Acetylation Status of Mitochondrial Chaperone TRAP1

Abstract

Parkinson’s disease (PD) is well-known neurodegenerative disorder characterized by dysfunction of dopaminergic neuron. The loss of dopaminergic neurons occurs for several reasons. Misfolded alpha-synuclein (aSYN) aggregation is well-known hallmark of disease of PD. Misfolded aSYN aggregates form Lewy bodies that preferentially bind to mitochondrial importers and suppress mitochondrial protein import. Reduced mitochondrial protein import impairs the mitochondrial complex formation, eventually leading to mitochondrial damage. The impairment of mitochondria complex I disrupts several mitochondrial pathway, including increase of reactive oxygen species (ROS), loss of membrane potential (MMP) and decrease of adenosine triphosphate (ATP) production. Mitochondrial targeted PD therapy is mitochondrial quality improvement, energy production and reduction of oxidative stress Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone which is involved in multiple mitochondria pathways including stabilization of mitochondrial complexes, regulation of ROS levels and maintenance of membrane potential. Sirtuin 3 (SIRT3) is an NAD+ dependent deacetylase enzyme that regulates mitochondrial protein through deacetylation activity. Previous research has shown SIRT3 activates TRAP1 through deacetylation, while TRAP1 simultaneously facilitates the folding SIRT3 through positive feedback loop. In this study, we evaluated four acetylation sites (K96, K332, K431 and K466) of TRAP1 by performing acetylation mimic mutants lysine(K)-to-glutamine(Q) and deacetylation mimic mutants lysine(K)-to-arginine(R). Through TRAP1 chaperone activity measurements using ATPase assay and SIRT3 activity assay. K96Q and K332Q exhibited decreased of TRAP1 activity and increased of client interaction. In cell-based experiments, Transfection of KQ and KR mutants to TRAP1 knock-out (KO) SH-SY5Y cells demonstrated the decrease of all mitochondrial complexes except K96R. The K96R showed reversible phenotypes in K96Q western blot analysis. The other mutants of lysine site (K332, K431 and K466) of TRAP1 showed minimal differences in western blot analysis. Transfection of TRAP1 K96Q increased sensitivity to rotenone-induced PD model which showed an increase of rotenone-induced cell viability, an increase of oxidative stress, a decrease of total ATP level and loss of membrane potential. Conversely, K96R mutant alleviated rotenone-induced cell viability, loss of ATP reduction, loss of membrane potential and oxidative stress. Taken together, TRAP1 is involved in multiple pathways of mitochondria under stress condition that alleviate ATP level reduction, MMP loss and ROS generation. These results suggest TRAP1 may have distinct therapeutic implication to PD treatment. Keywords: TRAP1, Acetylation, Parkinson’s disease, Mitochondria