valinomycin and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

Topics: Cytosol; HEK293 Cells; HeLa Cells; Humans; Leupeptins; Mitochondria; Mitochondrial Membranes; Mitophagy; Parkinson Disease; Proteasome Inhibitors; Protein Binding; Protein Interaction Domains and Motifs; Protein Kinases; Protein Transport; Proteolysis; Ubiquitin-Protein Ligases; Valinomycin

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.

Topics: Carbonyl Cyanide m-Chlorophenyl Hydrazone; Fibroblasts; GTP Phosphohydrolases; Humans; Hydrogen Peroxide; Leupeptins; Macrolides; Membrane Proteins; Membrane Transport Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Models, Biological; Mutation; Oligopeptides; Proteasome Endopeptidase Complex; Protein Kinases; Protein Transport; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Valinomycin

Mutations in the PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are associated with autosomal-recessive forms of Parkinson disease (PD). Both are involved in the maintenance of mitochondrial integrity and protection from multiple stressors. Recently, Parkin was demonstrated to be recruited to impaired mitochondria in a PINK1-dependent manner, where it triggers mitophagy. Using primary human dermal fibroblasts originating from PD patients with various PINK1 mutations, we showed at the endogenous level that (i) PINK1 regulates the stress-induced decrease of endogenous Parkin; (ii) mitochondrially localized PINK1 mediates the stress-induced mitochondrial translocation of Parkin; (iii) endogenous PINK1 is stabilized on depolarized mitochondria; and (iv) mitochondrial accumulation of full-length PINK1 is sufficient but not necessary for the stress-induced loss of Parkin signal and its mitochondrial translocation. Furthermore, we showed that different stressors, depolarizing or non-depolarizing, led to the same effect on detectable Parkin levels and its mitochondrial targeting. Although this effect on Parkin was independent of the mitochondrial membrane potential, we demonstrate a differential effect of depolarizing versus non-depolarizing stressors on endogenous levels of PINK1. Our study shows the necessity to introduce an environmental factor, i.e. stress, to visualize the differences in the interaction of PINK1 and Parkin in mutants versus controls. Establishing human fibroblasts as a suitable model for studying this interaction, we extend data from animal and other cellular models and provide experimental evidence for the generally held notion of PD as a condition with a combined genetic and environmental etiology.

Topics: Blotting, Western; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytosol; Fibroblasts; Humans; Hydrogen Peroxide; Ionophores; Leupeptins; Mitochondria; Mutation; Oligopeptides; Oxidants; Parkinson Disease; Protein Kinases; Protein Transport; RNA Interference; Transfection; Ubiquitin-Protein Ligases; Valinomycin