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

[This corrects the article DOI: 10.1371/journal.pone.0092993.].

Unveiling the molecular networks underlying cellular impairment in Saccharomyces cerevisiae: investigating the effects of magnesium oxide nanoparticles on cell wall integrity and endoplasmic reticulum stress response.

Nanoparticles, particularly magnesium oxide nanoparticles (MgO-NPs), are increasingly utilized in various fields, yet their potential impact on cellular systems remains a topic of concern. This study aimed to comprehensively investigate the molecular mechanisms underlying MgO-NP-induced cellular impairment in Saccharomyces cerevisiae, with a focus on cell wall integrity, endoplasmic reticulum (ER) stress response, mitochondrial function, lipid metabolism, autophagy, and epigenetic alterations. MgO-NPs were synthesized through a chemical reduction method, characterized for morphology, size distribution, and elemental composition. Concentration-dependent toxicity assays were conducted to evaluate the inhibitory effect on yeast growth, accompanied by propidium iodide (PI) staining to assess membrane damage. Intracellular reactive oxygen species (ROS) accumulation was measured, and chitin synthesis, indicative of cell wall perturbation, was examined along with the expression of chitin synthesis genes. Mitochondrial function was assessed through Psd1 localization, and ER structure was analyzed using dsRed-HDEL marker. The unfolded protein response (UPR) pathway activation was monitored, and lipid droplet formation and autophagy induction were investigated. Results demonstrated a dose-dependent inhibition of yeast growth by MgO-NPs, with concomitant membrane damage and ROS accumulation. Cell wall perturbation was evidenced by increased chitin synthesis and upregulation of chitin synthesis genes. MgO-NPs impaired mitochondrial function, disrupted ER structure, and activated the UPR pathway. Lipid droplet formation and autophagy were induced, indicating cellular stress responses. Additionally, MgO-NPs exhibited differential cytotoxicity on histone mutant strains, implicating specific histone residues in cellular response to nanoparticle stress. Immunoblotting revealed alterations in histone posttranslational modifications, particularly enhanced methylation of H3K4me. This study provides comprehensive insights into the multifaceted effects of MgO-NPs on S. cerevisiae, elucidating key molecular pathways involved in nanoparticle-induced cellular impairment. Understanding these mechanisms is crucial for assessing nanoparticle toxicity and developing strategies for safer nanoparticle applications.

Dissecting the role of mitogen-activated protein kinase Hog1 in yeast flocculation.

Living organisms are frequently exposed to multiple biotic and abiotic stress forms during their lifetime. Organisms cope with stress conditions by regulating their gene expression programs. In response to different environmental stress conditions, yeast cells activate different tolerance mechanisms, many of which share common signaling pathways. Flocculation is one of the key mechanisms underlying yeast survival under unfavorable environmental conditions, and the Tup1-Cyc8 corepressor complex is a major regulator of this process. Additionally, yeast cells can utilize different mitogen-activated protein kinase (MAPK) pathways to modulate gene expression during stress conditions. Here, we show that the high osmolarity glycerol (HOG) MAPK pathway is involved in the regulation of yeast flocculation. We observed that the HOG MAPK pathway was constitutively activated in flocculating cells, and found that the interaction between phosphorylated Hog1 and the FLO genes promoter region increased significantly upon sodium chloride exposure. We found that treatment of cells with cantharidin decreased Hog1 phosphorylation, causing a sharp reduction in the expression of FLO genes and the flocculation phenotype. Similarly, deletion of HOG1 in yeast cells reduced flocculation. Altogether, our results suggest a role for HOG MAPK signaling in the regulation of FLO genes and yeast flocculation.

Cdc73 is a major regulator of apoptosis-inducing factor 1 expression in Saccharomyces cerevisiae via H3K36 methylation.

Apoptosis-inducing factor 1 (AIF1) overexpression is intimately linked to the sensitivity of yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of AIF1 regulation in the cell would provide a significant understanding of the factors guiding yeast apoptosis. In this report, we show the time-dependent induction of AIF1 under hydrogen peroxide stress. Additionally, we find that AIF1 expression in response to hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another amino acid significantly abrogates AIF1 expression. However, substituting the lysine (K) in H3K4 or H3K79 with alanine (A) does not affect AIF1 expression level under hydrogen peroxide stress. Altogether, reduced AIF1 expression in cdc73Δ is plausibly due to reduced H3K36me3 levels in the cells.

