Effect of Different Models of Mitochondrial Dysfunction on Coenzyme Q10 Levels, Interaction of COQ Proteins, and Expression of COQ Genes and Proteins 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) of mitochondrial oxidative phosphorylation, but also the only endogenously synthesized lipid-soluble antioxidants. Although exogenous CoQ10 has been widely used in humans, the biosynthetic pathway of CoQ10 and its regulation in human cells is poorly understood. Nine COQ proteins, COQ1-COQ9, in which COQ1 consists of PDSS1 and PDSS2 subunits in humans, were found to be essential for the terminal biosynthesis of CoQ6 in the yeast. These proteins are mitochondrial proteins, in which most are matrix proteins, have some direct interaction among several COQ proteins, and form multi-subunit complex for biosynthesis of CoQ6 in the yeast, which has never been studied in human cells. Primary CoQ10 deficiency disease in humans is known to be associated with mutations of PDSS and COQ genes, but causes of many cases of secondary CoQ10 deficiency disease are still unknown. Our recent findings first demonstrated that oxidative stress induced by H2O2 elevated CoQ10 levels, while severe mitochondrial dysfunction caused by a complex III inhibitor, antimycin A (AA), mitochondrial DNA (mtDNA) depletion, or a chemical uncoupler, FCCP, resulted in suppression of CoQ10 levels, in human 143B cells even though AA and mtDNA depletion also increased levels of reactive oxygen species (ROS). However, both H2O2 and FCCP induced mRNA levels of several COQ genes. The results support our hypothesis that both ROS and mitochondrial energy deficiency may enhance common signaling pathways leading to alterations of expression of nuclear genes, but severe mitochondrial dysfunction might impede the import of mitochondrial proteins to form functional proteins, which requires mitochondrial membrane potential and ATP as energy sources. In order to further understand the relationship between various conditions of mitochondrial dysfunction and status of CoQ10 levels, protein and mRNA levels of PDSS or COQ, mitochondrial localization of PDSS and COQ proteins, and possible interaction COQ proteins, we will use different mitochondrial inhibitors, 143B-ρ0 cells without mtDNA, and a cybrid with mtDNA T8993G mutation, among which oxidative stress may be invovled, in this project to elucidate the above issues. Because several commercial antibodies for the detection of PDSS and COQ proteins are not useful, we will produce some self-made antibodies by purifying recombinant proteins for some of COQ proteins. On the other hand, we will express hemagglutinin (HA)-tagged PDSS or COQ fusion proteins in 143B cells in order to verify commercial or self-made antibodies against endogenous PDSS and COQ proteins, to examine whether these proteins are localized in the mitochondria in human cells, and to study possible protein interactions among COQ proteins in the human cells. Results from this study should contribute significantly to the understanding of the mechanism related to alterations of endogenous CoQ10 levels, COQ genes, and COQ proteins during mitochondrial dysfunction. That should be also highly relevant to secondary CoQ10 deficiency resulted from defects in oxidative phosphorylation.

Project ID:PC10007-0368
External Project ID:NSC100-2320-B182-014