Abstract
Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson’s disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.
Highlights
Parkinson’s disease (PD) is the second most common neurodegenerative disease
In order to determine if mitochondrial TNF receptor-associated protein-1 (TRAP1)/ Heat shock protein of 90kDa (HSP90) inhibition activates mitophagy, we first tested the ability of G-TPP to induce PTEN-induced putative kinase 1 (PINK1) stabilization and kinase activity
HeLa cells stably expressing untagged Parkin were treated for different time points and accumulation of PINK1 and Ub kinase activity were analyzed by western blot
Summary
Parkinson’s disease (PD) is the second most common neurodegenerative disease. Genetic analyses have revealed a number of genes associated with disease [1]. Early-onset forms of PD are frequently caused by mutations in the genes encoding for PINK1 and Parkin [2]. A major breakthrough for the field was that the activities of both, the mitochondrial kinase PINK1 and the cytosolic E3 ubiquitin (Ub) ligase Parkin, could be linked to mitochondrial quality control (mitoQC). [3, 4] and that loss of either gene function disrupts this stress-activated and neuroprotective pathway [4, 5]. In the absence of stress, PINK1 is constitutively imported through the translocase of the outer/inner membrane (TOM/TIM) machinery into mitochondria where it undergoes cleavage by the mitochondrial processing peptidase (MPP) in the matrix and the presenilin-associated rhomboid-like protease (PARL) in the inner mitochondrial membrane (IMM) [6,7,8]. N-terminally processed PINK1 is transported back to the cytoplasm where it is degraded by the proteasome [9]
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