AbstractMitophagy prevents damaged mitochondria from compromising cell survival and homeostasis. Various mitophagy pathways have been discovered with the PINK1-Parkin pathway being most investigated. PINK1-Parkin mediated mitophagy occurs in stress-induced conditions. Interestingly, another pathway, the Nrf2-Keap1 pathway, which acts as sensor for stress within the cell, is also activated and mediates several cytoprotective functions, including redox and intermediary metabolism homeostasis, and autophagy. Studies have also indicated crossover between Nrf2 and mitophagy but there is limited knowledge of the underlying mechanisms. The aim of this research was to gain a deeper understanding of the relationship between the Nrf2-Keap1 pathway and mitophagy.
Previous studies have shown PINK1 to be regulated by Nrf2 in conditions of stress. To elucidate the underlying mechanisms, experiments were carried out in wild-type and PINK1 knockout S-HeLa cells under basal and stress conditions induced by treatment with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation. PINK1 did not influence Nrf2 transcript, protein levels, half-life, subcellular localisation or transcriptional activity. However, FCCP induced Nrf2 at the transcript and protein levels, and upregulated the expression of Nrf2-target genes, regardless of the presence of absence of PINK1. A sub-population of the Nrf2-Keap1 protein complex is tethered to mitochondria due to a Keap1-PGAM5 interaction, and it has been shown that Keap1 mediates the ubiquitination and proteasomal degradation of PGAM5. PGAM5, a mitochondrial serine/threonine phosphatase, stabilises PINK1 for mitophagy. Thus, it was hypothesised that Keap1 knockdown would lead to PGAM5 and PINK1 stabilisation and increase mitophagy. However, our studies showed that this was not the case, despite an over two-fold increase in PINK1 transcript levels. Instead, PINK1 protein levels decreased. Overall, it was found that reduction in the Keap1 levels (consequent to FCCP treatment, Keap1 knockdown or high dose of the electrophilic cyanoenone, TBE-31, which modifies cysteine sensors in Keap1) correlated with a reduction in the levels of PINK1. The implications of these findings are not fully understood and further investigation is necessary.
|Date of Award
|Medical Research Council
|Albena Dinkova-Kostova (Supervisor) & Miratul Muqit (Supervisor)