Investigation of the acetic acid stress response in Saccharomyces cerevisiae with mutated H3 residues.

Enhanced levels of acetic acid reduce the activity of yeast strains employed for industrial fermentation-based applications. Therefore, unraveling the genetic factors underlying the regulation of the tolerance and sensitivity of yeast towards acetic acid is imperative for optimising various industrial processes. In this communication, we have attempted to decipher the acetic acid stress response of the previously reported acetic acid-sensitive histone mutants. Revalidation using spot-test assays and growth curves revealed that five of these mutants, viz., H3K18Q, H3S28A, H3K42Q, H3Q68A, and H3F104A, are most sensitive towards the tested acetic acid concentrations. These mutants demonstrated enhanced acetic acid stress response as evidenced by the increased expression levels of AIF1, reactive oxygen species (ROS) generation, chromatin fragmentation, and aggregated actin cytoskeleton. Additionally, the mutants exhibited active cell wall damage response upon acetic acid treatment, as demonstrated by increased Slt2-phosphorylation and expression of cell wall integrity genes. Interestingly, the mutants demonstrated increased sensitivity to cell wall stress-causing agents. Finally, screening of histone H3 N-terminal tail truncation mutants revealed that the tail truncations exhibit general sensitivity to acetic acid stress. Some of these N-terminal tail truncation mutants viz., H3 [del 1-24], H3 [del 1-28], H3 [del 9-24], and H3 [del 25-36] are also sensitive to cell wall stress agents such as Congo red and caffeine suggesting that their enhanced acetic acid sensitivity may be due to cell wall stress induced by acetic acid.

Mechanisms of DNA methylation and histone modifications.

The field of genetics has expanded a lot in the past few decades due to the accessibility of human genome sequences, but still, the regulation of transcription cannot be explicated exclusively by the sequence of DNA of an individual. The coordination and crosstalk between chromatin factors which are conserved is indispensable for all living creatures. The regulation of gene expression has been dependent on the methylation of DNA, post-translational modifications of histones, effector proteins, chromatin remodeler enzymes that affect the chromatin structure and function, and other cellular activities such as DNA replication, DNA repair, proliferation and growth. The mutation and deletion of these factors can lead to human diseases. Various studies are being performed to identify and understand the gene regulatory mechanisms in the diseased state. The information from these high throughput screening studies is able to aid the treatment developments based on the epigenetics regulatory mechanisms. This book chapter will discourse on various modifications and their mechanisms that take place on histones and DNA that regulate the transcription of genes.

Copper inhibits protein maturation in the secretory pathway by targeting the Sec61 translocon in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum. These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the endoplasmic reticulum. Here, using immunoblotting, microscopy, and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated proteins, Gas1 (glycophospholipid-anchored surface protein) and CPY (carboxypeptidase Y), in the cytosol. We further show that the copper-sensitive phenotype of sec61-deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Furthermore, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper-mediated accumulation of post-translationally translocated proteins is due to the inhibition of Sec61.

Heavy metal exposure induces Yap1 and Hac1 mediated derepression of GSH1 and KAR2 by Tup1-Cyc8 complex.

Heavy metal pollution is one of the most severe environmental problem. The toxicity of heavy metals is correlated with the production of increased reactive oxygen species and misfolded protein accumulation. Exposures of these metals even at low concentrations adversely affect human health. The Tup1-Cyc8 complex has been identified as a general repressor complex, is also involved in the derepression of few target genes in association with gene-specific activator proteins. Exposure to heavy metals activates the antioxidant defense mechanism, essential for cellular homeostasis. Here we present evidence that TUP1/CYC8 deleted cells are compromised to tolerate heavy metals exposure. Upon metal-induced oxidative stress, Yeast AP-1p (Yap1) recruits the Tup1-Cyc8 complex to the promoter of oxidative stress response gene GSH1 and derepresses its expression. We also found that the TUP1/CYC8 deficient cells have altered endoplasmic reticulum (ER) homeostasis and fail to activate the unfolded protein response pathway. In response to ER stress, the Tup1-Cyc8 complex, with the help of activated Hac1, binds to the promoter of ER chaperone KAR2 and activates its transcription. Altogether, our findings suggest that the Tup1-Cyc8 complex is crucial for the activation of genes that are involved in the mitigation of oxidative and ER stress during heavy metal exposure.

