Biology subjects

Tomar, R. S.

Publications and source records attributed to Tomar, R. S..

7 recordsLinked to original sources

bioRxiv2025

Lipid droplet-sphingolipid crosstalk regulates Doa1 activity and DNA damage response

Neutral lipid droplets (nLDs) are dynamic storage organelles that protect the cells from lipotoxicity. They store excess neutral lipids, sequester heavy metals and serve as membrane reservoirs. However, their role in genome integrity remains unclear. Previous studies have linked the shortened lifespan of LD deficient cells to impaired mitochondrial functions caused by defective sterol metabolism, but the molecular origin of this dysfunction remains elusive. In this study, we demonstrate that lack of nLDs inhibits DNA damage sensing mechanism, causing insensitivity to DNA damaging agents. Despite sustained DNA breaks, LD deficient cells unable to activate the checkpoint kinases or arrest cell cycle progression. Persistent insensitivity to double strand breaks results in accumulation of mutations, loss of nuclear as well as mitochondrial DNA integrity leading to accelerated cellular ageing. Through systematic genetic dissection of nLD synthesis genes, we identify a crucial role of lanosterol esterification in lipid droplet formation and their role in the regulation of DNA damage sensing and repair responses. Our study identifies a key regulatory protein, Doa1 that functionally links lipid droplet biosynthesis to DNA damage repair kinase signalling. Together, these findings establish a previously uncharacterized role of neutral lipid droplets in the maintaining the genome stability by acting not only as metabolic buffers but also as signalling platforms that coordinate DNA damage sensing and repair processes. Our work reveals a new mechanistic connection between cellular lipid homeostasis and genome maintenance, highlighting lipid droplets as critical determinants of healthy cellular aging.

cell biology↗

bioRxiv2025

Metabolic regulation of copper homeostasis governs the Sec61-dependent protein translocation process in Saccharomyces cerevisiae

Concentration of cellular labile pool of copper must be strictly regulated because disruption in copper homeostasis results in diseases. In Saccharomyces cerevisiae, elevated levels of labile copper impair cell viability by inhibiting Sec61-mediated protein translocation into the endoplasmic reticulum. We investigated how metabolic pathways, specifically mitochondrial respiration and autophagy, contribute to copper homeostasis and the translocation of secretory proteins. We show that copper selectively inhibits protein translocation in yeast cells grown in minimal medium but not in rich medium, highlighting a critical role of nutrients in modulating copper toxicity. Supplementation of specific amino acids suppresses the copper-induced defects in protein translocation and cell death, identifying amino acids suppressors of the copper toxicity. Using a panel of gene deletion mutants affecting mitochondrial functions, autophagy, peroxisomes, and lipid droplets, we demonstrate that metabolic pathways regulate sub-cellular distribution of copper and translocation of secretory proteins. Further, disruption of redox and pH homeostasis, and pharmacological inhibition of respiration, reveals that correct subcellular trafficking of copper is essential to prevent inhibitory effects on protein translocation process. Together, our findings provide mechanistic insights into how metabolic status influences cellular copper homeostasis and the secretory pathway of proteins, with broader implications for understanding diseases of copper metabolism.

cell biology↗

bioRxiv2024

Screening of histone mutants reveals a domain within the N-terminal tail of histone H3 that regulates the Tup1-independent repressive role of Cyc8 at the active FLO1

Yeast flocculation relies on cell surface flocculin proteins encoded by the FLO1 gene. The expression of FLO1 is antagonistically regulated by the Tup1-Cyc8 and the Swi-Snf complexes. The Post translational modifications of core histones regulate the transcription of Tup1-Cyc8-regulated genes. However, the mechanisms by which the physical presence of tail residues regulate FLO1 transcription process and flocculation is yet to be completely understood. Through screening we have identified a new region within the N-terminal tail of histone H3 regulating the transcription of FLO1 and FLO5. One of the histone H3 N-terminal truncation mutants H3{Delta}(17-24) showed higher FLO1 expression compared to wild-type H3. Results revealed that in absence of 17-24 stretch the occupancy of Cyc8 decreases from the upstream regions of FLO1. Additionally, analysis suggests that Hda1 is required for the Cyc8-mediated repression of FLO1. Altogether we demonstrate that 17-24 stretch is essential for the Tup1 independent binding of Cyc8 at the promoters assisted by Hda1, leading to the strong repression of FLO1 transcription. In the absence of the 17-24 stretch, Cyc8 cannot bind, resulting in uncontrolled transcription of FLO1.

molecular biology↗

bioRxiv2023

Cdc73 majorly regulates apoptosis-inducing factor (AIF1) in Saccharomyces cerevisiae via the H3K36 methylation

