Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast.
ABSTRACT: Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.
Project description:The amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of Escherichia coli contains genes that are important for diverse cellular functions, including DNA mismatch repair (mutL), tRNA modification (miaA), pleiotropic regulation (hfq), and proteolysis (hflX-hflK-hflC). We show that this superoperon contains three E simga(32)-dependent heat shock promoters, P(mutL)HS,P(miaA)HS, and P1(hfq)HS, in addition to four E sigma(70)-dependent promoters, P(mutL), P(miaA), P2(hfq), and P3(hfq). Transcripts from P(mutL)HS and P(miaA)HS were most prominent in vivo during extreme heat shock (50 degrees C), whereas P1(hfq)HS transcripts were detectable under nonshock conditions and increased significantly after heat shock at 50 degrees C. The P(mutL)HS, P(miaA)HS, and P1(hfq)HS transcripts were not detected in an rpoH null mutant. All three promoters were transcribed by E sigma (32) in vitro at 37 degrees C and contain -35 and -10 regions that resemble the E sigma(32) consensus. In experiments to assess the possible physiological relevance of the P(mutL)HS and P(miaA)HS promoters, we found that E. coli prototrophic strain MG 1655 increased in cell mass and remained nearly 100% viable for several hours at 50 degrees C in enriched media. In these cells, a significant fraction of mutL and hfq-hflA region transcripts were from P(mutL)HS and P1(hfq)HS, respectively, and the amounts of the miaA, hfq, hflX, hflK, and hflC transcripts increased in comparison with those in nonstressed cells. The cellular amounts of MutL and the hfq gene product (HF-I protein) were maintained during heat shock at 44 or 50 degrees C. Consistent with their expression patterns, miaA and hfq were essential for growth and viability, respectively, at temperatures of 45 degrees C and above. Together, these results suggest that there is a class of E sigma(32) promoters that functions mainly at high temperatures to ensure E. coli function and survival.
Project description:We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.
Project description:During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions.
Project description:Non-alcoholic fatty liver disease (NAFLD) is characterized by accumulation of triglycerides (TG) in hepatocytes, which may also trigger cirrhosis. The mechanisms of NAFLD are not fully understood, but insulin resistance has been proposed as a key determinant.To determine the TG content and long chain fatty acyl CoA composition profile in liver from obese non-diabetic insulin resistant (IR) and lean insulin sensitive (IS) baboons in relation with hepatic and peripheral insulin sensitivity.Twenty baboons with varying grades of adiposity were studied. Hepatic (liver) and peripheral (mainly muscle) insulin sensitivity was measured with a euglycemic clamp and QUICKI. Liver biopsies were performed at baseline for TG content and LCFA profile by mass spectrometry, and histological analysis. Findings were correlated with clinical and biochemical markers of adiposity and insulin resistance.Obese IR baboons had elevated liver TG content compared to IS. Furthermore, the concentration of unsaturated (LC-UFA) was greater than saturated (LC-SFA) fatty acyl CoA in the liver. Interestingly, LC-FA UFA and SFA correlated with waist, BMI, insulin, NEFA, TG, QUICKI, but not M/I. Histological findings of NAFLD ranging from focal to diffuse hepatic steatosis were found in obese IR baboons.Liver TG content is closely related with both hepatic and peripheral IR, whereas liver LC-UFA and LC-SFA are closely related only with hepatic IR in non-human primates. Mechanisms leading to the accumulation of TG, LC-UFA and an altered UFA: LC-SFA ratio may play an important role in the pathophysiology of fatty liver disease in humans.
Project description:Obesity-associated hepatic lipid accumulation and chronic low-grade inflammation lead to metabolic defects. Saturated fatty acids (SFA) are a risk factor for, whereas unsaturated fatty acids (UFA) are thought to be protective against, developing metabolic diseases. Sex differences exist in the regulation of metabolism. We tested the hypothesis that diets high in SFA, mono-UFA (MUFA), or poly-UFA (PUFA) had early, sex-distinct effects that differentially contribute to long-term metabolic disturbance such as fatty liver and insulin resistance. Metabolic changes including body and fat mass, circulating leptin and glucose levels, plasma lipid profile, hepatic lipid accumulation, expression levels of genes related to lipid metabolism and low-grade inflammation, and tissue insulin sensitivity were compared between male and female mice fed with a low-fat chow, or high-fat SFA, MUFA, or PUFA for a short period of four days. SFA and MUFA males increased adiposity associated with increased liver lipid accumulation and rapid activation of inflammation in adipose and muscle tissues, whereas PUFA males did not show lipid accumulation or tissue inflammation compared to chow males. All SFA and UFA males displayed tissue insulin resistance. In contrast, female high-fat diet groups had normal liver lipid content and maintained tissue insulin sensitivity without showing tissue inflammation. Therefore, sex differences existed during early phase of development of metabolic dysfunction. The beneficial effects of PUFA, but not MUFA, were corroborated in protection of obesity, hyperlipidemia, fatty liver, and low-grade inflammation. The benefit of MUFA and PUFA in maintaining tissue insulin sensitivity in males, however, was questioned.
