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.

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.

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.

Modulation of ruthenium anticancer drugs analogs with tolfenamic acid: Reactivity, biological interactions and growth inhibition of yeast cell.

We synthesized two ruthenium(II) complexes: trans,trans-[Ru(im)2(tfa)2] (1) and trans,trans­[Ru(in)2(tfa)2] (2) where im = 1H­imidazole, in = 1H­indazole and tfa = tolfenamic acid, a potential nonsteroidal anti-inflammatory drug (NSAID). The NSAID was opted as bioactive ligand to understand its synergistic therapeutic effect in structurally analogous Ru(II)-compounds with KP418 (imidazolium trans­[tetrachloridobis(1H­imidazole)ruthenate(III)]) and KP1019 (indazolium trans­[tetrachloridobis(1H­indazole)ruthenate(III)]). The complexes were studied using various analytical methods and structure was determined by X-ray crystallography. Both the complexes display discrete mononuclear Ru(II) center in {RuN4O2} distorted octahedral geometry. The reactivity of the complexes was tested with potentially important biomolecules involved in metabolism of cancer cells, viz. l­arginine, dl­methionine, glutathione and L(+)ascorbate. Such studies intended to provide deeper insights on intracellular speciation and kinetic substitution encountered by Ru-drugs to target alternative cell death pathways. The complexes demonstrate a preferential binding affinity with calf thymus DNA (Kb ~ 104 M-1) and human serum albumin (KHSA = 105 M-1). Both the complexes showed potent inhibition of wild type yeast cell growth in a dose-dependent manner. Yeast cells were used as a powerful model system to study the molecular mechanism of pathobiology which shares a high degree of conservation of both cellular and molecular processes with human cells for assessing toxicity potential of the complexes. Fluorescence imaging studies reveal the localization of both complexes to yeast mitochondria despite its rigid cell wall and induce mitochondrial damage and formation of reactive oxygen species (ROS). The Micrococcal nuclease assay revealed complexes do not alter global nucleosome occupancy and probably target specific regions of the genome.

Modifying Chromatin by Histone Tail Clipping.

Post-translational modifications (PTMs) of histone proteins play a crucial role in the regulation of chromatin structure and functions. Studies in the last few decades have revealed the significance of histone PTMs in key cellular processes including DNA replication, repair, transcription, apoptosis and cell cycle regulation. The PTMs on histones are carried out by chromatin modifiers, which are reversible in nature. The dynamic activity of chromatin modifiers maintains the levels of different PTMs on histones. The modified histones are recognized by reader proteins, which recruit effector proteins to regulate the function. The interplay between histone PTMs and chromatin dynamics plays a major role in the regulation of most of the cellular processes. Importantly, the perturbations in the histone PTMs by various intrinsic or extrinsic factors can cause defects in fundamental cellular processes leading to a wide range of diseases. The proteolytic clipping of histone proteins has also been shown to regulate many biological processes. Histone clipping has been observed from yeast to mammals, suggesting that this mechanism is a conserved epigenetic phenomenon. In this review, we have summarized the significance of histone clipping and provided future directions to comprehend the mechanism of this distinct and poorly understood epigenetic event.

A systematic assessment of chemical, genetic, and epigenetic factors influencing the activity of anticancer drug KP1019 (FFC14A).

KP1019 ([trans-RuCl4(1H-indazole)2]; FFC14A) is one of the promising ruthenium-based anticancer drugs undergoing clinical trials. Despite the pre-clinical and clinical success of KP1019, the mode of action and various factors capable of modulating its effects are largely unknown. Here, we used transcriptomics and genetic screening approaches in budding yeast model and deciphered various genetic targets and plethora of cellular pathways including cellular signaling, metal homeostasis, vacuolar transport, and lipid homeostasis that are primarily targeted by KP1019. We also demonstrated that KP1019 modulates the effects of TOR (target of rapamycin) signaling pathway and induces accumulation of neutral lipids (lipid droplets) in both yeast and HeLa cells. Interestingly, KP1019-mediated effects were found augmented with metal ions (Al3+/Ca2+/Cd2+/Cu2+/Mn2+/Na+/Zn2+), and neutralized by Fe2+, antioxidants, osmotic stabilizer, and ethanolamine. Additionally, our comprehensive screening of yeast histone H3/H4 mutant library revealed several histone residues that could significantly modulate the KP1019-induced toxicity. Altogether, our findings in both the yeast and HeLa cells provide molecular insights into mechanisms of action of KP1019 and various factors (chemical/genetic/epigenetic) that can alter the therapeutic efficiency of this clinically important anticancer drug.