and control parkin-mediated ubiquitination of a variety of substrates such as Pael-R, synphilin-1, Sept5, and PICK1 among others (24Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 25Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nature. 2000; 25: 302-305Google Scholar, 26Zhang 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 (842) Google Scholar, 27Doss-Pepe E.W. Chen L. Madura K. J. Biol. Chem. 2005; 280: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 28Imai 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 (427) Google Scholar, 29Lim K.L. Chew K.C. Tan J.M. Wang C. Chung K.K. Zhang Y. Tanaka Y. Smith W. Engelender S. Ross C.A. Dawson V.L. Dawson T.M. J. Neurosci. 2005; 25: 2002-2009Crossref PubMed Scopus (449) Google Scholar, 30Choi P. Snyder H. Petrucelli L. Theisler C. Chong M. Zhang Y. Lim K. Chung K.K. Kehoe K. D'Adamio L. Lee J.M. Cochran E. Bowser R. Dawson T.M. Wolozin B. Brain Res. Mol. Brain Res. 2003; 117: 179-189Crossref PubMed Scopus (109) Google Scholar, 31Joch M. Ase A.R. Chen C.X. MacDonald P.A. Kontogiannea M. Corera A.T. Brice A. Séguéla P. Fon E.A. Mol. Biol. Cell. 2007; 18: 3105-3118Crossref PubMed Scopus (121) Google Scholar). Other members of the RBR family include the human homolog of Drosophila Ariadne (HHARI), DORFIN, and HOIL-1, which share close domain architecture (32Moynihan T.P. Ardley H.C. Nuber U. Rose S.A. Jones P.F. Markham A.F. Scheffner M. Robinson P.A. J. Biol. Chem. 1999; 274: 30963-30968Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 33Ardley H.C. Tan N.G.S. Rose S.A. Markham A.F. Robinson P.A. J. Biol. Chem. 2001; 276: 19640-19647Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 34Niwa J. Ishigaki S. Doyu M. Suzuki T. Tanaka K. Sobue G. Biochem. Biophys. Res. Commun. 2001; 281: 706-713Crossref PubMed Scopus (71) Google Scholar, 35Yamanaka K. Ishikawa H. Megumi Y. Tokunaga F. Kanie M. Rouault T.A. Morishima I. Minato N. Ishimori K. Iwai K. Nat. Cell Biol. 2003; 5: 336-340Crossref PubMed Scopus (152) Google Scholar). Traditionally, RING domains coordinate two Zn2+ ions through a C3HC4 metal-binding consensus sequence. However, the RING2 domain of HHARI binds a single Zn2+ (36Capili A.D. Edghill E.L. Wu K. Borden K.L. J. Mol. Biol. 2004; 340: 1117-1129Crossref PubMed Scopus (85) Google Scholar), and because this is the only RING2 structure available for an RBR protein, it suggests that there may be variability in the number of Zn2+ ions coordinated by different RING domains. The recent three-dimensional structure of the parkin IBR domain (23Beasley S.A. Hristova V.A. Shaw G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3095-3100Crossref PubMed Scopus (79) Google Scholar) revealed a two-site zinc-binding motif with a novel fold compared with other zinc-binding motifs (37Matthews J.M. Sunde M. IUBMB Life. 2002; 54: 351-355Crossref PubMed Scopus (249) Google Scholar). However, despite the potential importance of zinc binding to the RING domains (or other portions) of parkin, the ability and capacity for zinc coordination or its impact on structure has not been identified for parkin. The N terminus of parkin comprises a ubiquitin-like domain (UblD) proposed to facilitate the delivery and degradation of ubiquitinated substrates by the 26 S proteosome via interactions with the S5a subunit (38Sakata E. Yamaguchi Y. Kurimoto E. Kikuchi J. Yokoyama S. Yamada S. Kawahara H. Yokosawa H. Hattori N. Mizuno Y. Tanaka K. Kato K. EMBO Rep. 2003; 4: 301-306Crossref PubMed Scopus (230) Google Scholar, 39Tashiro M. Okubo S. Shimotakahara S. Hatanaka H. Yasuda H. Kainosho M. Yokoyama S. Shindo H. J. Biomol. NMR. 2003; 25: 153-156Crossref PubMed Scopus (14) Google Scholar). The central ∼150 residues of parkin separating the UblD from the RBR region are referred to as the unique parkin domain (UPD). This segment of parkin is essential for function, and ARJP associated mutations within this region have been shown to lead to dysfunction of parkin E3 ligase activity (40Sato S. Chiba T. Sakata E. Kato K. Mizuno Y. Hattori N. Tanaka K. EMBO J. 2006; 25: 211-221Crossref PubMed Scopus (101) Google Scholar, 41Ren Y. Zhao J. Feng J. J. Neurosci. 