Project description:OBJECTIVES:Both heat shock (HS) and ionizing radiation have an impact on the cell cycle and may induce cell cycle arrest or apoptosis. Mutations of the p53 gene are observed at a high frequency in human tumours and are recognized in about half of all human cancers. Sensitivity to radiation, heat and anticancer agents has been observed in p53(+/+) cells, but not in mutated or p53-deficient cells. Moreover, enhancement of radiosensitivity by HS has been observed in wild-type p53 cells but not in p53-deficient cells. The molecular mechanism of the differential cell response to HS or ionizing radiation is not yet understood. MATERIALS AND METHODS:Differences in cellular response to radiation (200 kV X-ray, 1, 2, 5 Gy) and HS (39 degrees C, 41 degrees C and 43 degrees C for 30 min) on cell cycle progression of cultures of human p53 mutant cells were investigated by flow cytometry. In addition, the effects of stressors used on the expression of several heat shock genes (HSP27, HSP60, HSP70, HSC70, HSP75, HSP78, HSP90) were studied by reverse transcriptase-polymerase chain reaction. RESULTS AND CONCLUSIONS:Yet, with respect to HSP gene expression, different stressors produced similar effects. Combination of HS and radiation treatment significantly induced the transcription of the HSP70 gene above the level induced by each stressor alone. Cell cycle analysis, however, revealed striking differences in prolonged dynamics of cell division in response to each stressor. Thus, p53 status could be a useful indicator in predictive assays for hyperthermia cancer treatment in combination with radiation and/or chemotherapy.
Project description:Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription.We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes.HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.
Project description:Temperature is a ubiquitous environmental variable which can profoundly influence the physiology of living cells as it changes over time and space. When yeast cells are exposed to a sublethal heat shock, normal metabolic functions become repressed and the heat shock transcription factor Hsf1 is activated, inducing heat shock proteins (HSPs). Candida albicans, the most prevalent human fungal pathogen, is an opportunistic pathogen that has evolved as a relatively harmless commensal of healthy individuals. Even though C. albicans occupies thermally buffered niches, it has retained the classic heat shock response, activating Hsf1 during slow thermal transitions such as the increases in temperature suffered by febrile patients. However, the mechanism of temperature sensing in fungal pathogens remains enigmatic. A few studies with Saccharomyces cerevisiae suggest that thermal stress is transduced into a cellular signal at the level of the membrane. In this study, we manipulated the fluidity of C. albicans membrane to dissect mechanisms of temperature sensing. We determined that in response to elevated temperature, levels of OLE1, encoding a fatty acid desaturase, decrease. Subsequently, loss of OLE1 triggers expression of FAS2, encoding a fatty acid synthase. Furthermore, depletion of OLE1 prevents full activation of Hsf1, thereby reducing HSP expression in response to heat shock. This reduction in Hsf1 activation is attributable to the E3 ubiquitin ligase Rsp5, which regulates OLE1 expression. To our knowledge, this is the first study to define a molecular link between fatty acid synthesis and the heat shock response in the fungal kingdom.
Project description:To determine the effect of heat stress (HS) on adipose tissue metabolome, a combination of liquid chromatography-mass spectrometry-based metabolomics profiling approaches was applied to characterize changes of metabolite classes in adipocytes differentiated in culture (in vitro) and mesenteric adipose tissue of pigs exposed to HS (in vivo). Effect of HS on the composition of individual fatty acids in cultured adipocytes, mesenteric adipose tissue, and serum of animals was also investigated using gas chromatography analysis. In vitro, preadipocytes were differentiated either under control (37 °C) or HS (41.5 °C) temperature for 9 d. For the animal experiment, pigs were kept either in control (Con) environment (20 °C) with ad libitum feed intake, HS (35 °C) temperature with ad libitum feed intake (HS), or at 20 °C with pair feeding to the HS pigs. In cultured cells, HS increased triglyceride and decreased monoacylglycerol (P < 0.05) species accumulation compared with control. Phosphatidylinositol and phosphatidylserine were increased by HS, whereas phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol were decreased relative to control (P < 0.05). Heat-stressed adipocytes in culture also had higher concentrations of saturated (SFA) and monounsaturated fatty acids (P < 0.05) relative to control. Pathways of proline and biotin metabolism were elevated (P < 0.05) by HS in adipocytes. The metabolomics signatures in adipocytes cultured under HS indicates that pathways centered around diacylglycerol metabolism are impacted by HS. In adipose tissue from animals in the HS treatment, there was increased (P < 0.05) abundance of 4,8 dimethylnonanoyl carnitine (P < 0.05). Heat-stressed animals also had higher (P < 005) serum linoleic, total polyunsaturated fatty acids, and decreased total SFA than PF (P < 0.05). These results indicate that HS elevates lipogenic pathways while suppressing fatty acid oxidation and demonstrate the usefulness of metabolomics analysis as a tool for determining the impact of HS in pig tissues.
Project description:Bacillus subtilis grown at 37 degrees C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37 degrees C are transferred to 20 degrees C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a delta5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37 degrees C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37 degrees C, but a shift of cultures from 37 to 20 degrees C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.