Human OVCA2 and its homolog FSH3-induced apoptosis in Saccharomyces cerevisiae.

Mammalian ovarian tumor suppressor candidate 2 (OVCA2) gene belongs to the family of serine hydrolase (FSH). This study aimed to elucidate the functional similarities of OVCA2 with its yeast homolog genes (FSH1, FSH2, and FSH3) regarding apoptosis. We found that the expression of OVCA2 in Saccharomyces cerevisiae increased production of reactive oxygen species (ROS), decreased cell growth, disturbed mitochondrial morphology, reduced membrane potential, increased chromatin condensation, and externalization of phosphatidylserine (PS) (annexin V/propidium iodide staining) indicating induced apoptotic cell death in yeast. We also showed that complementation of OVCA2 in fsh3Δ cells reduced cell growth and increased the apoptotic phenotypes. Collectively, our results suggest that complementation of human OVCA2 in fsh3Δ cells induced apoptosis in S. cerevisiae.

Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae.

The endoplasmic reticulum (ER) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. The SEC59 gene encodes dolichol kinase, required for protein glycosylation in the ER. The mutation of sec59-1 caused a protein N-glycosylation defect mediated ER stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced. In the sec59-1∆ cell, the N-glycosylation of vacuolar carboxy peptidase-Y (CPY) was significantly reduced; whereas the ER stress marker Kar2p and unfolded protein response (UPR) were significantly increased. Increased levels of Triacylglycerol (TAG), sterol ester (SE), and lipid droplets (LD) could be attributed to up-regulation of DPP1, LRO1, and ARE2 in the sec 59-1∆ cell. Also, the diacylglycerol (DAG), sterol (STE), and free fatty acids (FFA) levels were significantly increased, whereas the genes involved in peroxisome biogenesis and Pex3-EGFP levels were reduced when compared to the wild-type. The microarray data also revealed increased expression of genes involved in phospholipid, TAG, fatty acid, sterol synthesis, and phospholipid transport resulting in dysregulation of lipid homeostasis in the sec59-1∆ cell. We conclude that SEC59 dependent N-glycosylation is required for lipid homeostasis, peroxisome biogenesis, and ER protein quality control.

FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential. When wild type cells were overexpressed with FSH1 there was up regulation of AIF1 level when compared to control cells suggesting that overexpression of FSH1 regulated cell death in yeast.

Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae.

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I- and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription. The WT cells, under I- significantly reduced the expression of GCR1 and PIS1, but increased the expression of KCS1 and de-repressed INO1. The Kcs1p expression was reduced in gcr1∆ cells; this reduced INO1 expression resulting in abnormal vacuolar structure and reduced autophagy in Saccharomyces cerevisiae.

FSH3 mediated cell death is dependent on NUC1 in Saccharomyces cerevisiae.

Family of Serine Hydrolases (FSH) members FSH1, FSH2 and FSH3 in Saccharomyces cerevisiae share conserved sequences with the human candidate tumor suppressor OVCA2. In this study, hydrogen peroxide (H2O2) exposure increased the expression of both mRNA and protein levels of FSH3 in wild-type (WT) yeast cells. The deletion of FSH3 improved the yeast growth rate under H2O2-induction as compared to WT control cells. The overexpression of FSH3 in WT yeast cells caused an apoptotic phenotype, including accumulation of reaction oxygen species, decreased cell viability and cell death. The double deletions fsh1Δ fsh2Δ, fsh1Δ fsh3Δ and fsh2Δ fsh3Δ displayed increased growth compared to WT cells. However, the overexpression of FSH3 effectively inhibited cell growth in all double deletions. Moreover, the overexpression of FSH3 in cells lacking NUC1 did not cause any growth defect in the presence or absence of H2O2. Our results suggest that FSH3 induced apoptosis of yeast in a NUC1 dependent manner.

Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in Saccharomyces cerevisiae.

The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca(2+)) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress.

Defect of zinc transporter ZRT1 ameliorates cadmium induced lipid accumulation in Saccharomyces cerevisiae.

Cadmium (Cd) is a non-essential divalent heavy metal that enters the cells by utilizing the transport pathways of the essential metals, like zinc (Zn), in Saccharomyces cerevisiae. This work focuses on Cd accumulation and its impact on deletion of Zn transporters Zrt1p and Zrt2p and lipid homeostasis. Cd exposure reduces the Zn levels in the mutant strains, and the effect was higher in zrt2Δ cells. Upon Cd exposure, the wild-type and zrt2Δ cells follow a similar pattern, but an opposite pattern was observed in zrt1Δ cells. The Cd influx and ROS levels were high in both wild-type cells and zrt2Δ cells but significantly reduced in zrt1Δ cells. Cd exposure led to accumulation of triacylglycerol and lipid droplets in wild-type cells and zrt2Δ cells but these levels were decreased in zrt1Δ cells. Hence, these studies suggest that the zrt1Δ cells provide resistance towards Cd and aid in the maintenance of lipid homeostasis in yeast cells.