2003; 23: 3316-3324Crossref PubMed Google Scholar). However, the absence of any sequence similarity to other proteins or the identification of a distinct domain within the UPD has made these experiments difficult to interpret. Other than the isolated UblD and IBR domains of parkin, there has been limited success with the purification and characterization of parkin, especially when lacking affinity tags in the final purified form. Bacterially expressed parkin typically shows a heterogeneous mixture of full-length and degraded protein species, making characterization of the protein difficult (42Rankin C.A. Joazeiro C.A. Floor E. Hunter T. J. Biomed. Sci. 2001; 8: 421-429Crossref PubMed Scopus (31) Google Scholar). In this work we have used purified parkin to identify a novel zinc-binding C4C3(C/H) domain upstream of the RBR region and within the UPD. We have used limited proteolysis and electrospray ionization mass spectrometry (ESI-MS) to show that this domain coordinates two Zn2+ ions in addition to six other Zn2+ ions in the RBR C terminus. The presence of a new parkin-specific zinc-binding domain provides insight into the structure of parkin and opens the door to establish the importance of this domain in ARJP for this new subclass of RBR E3 ligases. Recombinant Parkin—Human parkin was cloned from the Human Fetal Brain Marathon-Ready cDNA library (Clontech) and ligated into the PCR Blunt-TOPO vector using the Zero Blunt TOPO PCR kit from Invitrogen. The human parkin sequence was then transferred to the pGEX-4T3 bacterial expression vector. Rat parkin cloned into a pGEX-4T1 vector was received from Dr. Ted Fon (McGill University). Protein Expression and Purification—The pGEX-4T1 and pGEX-4T3 parkin constructs were overexpressed in Escherichia coli BL21 Codon Plus competent cells and grown at 37 °C in LB containing 50 mg/liter ampicillin until an A600 of 0.6 was recorded. Protein expression was induced by the addition of 50 μm isopropyl β-d-1-thiogalactopyranoside, supplemented by 500 μm ZnCl2. Following induction, the temperature was reduced to 16 °C, and the cells were grown overnight. The E. coli cells were harvested and lysed by sonication on ice in 20 mm Tris-HCl (pH 7.4), 120 mm NaCl, 1 mm dithiothreitol buffer supplemented with EDTA-free protease inhibitor mixture (Roche Applied Science). Triton X-100 was added to the cell lysate to a final concentration of 1%, and the sample was incubated at 4 °C for 30 min with rotation. Centrifugation at 20,000 rpm for 30 min was performed prior to collecting the supernatant. The remainder of the purification steps were carried out at 4 °C, unless indicated otherwise. GST-parkin was purified by fast protein liquid chromatography on a 20-ml GST Prep FF 16/10 affinity chromatography column (GE Healthcare) and eluted with 20 mm Tris-HCl (pH 7.4), 120 mm NaCl, 1 mm dithiothreitol, 20 mm glutathione. Purified parkin was dialyzed 300-fold against 20 mm Tris-HCl (pH 7.4), 120 mm NaCl, 1 mm dithiothreitol. The parkin sample was concentrated to a 5-ml volume using Amicon Ultra concentrators with a mass cut-off of 10 kDa (Millipore). The GST tag was cleaved from the construct with 1.5 μg of thrombin (Amersham Biosciences), with rotation at room temperature for 30 min. Thrombin was removed from the sample with p-aminobenzamidine-agarose beads (Sigma) by incubation with rotation at room temperature for 30 min. Removal of the GST tag from human and rat parkin cloned into the pGEX-4T3 and pGEX-4T1 vectors, respectively, resulted in short residual sequences preceding the N-terminal sequence of parkin. Human parkin is preceded by a GSPNS amino acid sequence, and a GSPGIPARPAATTLPVT sequence is found N-terminal to methionine at position one of rat parkin. Parkin was separated from the GST tag on the 20-ml GST Prep FF 16/10 affinity column, using a flow rate of 1 ml/min. The flow through was concentrated to 500 μl using Amicon Ultra concentrators with a 10-kDa cut-off (Millipore) and separated on a 120-ml Superdex HiLoad 16/60 gel filtration column (GE Healthcare) at a flow rate of 0.3 ml/min. In Vitro Ubiquitination Assay—Auto-ubiquitination assays of GST-parkin and parkin were carried out in a 20 mm Tris (pH 7.4), 120 mm NaCl, 1 mm TCEP buffer using standard ubiquitin conjugation reagents and enzymes (Boston Biochem) unless indicated otherwise. Bacterially expressed GST-parkin and parkin at concentrations of 25 μm each were incubated with 90 nm of E1 enzyme, 0.2 mm ubiquitin, 2.5 μm of Ubc7 (expressed and purified as a His6-Ubc7 protein from a pET28a construct), 4 mm MgATP, and 1× reaction buffer. The reactions were carried out at 37 °C for 1 h, inhibited with E1 inhibition buffer, and then heated to 100 °C with Laemmli sample buffer prior to analysis by SDS-PAGE gel electrophoresis. Ubiquitination was identified by Western blotting with anti-ubiquitin polyclonal antibody, used according to the manufacturers protocol. Limited Proteolysis—Parkin was subjected to limited proteolysis with both trypsin and V8 proteases. Six 140-μl samples of 50 μm parkin were incubated at 4 °C with 0.4 μg of trypsin or V8 each for 5, 10, 15, 30, 60, 90, or 120 min. Upon completion 20 μl from each sample were heated to 100 °C with Laemmli sample buffer and analyzed by SDS-PAGE with Coomassie Blue staining. Protease activity in the remainder of each reaction was inhibited with 5% formic acid; the sample was then desalted by centrifugation with protein desalting spin columns (Pierce). These samples were analyzed by ESI-MS (University of Western Ontario Biological Mass Spectrometry Laboratory). Mass fragments detected by ESI-MS were assigned to parkin sequences using the ProteinInfo amino acid sequence analysis tool PROWL. Bioinformatics—A comprehensive list of parkin orthologs was compiled from a search of the RefSeq nonredundant protein data base (Release 29) and the Uniprot databases, resulting in a list of proteins from 28 separate species based on human isoform 1. The multiple sequence alignment was originally created using ClustalW and manually edited using Jalview. The list was parsed based the exclusion of alternate isoforms, as well as sequences containing probable errors such as the Bos taurus parkin that possesses an extended C terminus. The full-length sequences for parkin from human, mouse, rat, fly, worm, and HOIL-1 were submitted for PONDR VSL1 analysis. Circular Dichroism Spectropolarimetry—Parkin samples in the range of 3–5 μm were analyzed using a Jasco J-810 circular dichroism spectropolarimeter. A 0.1-mm cell was used to measure protein absorbance in the UV range between 240 and 190 nm, keeping high tension voltage below 600 V. Fifty scans were collected per sample at 4 °C. EDTA was titrated into each parkin sample and equilibrated for 15 min after each addition prior to data collection. A CD spectrum of the buffer in the presence and absence of EDTA was collected at the beginning of each experiment and used for base-line correction. Zinc Analysis by ICP-AES and Quadropole Time-of-flight ESI-MS—Parkin, eluted from the Superdex HiLoad 16/10 gel filtration column, was submitted for ICP-AES Zn2+ analysis to determine the concentration of Zn2+ in the sample (Laboratory for Geochemical Analysis, University of Western Ontario). The concentration of parkin in the sample was