title: Mutations in PINK1 and Parkin impair ubiquitination of Mitofusins in human fibroblasts.
abstract: PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H(2)O(2) resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.
authors.affiliation: Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Rakovic
authors.forename: Anne
authors.initials: A
authors.lastname: Grünewald
authors.forename: Jan
authors.initials: J
authors.lastname: Kottwitz
authors.forename: Norbert
authors.initials: N
authors.lastname: Brüggemann
authors.forename: Peter P
authors.initials: PP
authors.lastname: Pramstaller
authors.forename: Katja
authors.initials: K
authors.lastname: Lohmann
authors.forename: Christine
authors.initials: C
authors.lastname: Klein
mesh_terms: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics*; D021381:Protein Transport / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D054875:Ubiquitination / Q000187:drug effects; D014634:Valinomycin / Q000494:pharmacology; D000099202:PTEN-Induced Putative Kinase
chemical_list: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D020558:GTP Phosphohydrolases; D006861:Hydrogen Peroxide; D007976:Leupeptins; D018942:Macrolides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex; D011494:Protein Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D014634:Valinomycin; D000099202:PTEN-Induced Putative Kinase; C057620:bafilomycin A; C111567:parkin protein; C470018:MFN2 protein, human; C470017:Mfn1 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin