RESUMO
Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians.
Assuntos
Proteínas de Choque Térmico Pequenas/genética , Filogenia , Ranidae/genética , Estresse Fisiológico/genética , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico Pequenas/fisiologia , Ranidae/fisiologia , Pele/metabolismoRESUMO
Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.
Assuntos
Células Epiteliais/efeitos dos fármacos , Isotiocianatos/farmacologia , Rim/citologia , Xenopus , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismoRESUMO
Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates.
Assuntos
Anfíbios/fisiologia , Aves/fisiologia , Peixes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP30/metabolismo , Répteis/fisiologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Anfíbios/crescimento & desenvolvimento , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Aves/crescimento & desenvolvimento , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixes/crescimento & desenvolvimento , Proteínas de Choque Térmico HSP30/química , Proteínas de Choque Térmico HSP30/genética , Corpos de Inclusão/metabolismo , Especificidade de Órgãos , Filogenia , Transporte Proteico , Répteis/crescimento & desenvolvimento , Proteínas de Répteis/química , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Especificidade da Espécie , Estresse Fisiológico , Terminologia como AssuntoRESUMO
To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.
Assuntos
Candida glabrata/genética , Candidíase/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Ácido Acético/toxicidade , Candida glabrata/metabolismo , Candida glabrata/patogenicidade , Candidíase/metabolismo , Candidíase/microbiologia , Candidíase/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Evolução Molecular , Feminino , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Proteínas de Choque Térmico HSP30/genética , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/genética , Transcriptoma/genética , Vagina/metabolismo , Vagina/microbiologiaRESUMO
The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.
Assuntos
Arsenitos/farmacologia , Cloreto de Cádmio/farmacologia , Heme Oxigenase-1/metabolismo , Rim/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Compostos de Sódio/farmacologia , Poluentes Químicos da Água/farmacologia , Proteínas de Xenopus/metabolismo , Animais , Arsenitos/toxicidade , Cloreto de Cádmio/toxicidade , Linhagem Celular , Estruturas Citoplasmáticas/efeitos dos fármacos , Estruturas Citoplasmáticas/metabolismo , Indução Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP30/agonistas , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Heme Oxigenase-1/química , Heme Oxigenase-1/genética , Temperatura Alta/efeitos adversos , Imuno-Histoquímica , Rim/citologia , Rim/metabolismo , Leupeptinas/farmacologia , Triterpenos Pentacíclicos , Transporte Proteico/efeitos dos fármacos , Compostos de Sódio/toxicidade , Testes de Toxicidade Aguda , Triterpenos/farmacologia , Poluentes Químicos da Água/toxicidade , Vitanolídeos/farmacologia , Proteínas de Xenopus/agonistas , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. In this study, we examined the pattern of accumulation of HSP30 and HSP70 in Xenopus laevis A6 kidney epithelial cells recovering from heat shock. Immunoblot analysis revealed the presence of elevated levels of HSP30 after 72h of recovery. However, the relative levels of HSP70 declined to near control levels after 24h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. This study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation.
Assuntos
Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Animais , Linhagem Celular , Cicloeximida/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP70/genética , Leupeptinas/farmacologia , Fosforilação , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteólise , Estabilidade de RNA , Xenopus laevis , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The genus Saccharomyces comprises seven single-genome species (S. arboricola, S. cerevisiae, S. eubayanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum) and two hybrid species - S. pastorianus (S. cerevisiae plus S. eubayanus) and S. bayanus (mostly S. uvarum plus S. eubayanus). Species-specific primers have already been developed for the identification of each of the single-genome species, and these primers can usually detect both genomes in hybrids. It would be advantageous if a single reaction could detect any member of the clade. We have investigated three potentially generic approaches to design genus-specific primers. Two methods that both use sequence alignment differences for primer design were only partly successful. A third method used synteny data to identify 136 target genes that are potentially present only in all species of the Saccharomyces clade. HSP30 (YCR021C) was fully successful; different primer pairs were developed with high G+C content for use at 63 °C. In < 3 h, using a robust colony-PCR followed by gel electrophoresis, the method can reliably detect any member of the genus. This novel approach still uses conventional sequence alignment mismatches but relies principally on the presence of the target gene only within the genus Saccharomyces.
Assuntos
Primers do DNA/genética , DNA Fúngico/genética , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Saccharomyces/classificação , Saccharomyces/genética , Sintenia , Composição de Bases , DNA Fúngico/química , Eletroforese , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP30/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Temperatura , Fatores de TempoRESUMO
Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more ß-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.
Assuntos
Escherichia coli K12/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP30/química , Dobramento de Proteína , Sequência de Aminoácidos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Temperatura Alta , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Temperatura Alta/efeitos adversos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Vitanolídeos/farmacologia , Actinas/metabolismo , Animais , Linhagem Celular , Quimotripsina/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Rim/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacos , Xenopus laevisRESUMO
Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.
