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HSF1-mediated oxidative stress response to menadione in Saccharomyces cerevisiae KNU5377Y3 by using proteomic approach  [PDF]
Il-Sup Kim, Hyun Kim, Young-Saeng Kim, Ingnyol Jin, Ho-Sung Yoon
Advances in Bioscience and Biotechnology (ABB) , 2013, DOI: 10.4236/abb.2013.41007
Abstract: The hat shock transcription factor HSF1 inthe yeast Saccharomyces cerevisiae regulates a wide range of genes and functions in diverse cellular reactions. To investigate the physiological response of HSF1 inthe presence of menadione (MD) in S. cerevisiae KNU 5377Y3, wild-type (k3wt) and isogenic hsf1 mutant (k3h1) cells were introduced. HSF1 was induced when k3wt cells were exposed to the superoxide-generating agent MD and k3h1 cells were hypersensitive to MD. Under MD stress, k3h1 cells down-regulated the expression of metabolic enzymes (Hxk, Fba1, Pgk1, Eno2, and Adh1), antioxidant enzymes (Trx2 and porin), and molecular chaperones and their cofactors (Hsp104, Ssb1, Hsp60, Hsp42, Hsp26, Hsp12, Cpr1, and Sti1). In addition, k3h1 cells increased cellular hydroperoxide levels and protein carbonylation under MD stress as compared to k3wt cells. However, there was a moderate difference in the wild-type (b3wt) and mutant (b3h1) cells derived from S. cerevisiae S288Cunder the same conditions. Thus, these results show that HSF1 is an important component of the stress response system, acting as an activator of cell rescue genes in S. cerevisiae KNU5377Y3, and its expression protects the cells from MD-induced oxidative damage by maintaining redox homeostasis and proteostasis in the presence of MD.
Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae  [PDF]
Frederico Augusto Vieira Castro, Diana Mariani, Anita Dolly Panek, Elis Cristina Araújo Eleutherio, Marcos Dias Pereira
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003999
Abstract: Background Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. Methodology/Principal Findings Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Δ and gtt2Δ) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. Conclusions/Significance These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.
Hsf1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis  [PDF]
Sricharan Bandhakavi, Hongwei Xie, Brennon O'Callaghan, Hiroshi Sakurai, Do-Hyung Kim, Timothy J. Griffin
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001598
Abstract: TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections.
Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae  [PDF]
Kuninori Suzuki, Shingo Nakamura, Mayumi Morimoto, Kiyonaga Fujii, Nobuo N. Noda, Fuyuhiko Inagaki, Yoshinori Ohsumi
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091651
Abstract: Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.
Proteomic Analysis of the Increased Stress Tolerance of Saccharomyces cerevisiae Encapsulated in Liquid Core Alginate-Chitosan Capsules  [PDF]
Johan O. Westman, Mohammad J. Taherzadeh, Carl Johan Franzén
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049335
Abstract: Saccharomyces cerevisiae CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT? labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.
Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata
Cuéllar-Cruz, Mayra;Casta?o, Irene;Arroyo-Helguera, Omar;De Las Pe?as, Alejandro;
Memórias do Instituto Oswaldo Cruz , 2009, DOI: 10.1590/S0074-02762009000400020
Abstract: candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. we are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. here we investigated c. glabrata, saccharomyces cerevisiae and candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (chp). in log-phase, in the presence of menadione, c. glabrata requires cta1p (catalase), while in a stationary phase (sp), cta1p is dispensable. in addition, c. glabrata is less resistant to menadione than c. albicans in sp. the s. cerevisiae laboratory reference strain is less resistant to menadione than c. glabrata and c. albicans; however s. cerevisiaeclinical isolates (cis) are more resistant than the lab reference strain. furthermore, s. cerevisiae cis showed an increased catalase activity. interestingly, in sp c. glabrata and s. cerevisiae are more resistant to chp than c. albicans and cta1p plays no apparent role in detoxifying this oxidant.
