Converging Evidence of Mitochondrial Dysfunction in a Yeast Model of Homocysteine Metabolism Imbalance

Abstract

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.

Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae

“…Currently, there is growing evidence that programmed cell death (PCD) is present in many unicellular organisms, such as protozoa, bacteria, slime moulds and yeasts. The presence of apoptotic phenotypes in Protista seems to confirm the hypothesis that apoptosis or some kind of it could have been (Madeo et al 1997;Allen et al 2006;Buttner et al 2007;Du et al 2008;Gao et al 2011) 2. a) Microscopy/FACS -TUNEL assay (Madeo et al 1997(Madeo et al , 1999(Madeo et al , 2002bGranot et al 2003;Bussche and Soares 2011;Ferreira et al 2011;Gao et al 2011;Guaragnella et al 2011;Izquierdo et al 2011;Kumar et al 2011;Liu et al 2011) b) DNA ladder -Electrophoresis (not used nowadays) c) Electron microscopy 3. a) Microscopy/FACS -DAPI staining; (Madeo et al 2002b;Buttner et al 2007;Du et al 2008;Aerts et al 2008;Bussche and Soares 2011;Izquierdo et al 2011;Ferreira et al 2011;Gao et al 2011) b) Electron microscopy (Madeo et al 1997;Gao et al 2011) 4. Fluorescence microscopy/FACS (Ludovico et al 2002) developed in unicellular organisms long before the evolutionary separation between fungi, plants and animals (Shemarova 2010;Semighini et al 2011).…”

The oxidative stress response of the filamentous yeastTrichosporon cutaneumR57 to copper, cadmium and chromium exposure

“…Transition metal copper is one of the most potent elements catalysing Fenton's reaction. Copper treatment induced ROS generation in yeasts S. cerevisiae [ 43 ] and fungi Podospora anserina ,[ 44 ] Humicola lutea [ 16 ] and aquatic hyphomycetes Varicosporium elodeae and Heliscus submersus . [ 45 ] Similar results have been reported for several plants [ 46 ] and human cells.…”

Suppressive Effects of Natural Compounds on Methionine Auxotrophy of a Cu,Zn-Superoxide Dismutase-Deficient Mutant of <i>Saccharomyces cerevisiae</i>

“…Several lines of evidence show that Hcy exerts an adverse effect on endothelial cells by increasing oxidative stress (Kolling et al, 2011;Leung et al, 2013). In yeast cells, high levels of Hcy can lead to mitochondrial dysfunction, which could potentially initiate proapoptotic pathways (Kumar et al, 2011). Met-O naturally occurs in protein as the oxidized form of Met residue as well as a free form amino acid, and free Met-O can be reversed by the methionine sulfoxide reductase family enzymes MsrA and fRMsr (Moskovitz et al, 2000;Le et al, 2009).…”