Assuntos
Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Triclosan (TCS) and triclocarban (TCC) are widely used broad spectrum bactericides that are common pollutants of waterways and soils. Methyl triclosan (mTCS) is the predominant bacterial TCS metabolite. Previous studies have shown that TCS disrupts thyroid hormone (TH) action; however, the effects of mTCS or TCC are not known. The present study uses the cultured frog tadpole tail fin biopsy (C-fin) assay and the TH-responsive rat pituitary GH3 cell line to assess the effects of these three chemicals (1-1000 nM) on TH signaling and cellular stress within 48 h. mRNA abundance of TH receptor ß, Rana larval keratin type I (TH-response), heat shock protein 30, and catalase (stress-response) was measured using quantitative real-time polymerase chain reaction in the C-fin assay. The TH-responsive gene transcripts encoding growth hormone, deiodinase I, and prolactin were measured in GH3 cells with the heat shock protein 70 transcript acting as a cellular stress indicator. We found alteration of stress indicators at a wide range of concentrations of TCS, mTCS, and TCC in both test systems. mTCS and TCC affected TH-responsive gene transcripts at the highest concentration in mammalian cells, whereas a modest effect included lower concentrations in the C-fin assay. In contrast, TCS did not affect TH-responsive transcripts. These results identify nontarget biological effects of these bacteriocides on amphibian and mammalian cells and suggest the TH-disrupting effects observed for TCS could be mediated through its metabolite.
Assuntos
Carbanilidas/toxicidade , Mamíferos/fisiologia , Ranidae/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Triclosan/análogos & derivados , Animais , Catalase/genética , Catalase/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Queratinas/genética , Queratinas/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase , Prolactina/genética , Prolactina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ranidae/genética , Ratos , Receptores beta dos Hormônios Tireóideos/efeitos dos fármacos , Receptores beta dos Hormônios Tireóideos/metabolismo , Triclosan/toxicidadeRESUMO
We investigated the mechanism underlying the natural variation in longevity within natural populations using the model budding yeast, Saccharomyces cerevisiae. We analyzed whole-genome gene expression in four progeny of a natural S. cerevisiae strain that display differential replicative aging. Genes with different expression levels in short- and long-lived strains were classified disproportionately into metabolism, transport, development, transcription or cell cycle, and organelle organization (mitochondrial, chromosomal, and cytoskeletal). With several independent validating experiments, we detected 15 genes with consistent differential expression levels between the long- and the short-lived progeny. Among those 15, SIR2, HSP30, and TIM17 were upregulated in long-lived strains, which is consistent with the known effects of gene silencing, stress response, and mitochondrial function on aging. The link between SIR2 and yeast natural life span variation offers some intriguing ties to the allelic association of the human homolog SIRT1 to visceral obesity and metabolic response to lifestyle intervention.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Ciclo Celular , Proliferação de Células , Perfilação da Expressão Gênica , Genes Fúngicos , Proteínas de Choque Térmico HSP30/genética , Peróxido de Hidrogênio/farmacologia , Viabilidade Microbiana , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para CimaRESUMO
Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein which is induced by various environmental stress conditions. However functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a hsp30 strain during heat stress. BY4741Deltahsp30 cells were found to be more sensitive compared to BY4741 cells when exposed to a lethal heat stress at 50 degrees Celsius. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that BY4741Deltahsp30 cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal stress induced changes in gene expression, the Deltahsp30mutant maintained elevated levels of Pdc1p, Trx1p and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.
Assuntos
Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico HSP30/química , Proteínas de Choque Térmico HSP30/genética , Temperatura Alta , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estresse FisiológicoRESUMO
In eukaryotes, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most proteins. Proteasome inhibition, which has been associated with various diseases, can cause alterations in various intracellular processes including the expression of heat shock protein (hsp) genes. In this study, we show that celastrol, a quinone methide triterpene and anti-inflammatory agent, inhibited proteasome activity and enhanced HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Treatment of cells with celastrol induced the accumulation of ubiquitinated protein and inhibited chymotrypsin-like activity. This was accompanied by a dose- and time-dependent accumulation of HSP30 and HSP70. Celastrol-induced HSP accumulation was mediated by HSF1-DNA binding activity since this response was inhibited by the HSF1 activation inhibitor, KNK437. Simultaneous exposure of cells with celastrol plus either mild heat shock or the proteasome inhibitor, MG132, produced an enhanced accumulation of HSP30 that was greater than the sum of the individual stressors alone. Immunocytochemical analysis revealed that celastrol-induced HSP30 accumulation occurred in the cytoplasm in a granular pattern supplemented with larger circular HSP30 staining structures. HSP30 was also noted in the nucleus with less staining in the nucleolus. In some cells, celastrol induced the collapse of the actin cytoskeleton and conversion to a rounder morphology. In conclusion, this study has shown that celastrol inhibited proteasome activity and induced HSF1-mediated expression of hsp genes in amphibian cells.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP70/genética , Inibidores de Proteassoma , Triterpenos/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Compostos Benzidrílicos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Leupeptinas/farmacologia , Triterpenos Pentacíclicos , Transporte Proteico/efeitos dos fármacos , Pirrolidinonas/farmacologia , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Northern and southern subspecies of the Atlantic killifish, Fundulus heteroclitus, differ in maximal thermal tolerance. To determine whether these subspecies also differ in their heat shock response (HSR), we exposed 20°C acclimated killifish to a 2 h heat shock at 34°C and examined gene expression in fish from both subspecies during heat shock and recovery using real-time quantitative PCR and a heterologous cDNA microarray designed for salmonid fishes. The heat shock proteins Hsp70-1, hsp27, and hsp30 were upregulated to a greater extent in the high temperature-tolerant southern subspecies than in the less tolerant northern subspecies, whereas hsp70-2 (which showed the largest upregulation of all the heat shock proteins) in both gill and muscle and hsp90α in muscle was upregulated to a greater extent in northern than in southern fish. These data demonstrate that differences in the HSR between subspecies cannot be due to changes in a single global regulator but must occur via gene-specific mechanisms. They also suggest that the role, if any, of hsps in establishing thermal tolerance is complex and varies from gene to gene. Heterologous microarray hybridization provided interpretable gene expression signatures, detecting differential regulation of genes known to be involved in the heat shock response in other species. Under control conditions, a variety of genes were differentially expressed in muscle between subspecies that suggest differences in muscle fiber type and could relate to previously observed differences between subspecies in the thermal sensitivity of swimming performance and metabolism.
