Intracellular thiols like cysteine, homocysteine and glutathione play a critical role in the regulation of important cellular processes. Alteration of intracellular thiol concentration results in many diseased states; for instance, elevated levels of homocysteine are considered to be an independent risk factor for cardiovascular disease. Yeast has proved to be an excellent model system for studying many human diseases since it carries homologues of nearly 40% of human disease genes and many fundamental pathways are highly conserved between the two organisms. In the present study, we demonstrate that cysteine and homocysteine, but not glutathione, inhibit yeast growth in a concentration-dependent manner. Using deletion strains (str2Delta and str4Delta) we show that cysteine and homocysteine independently inhibit yeast growth. Transcriptional profiling of yeast treated with cysteine and homocysteine revealed that genes coding for antioxidant enzymes like glutathione peroxidase, catalase and superoxide dismutase were down-regulated. Furthermore, transcriptional response to homocysteine did not show any similarity to the response to H2O2. We also failed to detect induction of reactive oxygen species in homocysteine- and cysteine-treated cells, using fluorogenic probes. These results indicate that homocysteine- and cysteine-induced growth defect is not due to the oxidative stress. However, we found an increase in the expression of KAR2 (karyogamy 2) gene, a well-known marker of ER (endoplasmic reticulum) stress and also observed HAC1 cleavage in homocysteine- and cysteinetreated cells, which indicates that homocysteine- and cysteine-mediated growth defect may probably be attributed to ER stress. Transcriptional profiling also revealed that genes involved in one-carbon metabolism, glycolysis and serine biosynthesis were up-regulated on exogenous addition of cysteine and homocysteine, suggesting that cells try to reduce the intracellular concentration of thiols by up-regulating the genes involved in their metabolism.

An elevated level of homocysteine, a thiol amino acid, is associated with various complex disorders. The cellular effects of homocysteine and its precursors S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) are, however, poorly understood. We used Saccharomyces cerevisiae as a model to understand the basis of pathogenicity induced by homocysteine and its precursors. Both homocysteine and AdoHcy but not AdoMet inhibited the growth of the str4⌬ strain (which lacks the enzyme that converts homocysteine to cystathioninemimicking vascular cells). Addition of AdoMet abrogated the inhibitory effect of AdoHcy but not that of homocysteine indicating that an increase in the AdoMet/AdoHcy ratio is sufficient to overcome the AdoHcy-mediated growth defect but not that of homocysteine. Also, the transcriptomic profile of AdoHcy and homocysteine showed gross dissimilarity based on gene enrichment analysis. Furthermore, compared with homocysteine, AdoHcy treatment caused a higher level of oxidative stress in the cells. However, unlike a previously reported response in wild type (Kumar, A., John, L., Alam, M. M., Gupta, A., Sharma, G., Pillai, B., and Sengupta, S. (2006) Biochem. J. 396, 61-69), the str4⌬ strain did not exhibit an endoplasmic reticulum stress response. This suggests that homocysteine induces varied response depending on the flux of homocysteine metabolism. We also observed altered expression of mitochondrial genes, defective membrane potential, and fragmentation of the mitochondrial network together with the increased expression of fission genes indicating that the imbalance in homocysteine metabolism has a major effect on mitochondrial functions. Furthermore, treatment of cells with homocysteine or AdoHcy resulted in apoptosis as revealed by annexin V staining and TUNEL assay. Cumulatively, our results suggest that elevated levels of homocysteine lead to mitochondrial dysfunction, which could potentially initiate pro-apoptotic pathways, and this could be one of the mechanisms underlying homocysteineinduced pathogenicity.

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