Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

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.

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

“…While it remains uncertain what exactly triggers activation of PINK1-PRKN-dependent mitoQC in vivo, organelle damage caused by mitochondrial DNA mutations, misfolded mitochondrial proteins and proteotoxic stress may be highly relevant factors [40][41][42]. In addition, it has become clear that the process is likely very localized and restricted to a few mitochondria or even submitochondrial regions at a time, as opposed to wholesale mitophagy observed in cell lines upon mitochondrial depolarization.…”

The different axes of the mammalian mitochondrial unfolded protein response

“…The following conditions causing mitochondrial protein misfolding have been shown to subsequently elicit the canonical UPR mt defined by chaperonin induction: (1) overexpression of misfolding-prone deletion mutants of OTCΔ; (2) inhibition of mitochondrial HSP90 [ 41 , 44 , 45 ]; (3) inhibition of lon peptidase 1 (LONP1) [ 41 , 46 ], which is crucial for the digestion of misfolded matrix proteins [ 12 ]; and (4) expression of another misfolding mitochondrial protein (EndoG, see below) [ 47 , 48 ]. Due to the high chaperonin protein levels under basal conditions [ 49 ], the analysis of changes in their levels has proven difficult as a readout for the UPR mt in mammalian cells, unless the UPR mt is induced chronically, or highly quantitative methods such as mass spectrometry are used [ 41 ].…”

“…However, there must also be destructive pathways activated upon failure to restore proteostasis. Strikingly, there have now been numerous reports of mitochondrial protein misfolding triggering LC3B lipidation, induction of autophagy genes, and mitophagy [ 41 , 44 , 48 , 94 , 95 ], suggesting that autophagy in general and the selective degradation of damaged mitochondria via this route might play a significant role in the UPR mt . However, substantiated evidence proving a causative relation between mitochondrial protein misfolding and autophagy is still lacking, and the same applies to insight into the mechanisms involved.…”

Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy

“…To test whether BAK ubiquitination by Parkin was restricted to AO treatment, we tested other mitophagy inducers. Treatment with the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or the mitochondrial targeted HSP90 inhibitor gamitrinibtriphenylphosphonium (GTPP; Munch & Harper, 2016;Fiesel et al, 2017) likewise induced significant ubiquitination of BAK ( Fig 3A).…”