Cantharidin downregulates PSD1 expression and inhibits autophagic flux in yeast cells.

Cantharidin is a terpenoid compound of insect origin, naturally produced by male blister beetles as an antipredatory mechanism. Cantharidin has anticancer properties, which are attributed to its ability to induce cell cycle arrest, DNA damage, MAPK signaling pathway, and apoptosis. Cantharidin has been reported to induce apoptosis in triple-negative breast cancer cells by suppressing autophagy via downregulation of Beclin 1 expression and autophagosome formation. However, it remains unclear which stage of the autophagic pathway is targeted by cantharidin. Herein, we report that yeast cells are sensitive to cantharidin, and external supplementation of ethanolamine (ETA) ameliorates the cytotoxicity. In addition, cantharidin downregulates phosphatidylserine decarboxylase 1 (PSD1) expression. We also report that cantharidin inhibits autophagic flux, and external administration of ETA could rescue this inhibition. Additionally, cotreatment with chloroquine sensitized the autophagy inhibitory effects of cantharidin. We conclude that yeast cells are sensitive to cantharidin due to inhibition of autophagic flux.

The H2A N-terminal tail is required to alleviate copper-induced stress in Saccharomyces cerevisiae.

Histone tail residues drive many biological processes by regulating genome-wide transcription. Functions of histone H3 and H4 tail residues in stress-responsive gene transcriptional programs have been extensively studied. The H2A tail residues have been shown to regulate DNA damage repair and oxidative stress response, but the involvement of N-terminal tail of H2A (H2ANtT) in proteostasis regulation is unknown. The unfolded protein response pathway (UPR) is an essential mechanism adopted by cells to prevent protein toxicity in response to ER stress. The disturbance in ER can occur by various factors such as heat stress, redox imbalance, exposure to xenobiotics and metals. Copper is utilized as a cofactor by cellular enzymes, but excessive copper affects ER homeostasis. We found that cells lacking 1-20 residues of H2ANtT are intolerant to copper stress, owing to the accumulation of misfolded proteins in the mutant cells. H2A 1-20 truncation also reduces the physiological UPR, and copper exposure further aggravates this effect. Furthermore, the expression of a spliced version of HAC1 mRNA in H2A∆(1-20) cells, encoding the downstream transcription factor of UPR signalling, rescues their growth under copper stress. Altogether these results provide evidence that H2ANtT reduces copper-induced ER stress by regulating UPR signalling.

The ATP-dependent SWI/SNF and RSC chromatin remodelers cooperatively induce unfolded protein response genes during endoplasmic reticulum stress.

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 function has been shown critical for inducing 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 in other pathways such as unfolded protein response (UPR) and protein quality control (PQC) is less known. This study shows that SWI/SNF occupies the promoters of UPR, HSP and PQC genes in response to unfolded protein stress, and its recruitment at UPR promoters depends on Hac1 transcription factor and other epigenetic factors like Ada2 and Ume6. Disruption of SWI/SNF's activity does not affect the remodeling of these promoters or gene expression. However, inactivation of RSC and SWI/SNF together diminishes induction of most of the UPR, HSP and PQC genes tested. Furthermore, RSC and SWI/SNF colocalize at these promoters, suggesting that these two remodelers functionally cooperate to induce stress-responsive genes under proteotoxic conditions.

Substitution of histone H3 arginine 72 to alanine leads to the deregulation of isoleucine biosynthesis in the budding yeast Saccharomyces cerevisiae.

Histone residues play an essential role in the regulation of various biological processes. In the present study, we utilized the H3/H4 histone mutant library to probe the functional aspects of histone residues in amino acid biosynthesis. We found that the histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of the arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in minimal medium. Histone mutant H3R72A requires external supplementation of either isoleucine, serine, or threonine for growth in minimal medium. We also observed that the H3R72 residue and leucine amino acid in synthetic complete medium might play a crucial role in determining the intake of isoleucine and threonine in yeast. Furthermore, gene deletion analysis of ILV1 and CHA1 in the H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in the budding yeast Saccharomyces cerevisiae.

