The Roles of Human COQ5 Protein in the Conditions of Mitochondrial Dysfunction and Oxidative Stress with Alterations in Endogenous Coenzyme Q10 Levels

Coenzyme Q (CoQ) is not only an essential mobile electron carrier in the mitochondrial electron transport chain (ETC), but also the only endogenously synthesized lipid-soluble antioxidant. Moreover, CoQ also serves as a cofactor for dihydroorotate dehydrogenase (DHODH), a mitochondrial enzyme for pyrimidine biosynthesis that can be inhibited by the complex III inhibitor antimycin A (AA). The biosynthetic pathway of CoQ10 and its regulation in human cells is poorly understood despite of many studies in yeast. Nine Coq proteins, Coq1p-Coq9p, were found to be essential for the terminal biosynthesis of CoQ6 in the yeast. Coq5p were shown to be a C-methyltransferase and critical for the stability of Coq3p and Coq4p. Those Coq proteins were found to be mitochondrial proteins and form a multi-subunit complex in the mitochondria of yeast. Coq1p in yeast corresponds to PDSS1 and PDSS2 subunits in humans. Mutations of some PDSS and COQ genes have been identified to cause primary CoQ10 deficiency disease in humans, but mechanisms of secondary CoQ10 deficiency disease caused by mitochondrial diseases are unknown. Our previous study showed that H2O2 (a reactive oxygen species) increased CoQ10 levels, whereas mtDNA depletion (ρ0 status), AA, or FCCP (a chemical uncoupler that dissipates proton gradient) suppressed CoQ10 levels in human 143B cells; however, both FCCP and H2O2 up-regulated several COQ genes, including COQ5. Currently the major difficulties in studying endogenous CoQ10 are the lack of validation on commercial antibodies for human PDSS and COQ proteins and the lack of commercial substrates for assaying enzymatic activities of those proteins. Recently we produced a self-made antibody that could specifically recognize both precursor and mature forms of human COQ5 and revealed that the mature form was only localized in the mitochondria and its maturation was suppressed by FCCP. Moreover, knockdown of COQ5 decreased not only CoQ10 levels, but also protein levels of cytochrome c, another mobile electron carrier in ETC. On the other hand, we found that the suppressive effect of AA on CoQ10 levels was enhanced under the condition of uridine supplementation, indicating the link between pyrimidine status and regulation of CoQ10 levels. The goals of this project are to investigate the roles of COQ5 gene or protein in the regulation of CoQ10 levels and cell survival under basal and different conditions of mitochondrial dysfunction and oxidative stress. The hypothesis to be tested is that human COQ5 is important for regulating CoQ10 levels in human cells in response to conditions of mitochondrial dysfunction and oxidative stress and might play protective roles independent of its enzymatic function. In this project, we will select stable clones from 143B cells for overexpression and knockdown of COQ5 to investigate the relationship between COQ5 levels and CoQ10 levels, expression of PDSS and other COQ genes, and expression of cytochrome c; investigate the possible interaction of COQ5 protein with other proteins, especially COQ proteins, and the existence of COQ protein complex containing COQ5; and examine the role of COQ5 in cytotoxicity and the alteration of levels of CoQ10, PDSS or COQ genes, mitochondrial status, and oxidative stress after the treatments of FCCP, AA, and H2O2. The effect of DHODH knockdown will be investigated during the study on AA. All the aims proposed have never been investigated in the literature. Results from this study will provide significant advances in the understanding of regulation of CoQ10 levels related to COQ5 protein in response to mitochondrial dysfunction and oxidative stress.

Project ID:PC10401-0162
External Project ID:NSC102-2320-B182-026-MY3