Book of Abstracts - New Frontiers 2022

Abstracts of oral presentations

HOW CELLS ARE ABLE TO ELIMINATE MITOCHONDRIA PRODUCING TOO MUCH REACTIVE OXYGEN SPECIES (ROS)?

A. Zeb 1 , V. Choubey 1 , R. Gupta 1 , V. Veksler 2 , A. Kaasik 1

1 Department of Pharmacology of Institute of Biomedicine and translational Medicine, University of Tartu, Tartu, Estonia; 2 University Paris-Saclay, INSERM UMR- S 1180, Châtenay -Malabry, France Although low ROS levels can be beneficial in normal physiological functions, the excessive ROS accumulation, often generated by defective mitochondria, can lead to cell oxidative damage. This is particularly important for cells like cardiomyocytes or neurons, which are very rich in mitochondria and where tissue regenerat ion is almost absent. Mitophagy is essential to remove “worn - out” mitochondria and to improve the mitochondrial population quality. It is well known how depolarized mitochondria that have reached the end of their life cycle are removed by Pink1/Parkin-dependent mitophagy. However, inner membrane depolarization is not the only prerequisite for mitophagy. Dysfunctional mitochondria could keep their membrane potential, and this potential favors excessive ROS production damaging the host cell. Thus, to survive, the cell should have a mechanism(s) to remove these internal killers, which are not yet depolarized. The present study unravels a mechanism of KEAP1-dependent mitophagy induced by even moderate cell ROS level. KEAP1 is one of the primary cellular ROS sensors. We tested various proteins that possess the KEAP1 binding site and were known to interact with or stabilize PINK1. Among them, only mitochondrial phosphatase PGAM5 did accumulate in response to moderate mitochondrial ROS production and, at the same time, induced PINK1/PRKN dependent mitophagy. PGAM5 is found in cells in two forms: a full-length and a short form. After being imported into mitochondria, PGAM5 is either inserted into the mitochondrial outer membrane (full-length form) or cleaved by the inner mitochondrial membrane (IMM)-resident proteases and then released back to the cytosol (short form). Only the full-length PGAM5 was co-immunoprecipitated with KEAP1, suggesting that KEAP1, being localized only in the cytosol, should interact with PGAM5 before it is imported to the mitochondria or/and when it is already inserted into the outer mitochondrial membrane. To dissect these two possibilities, we overexpressed fluorescently tagged PGAM5 and followed its fate in cells. Although the fluorescent PGAM5 was normally localized in the mitochondria, it started to accumulate in the cytosol when its proteasomal degradation was blocked. Western blot analysis demonstrated that it was specifically the full-length form of PGAM5 accumulating in the cytosol in response to proteasomal inhibition. Taken together, this suggests that KEAP1 controls the degradation of cytosolic full-length PGAM5 before it is inserted into the outer mitochondrial membrane. We speculate that PGAM5 might directly affect PINK1 processing. PINK1 and PGAM5 are both cleaved by the same IMM-resident proteases and competition between the substrates would favor PINK1 stabilization (followed by Parkin recruitment) when ROS favor PGAM5 accumulates. This mechanism serves as fine-tuned feedback allowing cells to sense when the quality of their mitochondrial population is declining and to trigger mitophagy.

Keywords: mitochondria, ROS, mitophagy, membrane potential

Funding: Estonian Research Council (PRG400) and the European Regional Development Fund (Project No. 2014-2020.4.01.15-0012). A.K. was supported by Chan Zuckerberg Initiative and A.K. and V.V. by Estonian - French Research Program PARROT.

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