ABC transporter Pdr5 is required for cantharidin resistance in Saccharomyces cerevisiae.

Cantharidin is a potent anti-cancer drug and is known to exert its cytotoxic effects in several cancer cell lines. Although we have ample knowledge about its mode of action, we still know a little about cantharidin associated drug resistance mechanisms which dictates the efficacy and cytotoxic potential of this drug. In this direction, in the present study we employed Sacharomyces cerevisiae as a model organism and screened mutants of pleiotropic drug resistance network of genes for their susceptibility to cantharidin. We show that growth of pdr1Δ and pdr1Δpdr3Δ was severely reduced in presence of cantharidin whereas that of pdr3Δ remain unaffected when compared to wildtype. Loss of one of the PDR1 target genes PDR5, encoding an ABC membrane efflux pump, rendered the cells hypersensitive whereas overexpression of it conferred resistance. Additionally, cantharidin induced the upregulation of both PDR1 and PDR5 genes. Interestingly, pdr1Δpdr5Δ double deletion mutants were hypersensitive to cantharidin showing a synergistic effect in its cellular detoxification. Furthermore, transcriptional activation of PDR5 post cantharidin treatment was majorly dependent on the presence of Pdr1 and less significantly of Pdr3 transcription factors. Altogether our findings suggest that Pdr1 acts to increase cantharidin resistance by elevating the level of Pdr5 which serves as a major detoxification safeguard under CAN stress.

The N-Terminal Tail of Histone H3 Regulates Copper Homeostasis in Saccharomyces cerevisiae.

Copper homeostasis is crucial for various cellular processes. The balance between nutritional and toxic copper levels is maintained through the regulation of its uptake, distribution, and detoxification via antagonistic actions of two transcription factors, Ace1 and Mac1. Ace1 responds to toxic copper levels by transcriptionally regulating detoxification genes CUP1 and CRS5 Cup1 metallothionein confers protection against toxic copper levels. CUP1 gene regulation is a multifactorial event requiring Ace1, TATA-binding protein (TBP), chromatin remodeler, acetyltransferase (Spt10), and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of the H3 tail in CUP1 transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, and Δ29-32) that reduce CUP1 expression. We detected reduced Ace1 occupancy across the CUP1 promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28, and Δ28-31 mutations correlating with the reduced CUP1 transcription. The majority of these mutations affect TBP occupancy at the CUP1 promoter, augmenting the CUP1 transcription defect. Additionally, some mutants displayed cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of the H3 N-terminal tail and their modifications in CUP1 regulation.

The mechanisms of action of chromatin remodelers and implications in development and disease.

The eukaryotic genetic material is packaged in the form of chromatin by wrapping DNA around nucleosomes. Cells maintain chromatin in a dynamic state by utilising various ATP-dependent chromatin remodelling complexes which can induce structural transformations in the chromatin. All chromatin remodelers contain an ATP hydrolysing-DNA translocase motor which facilitates nucleosomal DNA translocation. By DNA translocation ISWI and CHD subfamily remodelers slide nucleosomes and arrange them in a regularly spaced array. While SWI/SNF subfamily remodelers evict or displace nucleosomes from chromatin, which promotes recruitment of transcription machinery and DNA repair factors on the DNA. Besides DNA translocation, ISWI, CHD and INO80 subfamily remodelers escort nucleosome organisation and editing. In this review; we discuss different mechanisms by which chromatin remodelers regulate chromatin accessibility, nucleosome assembly and nucleosome editing. We attempt to elucidate how their action mediates various cellular and developmental processes, and their deregulation leads to disease pathogenesis. We emphasised on their role in cancer progression and potential therapeutic implications of these complexes. We also described the drugs and strategies which are being developed to target different subunits of remodelling complexes, histone modifying enzymes and polycomb repressive complex. This includes ATPase inhibitors, EZH2 (enhancer of zeste homolog 2) inhibitors, BET (bromodomain and extra terminal) inhibitors, PROTAC (proteolysis targeting chimaera) and inhibitors of protein-protein interaction.