AIF1 overexpression is intimately linked to the sensitivity of the yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of its regulation in the cell would provide a significant understanding of the factors ultimately guiding yeast apoptosis. In this report, we establish the time-dependent induction of AIF1 in hydrogen peroxide stress. Additionally, the AIF1 expression in hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another significantly abrogates the AIF1 expression. However, substituting H3K4 or H3K79 with A does not affect the AIF1 expression level under hydrogen peroxide stress. Altogether, the significant reduction of AIF1 expression in cdc73{Delta} cells plausibly reflects the reduced H3K36me3 modification and is independent of the H3K4me3 modification.

genetics↗

bioRxiv2021

The SWI/SNF and RSC Cooperatively Remodel the Promoters of Unfolded Protein Response Targets and Heat Shock Genes

The ATP-dependent chromatin remodelling complexes maintain the chromatin dynamics, enabling the gene expression or its silencing. The SWI/SNF subfamily remodelers (SWI/SNF and RSC) generally promote gene expression by displacing or evicting nucleosomes at the promoter regions. Their action creates a nucleosome-depleted region where transcription machinery accesses the DNA. Their involvement has been shown critical for the induction of stress-responsive transcription programs. Although the role of SWI/SNF and RSC complexes in transcription regulation of heat shock responsive genes is well studied, their involvement at other pathway genes such as UPR, HSP and PQC is less known. In this study, we showed that the SWI/SNF occupies promoters of UPR, HSP and PQC genes in response to the unfolded protein stress, and its recruitment at UPR promoters is dependent on the Hac1 transcription factor and other epigenetic factors like Ada2 and Ume6. Disruption of SWI/SNFs activity does not affect the remodelling of these promoters or gene expression. However, inactivation of both RSC and SWI/SNF complexes diminishes expression of most of the UPR, HSP and PQC genes tested. Altogether these results suggest that these two remodelers work together or one compensates the loss of the other to ensure optimal induction of the stress-responsive genes.

molecular biology↗

bioRxiv2018

A novel link between Cell Wall Integrity pathway and Flocculation, regulated by yeast Sen1 dependent interplay between Rlm1 and Tup1

The budding yeast, Saccharomyces cerevisiae is one of the most studied organisms used for the synthesis of products, to explore the human diseases and eukaryotic gene expression mechanisms. The yeast cells with flocculation property are in high demand for industrial applications. However, the pathogenic yeast becomes drug-resistant due to flocculation/biofilm phenotype. The flocculation property of yeast depends on the expression of specific FLO genes. Genetic and epigenetic factors have been suggested to induce the expression of FLOs and flocculation, an evolutionarily conserved process. The present study was undertaken to identify a molecular link between stress caused by genetic and epigenetic factors and expression of FLOs. We utilized flocculating yeast strains to study the regulation of FLO genes and flocculation phenotype. We found rough surface morphology and constitutive activation of Slt2 in flocculating cells. The external cell wall stress factors as well as specific mutations in Sen1 and histone proteins strongly correlated with the induction of FLO genes whereas deletion of SLT2/RLM1, suppressed the expression and flocculation phenotype. We detected constitutive binding of Rlm1 and eviction of Tup1 from the promoters of FLO1 and FLO5 genes in flocculating cells. Thus we provide evidence for the CWI pathway dependent flocculation of yeast, regulated by Sen1 mediated interplay between Tup1 and Rlm1.

molecular biology↗

bioRxiv2018

Cantharidin alters GPI-anchored protein sorting by targeting Cdc1 mediated remodeling in Endoplasmic Reticulum

Cantharidin (CTD) is a potent anticancer small molecule produced by several species of blister beetle. It has been a traditional medicine for the treatment of warts and tumors for many decades. CTD suppresses the tumor growth by inducing apoptosis, cell cycle arrest, and DNA damage. It is a known inhibitor of PP2A and PP1. In this study, we identified new molecular targets of CTD using Saccharomyces cerevisiae as a model organism which expresses a Cantharidin Resistance Gene (CRG1). CRG1 encodes a SAM-dependent methyltransferase that inactivates CTD by methylation. CTD alters lipid homeostasis, cell wall integrity, endocytosis, adhesion, and invasion in yeast cells. We found that CTD specifically affects the phosphatidylethanolamine (PE) associated functions which can be rescued by supplementation of ethanolamine (ETA) in the growth media. CTD also perturbed ER homeostasis and cell wall integrity by altering the GPI-anchored protein sorting. The CTD dependent genetic interaction profile of CRG1 revealed that Cdc1 activity in GPI-anchor remodeling is the key target of CTD, which we found to be independent of PP2A and PP1. Furthermore, our experiments with human cells suggest that CTD functions through a conserved mechanism in higher eukaryotes as well. Altogether, we conclude that CTD induces cytotoxicity by targeting Cdc1 activity in GPI-anchor remodeling in the endoplasmic reticulum (ER).

cell biology↗