Quantitative proteomic analysis of Parkin substrates in Drosophila neurons

Aitor Martinez, Benoit Lectez, Juanma Ramirez, Oliver Popp, James D. Sutherland, Sylvie Urbé, Gunnar Dittmar, Michael J. Clague*, Ugo Mayor

*Corresponding author for this work

    Research output: Contribution to journalArticleResearchpeer-review

    43 Citations (Scopus)

    Abstract

    Background: 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. Methods: 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. Results: 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. Conclusions: Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.

    Original languageEnglish
    Article number29
    JournalMolecular Neurodegeneration
    Volume12
    Issue number1
    DOIs
    Publication statusPublished - 11 Apr 2017

    Keywords

    • Alzheimer's Disease (AD)
    • Drosophila melanogaster
    • In vivo
    • Label Free Quantification (LFQ)
    • Neurodegeneration
    • Parkin (PARK2)
    • Parkin substrates
    • Parkinson's Disease (PD)
    • Ubiquitination
    • VPS35

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