Identification of Histone H3 and H4 Amino Acid Residues Important for the Regulation of Arsenite Stress Signaling in Saccharomyces cerevisiae.

Arsenic is an environmental carcinogen that causes many diseases in humans, including cancers and organ failures, affecting millions of people in the world. Arsenic trioxide is a drug used for the treatment of acute promyelocytic leukemia (APL). In the present study, we screened the synthetic histone H3 and H4 library in the presence of arsenite to understand the role of histone residues in arsenic toxicity. We identified residues of histone H3 and H4 crucial for arsenite stress response. The residues H3T3, H3G90, H4K5, H4G13, and H4R95 are required for the activation of Hog1 kinase in response to arsenite exposure. We showed that a reduced level of Hog1 activation increases the intracellular arsenic content in these histone mutants through the Fps1 channel. We have also noticed the reduced expression of ACR3 exporter in the mutants. The growth defect of mutants caused by arsenite exposure was suppressed in hyperosmotic conditions, in a higher concentration of glucose, and upon deletion of the FPS1 gene. The arsenite sensitive histone mutants also showed a lack of H3K4 methylation and reduced H4K16 acetylation. Altogether, we have identified the key residues in histone H3 and H4 proteins important for the regulation of Hog1 signaling, Fps1 activity, and ACR3 expression during arsenite stress.

SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomyces cerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance.

The Saccharomyces cerevisiae genome contains 6572 ORFs, of which 680 ORFs are classified as dubious ORFs. A dubious ORF is a small, noncoding, nonconserved ORF that overlaps with another ORF of the complementary strand. Our study characterizes a dubious/nondubious ORF pair, YPR099C/MRPL51, and shows the transcript and protein level expression of YPR099C. Its subcellular localization was observed in the mitochondria. The overlapping ORF, MRPL51, encodes a mitochondrial ribosomal protein of large subunit. Deletion of any ORF from YPR099C/MRPL51 pair induces common phenotypes, i.e. loss of mtDNA, lack of mitochondrial fusion and lack of respiratory growth, due to the double deletion (ypr099cΔ/Δmrpl51Δ/Δ) caused by sequence overlap. Hence, we created the single deletions of each ORF of the YPR099C/MRPL51 pair by an alternative approach to distinguish their phenotypes and identify the specific functions. Both the ORFs were found essential for the functional mitochondria and respiratory growth, but MRPL51 showed its specific requirement in mtDNA stability. The mechanism of mtDNA maintenance by Mrpl51 is probably Mhr1 dependent that physically interacts with Mrpl51 and also regulates mtDNA repair. Overall, our study provides strong evidence for the protein level expression of a dubious ORF YPR099C and the bifunctional role of Mrpl51 in mtDNA maintenance.

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway.

Flocculation is an essential characteristic of yeast cells required for survival under adverse conditions. The multicellular structure (flocs) of yeast provides a suitable microenvironment to enhance the chances of survival during stress conditions. Although the signaling events triggering flocculation have been studied earlier, molecular mechanisms remain elusive. In the present study, we used flocculating sen1 mutants to identify the mechanism of flocculation. Based on the abnormal cell surface morphology and constitutive phosphorylation of Slt2p in flocculating sen1 mutant cells, we hypothesized if flocculation was regulated by the cell wall integrity (CWI) pathway. Up-regulation of FLO genes in wild-type cells was observed upon the activation of CWI pathway either by chemical treatment or by deleting Slt2 phosphatase (Msg5). Our study with Slt2 mutants reveals that the active state of Slt2 is indispensable for flocculation. Deletion of either SLT2 or RLM1 leads to reduced flocculation. Furthermore, we observed overlapping binding sites for Rlm1 and Tup1 at the promoters of almost all the FLO genes. Finally, we show higher Rlm1 and lower Tup1 occupancy at the promoters of FLO1 and FLO5 in flocculating cells. Altogether we demonstrate that CWI MAPK (Slt2) pathway uses a non-catalytic mechanism to activate the transcription of FLO genes.