title: Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.
abstract: Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.
authors.affiliation: Section of Ophthalmology and Neuroscience, Leeds Institute for Molecular Medicine, Wellcome Trust Brenner Building, St. James's University Hospital, Leeds, UK.
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Davison
authors.forename: Kyla
authors.initials: K
authors.lastname: Pennington
authors.forename: Chao-Chun
authors.initials: CC
authors.lastname: Hung
authors.forename: Jianhe
authors.initials: J
authors.lastname: Peng
authors.forename: Rumana
authors.initials: R
authors.lastname: Rafiq
authors.forename: Antje
authors.initials: A
authors.lastname: Ostareck-Lederer
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Ostareck
authors.forename: Helen C
authors.initials: HC
authors.lastname: Ardley
authors.forename: Rosamonde E
authors.initials: RE
authors.lastname: Banks
authors.forename: Philip A
authors.initials: PA
authors.lastname: Robinson
mesh_terms: D002460:Cell Line; D018834:Chaperonin 60 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D006801:Humans; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012261:Ribonucleoproteins / Q000378:metabolism; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C072074:HNRNPK protein, human; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein