title: Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.
abstract: Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.
  
  
  
  Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.
  
  
  
  We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.
  
  
  
  Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.|Functional Genomics Unit, CIC bioGUNE, Derio, Spain.|Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
authors.forename: Aitor
authors.initials: A
authors.lastname: Martinez
authors.affiliation: Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.
authors.forename: Benoit
authors.initials: B
authors.lastname: Lectez
authors.affiliation: Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.|Functional Genomics Unit, CIC bioGUNE, Derio, Spain.
authors.forename: Juanma
authors.initials: J
authors.lastname: Ramirez
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Popp
authors.affiliation: Functional Genomics Unit, CIC bioGUNE, Derio, Spain.
authors.forename: James D
authors.initials: JD
authors.lastname: Sutherland
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Urbé
authors.affiliation: Department of Oncology, Luxembourg Institute of Health, Luxembourg City, Luxembourg.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. clague@liverpool.ac.uk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Clague
authors.affiliation: Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain. ugo.mayor@ehu.eus.|Functional Genomics Unit, CIC bioGUNE, Derio, Spain. ugo.mayor@ehu.eus.|Ikerbasque, Basque Foundation for Science, Bilbao, Bizkaia, Spain. ugo.mayor@ehu.eus.
authors.forename: Ugo
authors.initials: U
authors.lastname: Mayor
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004331:Drosophila melanogaster; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D024101:Mitochondrial Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D011494:Protein Kinases
keywords: Alzheimer’s Disease (AD); Drosophila melanogaster; In vivo; Label Free Quantification (LFQ); Neurodegeneration; Parkin (PARK2); Parkin substrates; Parkinson’s Disease (PD); Ubiquitination; VPS35