identified by the guanidine hydrochloride denaturation method and amino acid analysis done at the Amino Acid Analysis Facility at the Hospital for Sick Children in Toronto, Canada. The total number of Zn2+ ions bound to parkin was determined by quadropole time-of-flight ESI-MS (University of Western Ontario Biological Mass Spectrometry Laboratory) done on full-length parkin eluted from the Superdex HiLoad 16/10 column and dialyzed 1000-fold against 120 mm ammonium acetate at a pH of 7.4. Differential Stability of Rat and Human Parkin—Stability has proven to be the major limitation in the large scale purification of bacterially expressed parkin (42Rankin C.A. Joazeiro C.A. Floor E. Hunter T. J. Biomed. Sci. 2001; 8: 421-429Crossref PubMed Scopus (31) Google Scholar). To identify the source of instability in the parkin protein, the human and rat forms of the protein were expressed as GST fusion proteins (pGEX-4T3 and pGEX-4T1) in E. coli BL21 Codon Plus cells. Following GST-glutathione affinity chromatography, thrombin cleavage, and a second GST-glutathione affinity step, the protein was analyzed by gel filtration chromatography. SDS-PAGE analysis of the elution profile showed the presence of bands near 52 kDa (supplemental Fig. S1) that were confirmed by ESI-MS as the rat protein (observed molecular mass, 53165.1 ± 2.8 Da; calculated molecular mass, 53167.6 Da) and human protein (observed molecular mass, 52063.6 ± 2.5 Da; calculated molecular mass, 52063.0). Although the rat form of parkin reproducibly eluted as a single purified form, the human protein consistently showed the presence of other bands, notably a band near 12 kDa. ESI-MS analysis showed this band had a mass of 11980.1 ± 0.2 Da, very close to that calculated for a C-terminal cleavage at residue Thr103 within the UPD of human parkin by an unspecified source. The presence of this cleavage product is very similar to that previously observed by Rankin and co-workers (42Rankin C.A. Joazeiro C.A. Floor E. Hunter T. J. Biomed. Sci. 2001; 8: 421-429Crossref PubMed Scopus (31) Google Scholar). This fragment incorporated the entire UblD (residues 1–75) and the first 28 residues from the UPD. This region is the most divergent between the rat and human orthologs possessing only 38/53 (71%) similarity between residues Asp87 and Arg140. Further, sequence analysis of parkin using the program PONDR predicted a disordered state for residues 75–141 of the UPD where cleavage was noted. Based on our results, the sequence difference in rat parkin within this region prevents its cleavage and allows for the purification of milligram levels of full-length parkin lacking fusion protein tags. Ubiquitin Ligase Activity of in Vitro Purified Parkin—Parkin is a ubiquitin ligase capable of auto-ubiquitination, as well as ubiquitination of other substrate proteins including PAEL-R, synphilin, and Sept5 (26Zhang 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 (842) Google Scholar, 28Imai 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 (427) Google Scholar, 29Lim K.L. Chew K.C. Tan J.M. Wang C. Chung K.K. Zhang Y. Tanaka Y. Smith W. Engelender S. Ross C.A. Dawson V.L. Dawson T.M. J. Neurosci. 2005; 25: 2002-2009Crossref PubMed Scopus (449) Google Scholar). A number of studies have demonstrated auto-ubiquitination of parkin and various ARJP parkin mutants in vitro (13Hampe C. Ardila-Osorio H. Fournier M. Brice A. Corti O. Hum. Mol. Genet. 2006; 15: 2059-2075Crossref PubMed Scopus (192) Google Scholar, 26Zhang 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 (842) Google Scholar, 31Joch M. Ase A.R. Chen C.X. MacDonald P.A. Kontogiannea M. Corera A.T. Brice A. Séguéla P. Fon E.A. Mol. Biol. Cell. 2007; 18: 3105-3118Crossref PubMed Scopus (121) Google Scholar, 43Fallon L. Bélanger C.M. Corera A.T. Kontogiannea M. Regan-Klapisz E. Moreau F. Voortman J. Haber M. Rouleau G. Thorarinsdottir T. Brice A. van Bergen En Henegouwen P.M. Fon E.A. Nat. Cell Biol. 2006; 8: 834-842Crossref PubMed Scopus (315) Google Scholar, 44Yao D. Gu Z. Nakamura T. Shi Z.Q. Ma Y. Gaston B. Palmer L.A. Rockenstein E.M. Zhang Z. Masliah E. Uehara T. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10810-10814Crossref PubMed Scopus (470) Google Scholar, 45Chung K.K. Thomas B. Li X. Pletnikova O. Troncoso J.C. Marsh L. Dawson V.L. Dawson T.M. Science. 2004; 304: 1328-1331Crossref PubMed Scopus (675) Google Scholar) using GST, maltose-binding protein, and a variety of other N-terminally tagged parkin proteins. The availability of full-length parkin lacking a fusion tag enabled us to compare in vitro auto-ubiquitination of GST-parkin and parkin to determine whether the bacterially purified protein was properly folded in the absence of an N-terminal carrier protein. Auto-ubiquitination reactions using GST-parkin and parkin were assessed using different combinations of ubiquitin, E1 enzyme, and the E2 ubiquitin conjugation enzyme Ubc7. For both GST-parkin and parkin a broad high molecular mass ladder was observed above the parent protein as monitored by either SDS-PAGE or Western blot analysis (Fig. 1). This ladder is characteristic of covalent modification by ubiquitin resulting in a distribution of multiple ubiquitinated forms of either GST-parkin or parkin proteins. In the absence of E1, ubiquitin, or parkin, the higher molecular mass species corresponding to a ubiquitinated parkin protein was not observed. These results indicate that both the bacterially expressed GST-parkin and parkin generated in this study are functional and capable of carrying out auto-ubiquitination in vitro. Although several studies have shown that bacterially expressed parkin carrying an N-terminal tag is functional in auto-ubiquitination assays, this work is the first to show that the untagged protein also participates in this function. This observation demonstrates that full-length parkin lacking an N-terminal tag adopts a correctly folded structure that can be used for further characterization of this E3 ligase protein. A New Domain in Parkin Identified by V8 and Trypsin Limited Proteolysis—Sequence similarity and alignment methods have identified the N-terminal UblD (1–75) and C-terminal RBR domains (238–465) in parkin, whereas the central UPD (76–238) conforms to no known domain sequence or structural motif (22Morett E. Bork P. Trends Biochem. Sci. 1999; 24: 229-231Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 46Upadhya S.C. Hegde A.N. Trends Biochem. Sci. 2003; 28: 280-283Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, to date only the UblD and IBR domains (23Beasley S.A. Hristova V.A. Shaw G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3095-3100Crossref PubMed Scopus (79) Google Scholar, 38Sakata E. Yamaguchi Y." @default.
• W2080806128 created "2016-06-24" @default.
• W2080806128 creator A5012246358 @default.
• W2080806128 creator A5018971090 @default.
• W2080806128 creator A5071060262 @default.
• W2080806128 creator A5089681362 @default.
• W2080806128 date "2009-05-01" @default.
• W2080806128 modified "2023-10-14" @default.
• W2080806128 title "Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin" @default.
• W2080806128 cites W1821174618 @default.
• W2080806128 cites W1877873625 @default.
• W2080806128 cites W1963962397 @default.
• W2080806128 cites W1966936049 @default.
• W2080806128 cites W1969950937 @default.
• W2080806128 cites W1977142694 @default.
• W2080806128 cites W1986091023 @default.
• W2080806128 cites W1986188355 @default.
• W2080806128 cites W1986896647 @default.
• W2080806128 cites W1989946157 @default.
• W2080806128 cites W1991231925 @default.
• W2080806128 cites W1998851985 @default.
