Investigating the mechanism of cellular toxicity during aberrant branched-chain amino acid metabolism

Amino acids, apart from being building blocks of proteins, serve various cellular and metabolic functions1,2. Changes in amino acid handling have been observed in a wide range of human pathologies, including diabetes and various metabolic disorders (aminoacidopathies)3–5. Saccharomyces cerevisiae is used as a model to investigate how increase in amino acid content (in the form of amino acid dropout mix: AAM) in growth medium influences cell growth. Intriguingly, it was observed that increasing the concentration of AAM in the media (double or triple times; 2 X AAM and 3 X AAM respectively), severely affects the growth of auxotrophic but not of prototrophic yeast strains in presence of glucose as carbon substrate. Increased concentration of Ehrlich amino acids, which are degraded to fusel acidic/alcoholic compounds, induced the observed slow growth phenotype of BY4742. These phenotypes can be rescued by either re-establishing the functional leucine biosynthetic pathway in BY4742 (leucine auxotroph) or increasing leucine in proportion to the increased AAM. Interestingly, the amino acid dependent growth phenotypes are absent when cells grow in media containing non-fermentable carbon sources. Furthermore, the deletion of ILV2 or ILV3 (genes encoding enzymes involved in the leucine biosynthetic pathway) also rescues the growth phenotype of BY4742 on 2 X AAM and 3 X AAM growth media. It was found that Ilv3 is the potential switching point and links cellular growth to redox homeostasis. The possibility of leucine limitation per se or transport competition between different Ehrlich amino acids and leucine, as a cause for the observed phenotypes, is ruled out. Upregulation of the branched-chain amino acid pathway inhibits cell growth of BY4742 on 2 X AAM. Although we could not detect KIV, the α-keto acid intermediate formed by the Ilv3. It is proposed that KIV itself (or its unknown downstream product) leads to the onset of the observed phenotypes. Different studies suggest that oxidative stress (due to accumulation of branched-chain amino acids (BCaa) and their α-keto acids) contributes to the neurological damage of MSUD patients6–9. It was also observed that the trigger of the BCaa bio-synthesis pathway on 2 X AAM growth conditions also contributes to the significant oxidative stress in the cell. In conclusion, we propose that yeast can be used as a suitable model system to study how accumulation of BCaa and their α-keto acids are lead to oxidative stress that is potentially toxic to cells. Further, this knowledge and the underlying molecular mechanisms will enhance our understanding of MSUD in humans