Vikash Singh

Ebselen induces reactive oxygen species (ROS)‐mediated cytotoxicity in Saccharomyces cerevisiae with inhibition of glutamate dehydrogenase being a target

Ebselen is a synthetic, lipid‐soluble seleno‐organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting) assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH) or N‐acetyl‐l‐cysteine (NAC). Interestingly, a significant increase in ROS levels was noticed in gdh3‐deleted cells compared to wild‐type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two‐dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homolog of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis.

Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in Saccharomyces cerevisiae

Curcumin, a naturally occurring polyphenolic compound, is known to possess diverse pharmacological properties. There is a scarcity of literature documenting the exact mechanism by which curcumin modulates its biological effects. In the present study, we have used yeast as a model organism to dissect the mechanism underlying the action of curcumin. We found that the yeast mutants of histone proteins and chromatin modifying enzymes were sensitive to curcumin and further supplementation of iron resulted in reversal of the changes induced by curcumin. Additionally, treatment of curcumin caused the iron starvation induced expression of FET3, FRE1 genes. We also demonstrated that curcumin induces degradation of Sml1p, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. The degradation of Sml1p was mediated through proteasome and vacuole dependent protein degradation pathways. Furthermore, curcumin exerts biological effect by altering global proteome profile without affecting chromatin architecture. These findings suggest that the medicinal properties of curcumin are largely contributed by its cumulative effect of iron starvation and epigenetic modifications.

Sen1p contributes to genomic integrity by regulating expression of ribonucleotide reductase 1 (RNR1) in Saccharomyces cerevisiae.

Gene expression is a multi-step process which requires recruitment of several factors to promoters. One of the factors, Sen1p is an RNA/DNA helicase implicated in transcriptional termination and RNA processing in yeast. In the present study, we have identified a novel function of Sen1p that regulates the expression of ribonucleotide reductase RNR1 gene, which is essential for maintaining genomic integrity. Cells with mutation in the helicase domain or lacking N-terminal domain of Sen1p displayed a drastic decrease in the basal level transcription of RNR1 gene and showed enhanced sensitivity to various DNA damaging agents. Moreover, SEN1 mutants [Sen1-1 (G1747D), Sen1-2 (Δ1-975)] exhibited defects in DNA damage checkpoint activation. Surprisingly, CRT1 deletion in Sen1p mutants (Sen1-1, Sen1-2) was partly able to rescue the slow growth phenotype upon genotoxic stress. Altogether, our observations suggest that Sen1p is required for cell protection against DNA damage by regulating the expression of DNA repair gene RNR1. Thus, the misregulation of Sen1p regulated genes can cause genomic instability that may lead to neurological disorders and premature aging.

Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae

KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N‐terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109Δ mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double‐strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.

Assessment of the Biological Pathways Targeted by Isocyanate Using N-Succinimidyl N-Methylcarbamate in Budding Yeast Saccharomyces cerevisiae

Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates.

Anti-cancer drug KP1019 induces Hog1 phosphorylation and protein ubiquitylation in Saccharomyces cerevisiae.

Ruthenium-based anti-cancer drugs have attracted increasing interest in the last 20 years. KP1019 is one of the ruthenium-containing compounds that has demonstrated anti-tumor activity against various cancers, and has been tested in several clinical trials. Despite its success, the mode of action of KP1019 is not well described. In the present study, we have used budding yeast Saccharomyces cerevisiae to elucidate the action of KP1019. We have found that KP1019 causes dose-dependent cell arrest in the S-phase of cell cycle. Furthermore, we have demonstrated for the first time that the yeast mitogen-activated protein (MAP) kinase Hog1 is essential for the cells in response to KP1019. Hog1 is rapidly phosphorylated upon treatment with KP1019, and the deletion of the HOG1 gene potentiates the growth inhibition effect of KP1019. Moreover, we also observed the up-regulation of glycerol-3-phosphate dehydrogenase 1 (GPD1) mRNA in response to KP1019 treatment, a factor that is essential for the hyperosmotic stress response. Our results also reveal that membrane-bound sensor proteins of high osmolarity glycerol (HOG) pathway are crucial for Hog1 phosphorylation in response to KP1019-induced stress. Furthermore, KP1019 has also been found to increase the accumulation of ubiquitinated proteins and deletion of several members of ubiquitination pathways conferred sensitivity for KP1019. The findings presented here strongly suggest the ability of KP1019 to activate Hog1 MAP kinase and induce protein ubiquitination, which may underlie the therapeutic potential of this compound. In summary, we have disclosed a novel mechanism of KP1019 activity.

Mitogen-activated protein kinase Hog1 is activated in response to curcumin exposure in the budding yeast Saccharomyces cerevisiae

BackgroundCurcumin (CUR), an active polyphenol derived from the spice turmeric, has been traditionally used for centuries in ancient Indian medicine to treat a number of diseases. The physiological effects of CUR have been shown to be diverse; however, the target molecules and pathways that CUR affects have yet to be fully described.ResultsHere, we demonstrate for the first time that the budding yeast mitogen-activated protein kinase (MAPK) Hog1 is essential for the response to CUR. Moreover, CUR-induced Hog1 phosphorylation was rescued by supplementation of iron to the growth medium. Hog1 was rapidly phosphorylated upon CUR treatment, but unlike the response to hyperosmotic shock (0.8 M NaCl), it remains activated for an extended period of time. A detailed analysis of HOG pathway mutants revealed that Pbs2p, Ptc2p, and Ssk2p are required for optimal CUR-induced Hog1 phosphorylation. We also observed a Hog1 dependent transcriptional response to CUR treatment that involved the up-regulation of glycerol-3-phosphate dehydrogenase 1 (GPD1), a factor that is essential for the hyperosmotic stress response.ConclusionsOur present finding revealed the role of Hog1 MAPK in regulation of CUR-induced transcriptional response. We anticipate that our finding will enhance the understanding on the molecular mode of action of CUR on S. cerevisiae.

Signaling of Chloroquine-Induced Stress in the Yeast Saccharomyces cerevisiae Requires the Hog1 and Slt2 Mitogen-Activated Protein Kinase Pathways

ABSTRACT Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and the TOR pathway in Saccharomyces cerevisiae.

Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N‐terminal protein‐interaction and C‐terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome‐wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N‐terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin.

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N‐terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell‐wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis.