Exploring the impact of new USP14 inhibitors on mitophagy enhancement and identification of mitochondrial ubiquitination targets in cellular models

To maintain mitochondrial homeostasis, cells have evolved various mitochondrial quality control (MQC) mechanisms. Disruption of these mechanisms, leading to the accumulation of dysfunctional mitochondria, is strongly linked to the onset of neurodegenerative disorders. Mitophagy, a key component of MQC, refers to the selective degradation of damaged mitochondria via autophagy. Ubiquitin-mediated mitophagy relies on the antagonist actions of E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). Specific E3 ubiquitin ligases attach ubiquitin chains to Outer Mitochondrial Membrane (OMM) proteins, marking damaged mitochondria for degradation. In contrast, specific DUBs remove ubiquitin chains from the mitochondrial surface preventing organelle degradation. Inhibiting specific DUBs can, therefore, enhance mitophagy. Among these DUBs, USP14 is of particular interest due to its ability to regulate both autophagy and the ubiquitin-proteasome system. USP14 inhibition extends lifespan and rescue mitochondrial impairment in Drosophila model of Parkinson’s Disease (PD). Its inhibition enhances MARCH5-dependent mitophagy. Potent and highly specific inhibitors of USP14, such as IU1 and its derivatives IU1-47 and GNE-5997, are available. In this work, we showed an accumulation of ubiquitin and autophagic markers on mitochondria following USP14 inhibition with IU1-47 in HEK293 cells, indicative of on-going mitophagy in this condition. Additionally, we characterized the mitophagic effect of newly available inhibitor of USP14, GNE-5997, which has never been tested before. Treatment with GNE-5997 induces mitophagy and leads to a pattern in the accumulation of autophagic markers in iNeurons. Finally, we established conditions for the evaluation of the ubiquitination status of mitochondrial mitophagic targets, MFN2 and VDAC1.