The Effects Of Menadione On Rat Glial Cell Proliferation  [PDF]
Pinar Oztopcu VATAN,Selda KABADERE
Journal of Neurological Sciences , 2007,
Abstract: Background: Menadione inhibits cell growth of rodent and human cell lines. We reported that menadione had potential antiproliferative effect doses depend on both rat glioma (C6) and low passage human glioma cells in vitro. In this study, we examine menadione toxicity on primary glial cell culture cells in vitro.Material and Methods: Glial cells were obtained from 1-3 day old rat brain and cultured in humidified atmosphere of 5 % CO2, at 37 oC in flask. The glial cells were seeded in 96 well plates and incubated for 24 hours. Then cells were exposed 10, 25, 50, 75 ,and 100 μM menadione doses. After 24 hours, menadione toxicity was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) assay.All statistical analyses were performed by one-way analysis of variance (ANOVA) and followed by Tukey’s multiple comparison tests.Results: When the compared to the control, 10 and 25 μM menadione doses did not shown any activity on glial cell proliferation (p>0.05). However, 50, 75 ,and 100 μM menadione doses reduced 49, 61, 63 % in glial cells viability respectively (p<0.0001). Fifty % growth inhibitory concentration (IC50) was calculated as 48 μM.Conclusion: Our finding suggested that rat glial cells susceptible to menadione toxicity. In addition menadione has growth inhibitory effects in a dose depend manner on rat glial cells like rat glioma cell in vitro.
Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae
Menggen Ma, Z Lewis Liu
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-660
Abstract: We identified 365 candidate genes and found at least 3 significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1, and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strain Δrpn4 was unable to recover the growth in the presence of HMF.Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor HMF. Both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the HMF stress during the lag phase of growth. Transcription factor genes YAP1, PDR1, PDR3, RPN4, and HSF1 appeared to play key regulatory rules for global adaptation in the yeast S. cerevisiae.Bioethanol production from lignocellulosic biomass including agricultural and forestry residues has attracted increased attention worldwide [1-8]. Lignocellulosic biomass needs to be depolymerized into simple sugars in order to be utilized for microbial fermentation. The commonly applied dilute acid pretreatment generat
Menadione-induced apoptosis and its mechanism in plants
Yingli Sun,Jun Zhou,Yaoren Dai,Zhonghe Zhai
Chinese Science Bulletin , 2000, DOI: 10.1007/BF02909767
Abstract: Menadione can induce apoptosis in tobacco protoplasts. Typical characteristics are detected including the condensation of chromatin, the formation of apoptotic bodies and the degradation of genomic DNA into “DNA ladder”. Specific DNase is activated during this process. Ca2+ and Mg2+ are necessary for its activation, while Zn2+ and EDTA (Ethylenediaminetetraacetic acid) can inhibit its activation. The fragmentation of DNA and lamin can be inhibited by DEVD (Ac-Asp -Glu- Val- Asp-aldehyde). The fragmentation of lamin can also be inhibited by PMSF (Phenylmethylsulfonyl fluoride) and CH (Cyclohexinide). These results show that activation of specific DNase and proteases is involved in menadion-induced apoptosis in plants.
雄性hsf1基因缺陷小鼠的行为改变  [PDF]
心理学报 , 2007,
Abstract: ?为研究hsf1基因缺陷小鼠的行为特征,探索hsf1基因在小鼠行为表现中的作用。选取6~7个月大雄性hsf1基因缺陷小鼠39只及野生型小鼠36只进行情绪性评分、旷场实验、高架十字迷宫实验、简易迷津实验、t-cat实验、独木桥实验和悬挂实验以观察其情绪性唤醒水平、焦虑水平、探索行为、工作记忆能力和运动能力。结果表明hsf1基因缺陷小鼠的情绪唤醒水平和焦虑水平较低、探索行为减少、t-cat中转换率较低,提示小鼠的情绪、探索动机和工作记忆受hsf1基因的调控
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