• W2080806128 cites W2001324053 @default.
• W2080806128 cites W2009417817 @default.
• W2080806128 cites W2011314862 @default.
• W2080806128 cites W2012844038 @default.
• W2080806128 cites W2013558716 @default.
• W2080806128 cites W2013599007 @default.
• W2080806128 cites W2024794315 @default.
• W2080806128 cites W2025748949 @default.
• W2080806128 cites W2025840115 @default.
• W2080806128 cites W2026702943 @default.
• W2080806128 cites W2028267102 @default.
• W2080806128 cites W2036058336 @default.
• W2080806128 cites W2039824115 @default.
• W2080806128 cites W2040140774 @default.
• W2080806128 cites W2042201192 @default.
• W2080806128 cites W2047766237 @default.
• W2080806128 cites W2048332510 @default.
• W2080806128 cites W2051656237 @default.
• W2080806128 cites W2054580886 @default.
• W2080806128 cites W2061956914 @default.
• W2080806128 cites W2063127941 @default.
• W2080806128 cites W2068806054 @default.
• W2080806128 cites W2097062199 @default.
• W2080806128 cites W2098525070 @default.
• W2080806128 cites W2103022330 @default.
• W2080806128 cites W2104382960 @default.
• W2080806128 cites W2105249930 @default.
• W2080806128 cites W2105970121 @default.
• W2080806128 cites W2110780830 @default.
• W2080806128 cites W2111542278 @default.
• W2080806128 cites W2114876295 @default.
• W2080806128 cites W2114881821 @default.
• W2080806128 cites W2116786855 @default.
• W2080806128 cites W2117335809 @default.
• W2080806128 cites W2131079894 @default.
• W2080806128 cites W2135001691 @default.
• W2080806128 cites W2136000143 @default.
• W2080806128 cites W2136388422 @default.
• W2080806128 cites W2145683656 @default.
• W2080806128 cites W2150157946 @default.
• W2080806128 cites W2152395991 @default.
• W2080806128 cites W2154042815 @default.
• W2080806128 cites W2158674636 @default.
• W2080806128 cites W2160473598 @default.
• W2080806128 cites W2171483783 @default.
• W2080806128 cites W2232602227 @default.
• W2080806128 cites W2317804643 @default.
• W2080806128 cites W4297062289 @default.
• W2080806128 doi "https://doi.org/10.1074/jbc.m808700200" @default.
• W2080806128 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2685680" @default.
• W2080806128 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19339245" @default.
• W2080806128 hasPublicationYear "2009" @default.
• W2080806128 type Work @default.
• W2080806128 sameAs 2080806128 @default.
• W2080806128 citedByCount "118" @default.
• W2080806128 countsByYear W20808061282012 @default.
• W2080806128 countsByYear W20808061282013 @default.
• W2080806128 countsByYear W20808061282014 @default.
• W2080806128 countsByYear W20808061282015 @default.
• W2080806128 countsByYear W20808061282016 @default.
• W2080806128 countsByYear W20808061282017 @default.
• W2080806128 countsByYear W20808061282018 @default.
• W2080806128 countsByYear W20808061282019 @default.
• W2080806128 countsByYear W20808061282020 @default.
• W2080806128 countsByYear W20808061282021 @default.
• W2080806128 countsByYear W20808061282022 @default.
• W2080806128 countsByYear W20808061282023 @default.
• W2080806128 crossrefType "journal-article" @default.
• W2080806128 hasAuthorship W2080806128A5012246358 @default.
• W2080806128 hasAuthorship W2080806128A5018971090 @default.
• W2080806128 hasAuthorship W2080806128A5071060262 @default.
• W2080806128 hasAuthorship W2080806128A5089681362 @default.
• W2080806128 hasBestOaLocation W20808061281 @default.
• W2080806128 hasConcept C104317684 @default.
• W2080806128 hasConcept C115961737 @default.
• W2080806128 hasConcept C116834253 @default.
• W2080806128 hasConcept C126322002 @default.

• next