Detection of 6-demethoxyubiquinone in CoQ Deficiency Disorders: Insights into Enzyme Interactions and Identification of Potential Therapeutics

Coenzyme Q (CoQ) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency.