DEVELOPMENT OF MODELS FOR ELUCIDATING THE GENETIC BASIS AND PATHOGENESIS OF MITOCHONDRIAL DISEASES AND RELATED CONDITIONS

The yeast Saccharomyces cerevisiae is a cheap, time-saving, and versatile tool to study human genetic diseases, since a great number of genes and biological processes are evolutionarily conserved. Moreover, residues where mutations occur in human are often conserved in yeast. Not least, yeast can grow either by mitochondrial-dependent respiration or by ethanol fermentation, thus permitting to study mitochondrial diseases. We utilized this model organism to assess the functional impact of mutations in STRADA, SGPL1, and COQ genes, which are linked to primary CoQ10 deficiency, and where our goal was also to explore and enhance our understanding of their involvement in CoQ biosynthesis. Loss of function in the STRADA gene leads to PMSE syndrome (MIM #611087), a rare neurodevelopmental disorder characterized by drug-resistant epilepsy and severe developmental delays. Treatment with sirolimus has been effective. We employed a yeast model to evaluate the missense variants Ser264Arg in STRADA and its complete deletion, underscore the significance of timely molecular diagnosis for these patients and highlight the utility of yeast as a straightforward yet effective tool for validating the pathogenicity of missense variants. The SGPL1 gene encodes an enzyme involved in sphingolipid metabolism. Yeast's similarity to the human pathway makes it suitable for studying diseases like SPLIS (MIM # 617575), an autosomal recessive syndrome characterized by steroid-resistant nephrotic syndrome and multisystemic manifestations, resembling CoQ deficiency. Vitamin B6 shows promise in mitigating these diseases. Our study focused on the Gly210Glu mutation, which is situated at the pyridoxal 5'-phosphate (PLP) binding site (active form of vitamin B6) and revealed a potential role for the yeast gene GAD1. Coenzyme Q (CoQ) biosynthesis involves multiple genes. We investigated PDSS1, PDSS2, COQ4, and COQ5 in yeast. In yeast, the COQ1 gene controls the length of the polyprenyl chain and the number of isoprene units in CoQ synthesis. In contrast, humans and mice rely on PDSS1 and PDSS2 subunits to form heterotetramers for CoQ biosynthesis. This study aimed to clarify the roles of polyprenyl diphosphate genes and explore hybridization between human and murine sequences in determining isoprene chain length in S.cerevisiae, where the orthologous protein typically forms homodimeric or II homotetrameric structures. We used a yeast model and revealed that both murine Pdss1 and Pdss2 effectively complemented the absence of the coq1 gene in S.cerevisiae. Thus, we also validated the pathogenicity of 12 human missense mutations, 7 in PDSS1 (His78Asn, Glu123Gly, Val239Gly, Gln245His, Gly296Arg, Asp308Glu, Ser370Arg) and 5 in PDSS2 (Val116Met, His162Arg, Ser382Leu, Ala384Asp, Ile391Thr) genes. Additionally, we investigated mitochondrial import capabilities of murine PDSS1 vs. human PDSS1, revealing the crucial role of the mPDSS1 Nterminal in mitochondrial import in S.cerevisiae. Finally, we quantified CoQ levels in hybrid strains using HPLC, confirming that PDSS1 subunit 1 primarily determines the number of isoprene units in the Q-aromatic ring. COQ4's role in CoQ synthesis remains unclear. We created a yeast model with coq4 deletion and identified the importance of its metal-binding site, through the mutant Asp164Ala, and its catalytic activity. We expressed wild-type and mutant COQ4 proteins in bacteria for future enzymatic assays. COQ5 is crucial for CoQ synthesis and interacts with the CoQ-synthome. We confirmed genotypephenotype correlations using yeast and studied severe and milder patient mutations, as Gly118Ser and Arg123Trp. Saccharomyces cerevisiae, a simple eukaryotic model, offers a well-annotated genome, rapid generation times, and a versatile toolkit. These attributes position yeast as an optimal model for understanding genes and mutations associated with human diseases.