Human ADCK proteins in Coenzyme Q biosynthesis and mitochondrial homeostasis

Among mitochondrial diseases, primary Coenzyme Q (CoQ10) deficiencies are a heterogeneous group of rare severe genetic disorders, associated with a broad spectrum of phenotypes. They are caused by pathogenic variants in genes involved in CoQ10 biosynthesis, leading to CoQ10 deficiency in organs and tissues. To date, the exogenous CoQ10 supplementation is the only effective therapeutic option for CoQ10 deficiency treatment but it must be noticed that still very little is known about molecular mechanisms related to these diseases and in particular about CoQ10 biosynthesis and functioning. This further confirms the need for in-depth understanding to create more specific and effective therapies. While Mcp2 and Coq8p in yeast are related to mitochondrial lipid homeostasis and the synthesis, morphology and stability of respiratory chain complexes, the specific roles of their human orthologs ADCK1 and ADCK5, COQ8A (ADCK3) and COQ8B (ADCK4) are still unknown. This study aims to investigate the physiological roles of Aarf Domain-Containing Kinase (ADCK) proteins and their possible involvement in CoQ10 biosynthesis and mitochondrial homeostasis and whether the paralogue pairs ADCK1-ADCK5 and COQ8A-COQ8B have redundant or divergent roles in mitochondria. To achieve this, it was employed CRISPR/Cas9 technology in Human Embryonic Kidney (HEK) 293 cell line to generate knockout (KO) cell lines: ADCK1 -/-, ADCK5 -/-, ADCK1/5 -/-, COQ8A -/-, COQ8B -/- and COQ8A/B -/-. KO cell lines were then functionally characterized. In our ADCK1 -/-, ADCK5 -/-, ADCK1/5 -/- cellular models we didn’t find significant differences in CoQ10 level and enzymatic activity of respiratory chain complexes and organization of supercomplexes. Contrary to what is reported in literature, our findings suggest that neither ADCK1 nor ADCK5 are involved in the biosynthesis of CoQ10 but have other roles, partly with opposite effects on the control of respiration and mitochondrial morphology. As expected, we found a reduction on CoQ10 levels, respiration, activity of the respiratory chain complexes, the organization of super-complexes and the absence of the COQ proteins only in COQ8A/B-/-. Moreover, metabolomics analysis showed a general metabolic shutdown in COQ8A/B-/- cells, while some interesting differences were found in COQ8A-/- and COQ8B-/- that reflect also at transcriptomic level. Overall, these findings identified specific roles for the proteins COQ8A and COQ8B in biosynthesis of CoQ10 and lead to new hypothesis in the control of mitochondrial metabolism.