Regulation of Mitochondrial Autophagy by SUMO Proteases in Human Cells under Hypoxia

Hypoxia is implicated in several conditions including ischemia and age-related diseases like neurodegenerative disorders and cancer. Mitochondrial dysfunction and disturbances in mitophagy accompany hypoxia and can ultimately lead to cell death. HIF-1 is the master transcription factor in regulating transcription of hypoxic response genes, including those involved in autophagy and mitophagy. Hypoxia-induced mitophagy (HIM) clears defective or excess mitochondria when the cell undergoes hypoxic cell stress. The reversible post-translational modification, SUMOylation, regulates the HIF-1 pathway. SUMOylation itself is regulated by deSUMOylation enzymes such as SENP1 and SENP3. While SENP1 and SENP3 regulate aspects of the HIF-1 pathway and hypoxic response, it is unknown whether they regulate HIM. Understanding the mechanisms that regulate HIM is crucial in developing therapeutics for diseases linked to hypoxia. To investigate the regulation of HIM through the activity of SENP1 and SENP3, we performed RNAi-mediated knockdowns of proteins of interest and utilized the dual-fluorescent probe, Mito-pHfluorin, in immunofluorescence microscopy to quantify and compare the levels of HIM in HeLa cells between different experimental conditions. In this thesis, we uncovered molecular mechanisms that provide evidence of a TBC1D17-mediated SENP3-Fis1 axis that regulates HIM. SENP3, but not SENP1, is required for HIM and deSUMOylates the mitochondrial fission protein Fis1. Furthermore, Fis1 is also necessary for HIM. The SUMOylation status of Fis1 regulates HIM levels: deSUMOylated Fis1 induces mitophagy under hypoxia, whereas Fis1 that cannot be deSUMOylated reduces HIM. SUMOylation overall, especially SUMO-2/3-ylation, suppresses HIM. TAK-981, a potent inhibitor of global SUMOylation, significantly increases HIM. The Rab-GAP TBC1D17 suppresses HIM likely through its interaction with Fis1. However, whether the mechanism behind TBC1D17-dependent Fis1-mediated HIM involves SUMO remains unknown. Additionally, this study uncovered the capability of TAK-981 in upregulating mitophagy and MAM formation, leading to the discovery of a novel form of mitophagy, SUMOylation inhibition-induced mitophagy.