The Study on the Mechanisms of Coenzyme Q10 Deficiency Induced by Inhibitors of Mitochondrial Oxidative Phosphorylation in Human Cells

Dysfunction of mitochondrial oxidative phosphorylation, which is often accompanied by oxidative stress, plays an important role in the pathogenesis of human diseases and toxicity of xenobiotics. Coenzyme Q (CoQ) is not only an essential mobile electron carrier in the electron transport chain (ETC), but also the only endogenously synthesized lipid-soluble antioxidant. The biosynthetic pathway of CoQ10 and its regulation in human cells is poorly understood despite of many studies in yeast. Nine COQ proteins, COQ1-COQ9, were found to be essential for the terminal biosynthesis of CoQ6 in the yeast. COQ1 in humans consists of PDSS1 and PDSS2 subunits. These COQ proteins are mitochondrial proteins and there is direct interaction among some COQ proteins to form multi-subunit complex possibly required for biosynthesis of CoQ6 in the yeast. Primary CoQ10 deficiency disease is known to be caused by mutations of PDSS and COQ genes, but mechanisms of secondary CoQ10 deficiency disease caused by mitochondrial diseases are unknown. Our recent published results demonstrated that oxidative stress induced by H2O2 elevated CoQ10 levels, whereas antimycin A (AA), a complex III inhibitor, or FCCP, a chemical uncoupler, resulted in suppression of CoQ10 levels, in human 143B cells even though AA also increased levels of reactive oxygen species (ROS). In the current ongoing NSC project, we also found that oligomycin, a complex V inhibitor that can increase ROS from ETC due to increased proton gradient, decreased CoQ10 levels. We have also produced specific antibody for human COQ5 protein and found its localization in mitochondria. The goals of this new project are to investigate mechanisms involved in the down-regulation of CoQ10 levels in human cells induced by AA, FCCP, and oligomycin; and to explore the existence of multiple-subunit COQ protein complex or interaction among multiple COQ proteins. The hypothesis to be tested is that defective oxidative phosphorylation may decrease CoQ10 biosynthesis in human cells by (1) decreasing mRNA or protein levels of PDSS or COQ genes; (2) inhibiting import of mitochondrial PDSS and COQ proteins into mitochondrial matrix for subsequent maturation or interaction and assembly into a putative COQ protein complex for CoQ10 biosynthesis due to energy deficiency; or (3) oxidative damage to mitochondrial proteins. We will complete the investigation on oligomycin in 143B cells and compare effects of those three inhibitors on levels of CoQ10, ROS, ATP, and PDSS/COQ proteins in normal human WI38 cells and transformed WI38 (VA13) cells. We also need to verify commercial antibodies for human PDSS and COQ proteins by overexpressing these proteins with and without HA tag in cells. Furthermore, we will examine possible interaction between COQ proteins, putative COQ protein complex, and effect of mitochondrial inhibitors on these events. Finally, we will use novel superoxide dismutase mimics to investigate the role of oxidative damage to mitochondria in down-regulation of CoQ10 levels by AA and oligomycin in human cells. The significance of this study is better understanding on the mechanisms of secondary CoQ10 deficiency caused by mitochondrial dysfunction and the first investigation on putative interaction or assembly of COQ proteins into a supercomplex.

Project ID:PC10108-0812
External Project ID:NSC101-2320-B182-028