Assuntos
Proteínas de Peixes/genética , Fundulidae/genética , Perfilação da Expressão Gênica/métodos , Estudos de Associação Genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Análise de Sequência com Séries de Oligonucleotídeos , Aclimatação/genética , Animais , Proteínas de Peixes/metabolismo , Fundulidae/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP30 , Proteínas de Choque Térmico HSP70 , Inibidores de Proteassoma , Temperatura , Xenopus laevis , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Animais , Linhagem Celular , Inibidores de Cisteína Proteinase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP30/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Leupeptinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In order to find a promoter that could be influenced by temperature shift, we explored and isolated an Aspergillus oryzae gene expressed at high temperatures (37-42 degrees C) by the cDNA subtraction method. Of the 96 cDNA clones isolated from the subtraction library, one cDNA clone showed 73% identity with Aspergillus nidulans heat shock protein 30 (hsp30). Based on this, we designated the isolated gene hsp30 of A. oryzae. A. oryzae hsp30 was weakly expressed at 30 degrees C, but strongly at 40 degrees C. We showed that the promoter of this hsp30 induced heterologous gene expression at high temperatures using beta-glucuronidase (GUS) gene as a reporter. Regarding elucidation of the region essential for heat shock response, we showed that the minimum length of the promoter region that was essential for heat shock response was located between -388 and -272 (+1 indicated the first position of the translation initiation codon) of the hsp30 promoter. This promoter region harbors several putative transcription factor binding sites, including heat shock elements (HSEs), a CCAAT box, and a TATA box. Furthermore, site-directed mutagenesis of this promoter revealed that HSE1 (aTTCgtcGAAacgcccaGAAa) and HSE2 (cGAAagTTCtcGACg), located between -342 and -272 of the hsp30 promoter, were its cis-acting elements for heat shock response.
Assuntos
Aspergillus oryzae/genética , Regiões Promotoras Genéticas , Deleção de Sequência , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Códon/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP30/genética , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Mensageiro/genética , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1-10 microM sodium arsenite at a mild heat shock temperature of 30 degrees C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 microM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 degrees C with larger responses at 28 and 30 degrees C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 microM) and heat shock (30 degrees C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 degrees C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.
Assuntos
Adaptação Fisiológica , Arsenitos/farmacologia , Proteínas de Choque Térmico HSP30/genética , Proteínas de Choque Térmico HSP70/genética , Temperatura Alta , Rim/efeitos dos fármacos , Compostos de Sódio/farmacologia , Animais , Northern Blotting , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imuno-Histoquímica , Rim/citologia , Rim/metabolismo , Rim/fisiologia , Microscopia Confocal , XenopusRESUMO
Penicillium marneffei is a dimorphic fungus that can cause disseminated mycosis, especially in AIDS patients. The role of heat shock proteins and stress response-related proteins in P. marneffei remains unknown. In this study, we isolated a cDNA encoding for heat shock protein 30 (Hsp30) of P. marneffei using an antibody screening method. The DNA sequence and deduced amino acid sequence analysis showed high homology to other fungal hsp30 genes. Expression of P. marneffei hsp30 in response to temperature increase was determined by Northern blot analysis. A high level of hsp30 transcript was detected in yeast cells grown at 37 degrees C, whereas a very low or undetectable transcript level was observed in mycelial cells at 25 degrees C. A recombinant Hsp30 protein was produced and tested preliminarily for its immunoreactivity with sera from P. marneffei-infected AIDS patients using Western blot analysis. The positive immunoblot result, with some serum samples, confirmed the antigenic property of the Hsp30. Collectively, the high response of hsp30 to temperature increase could indicate it may play a role in heat stress response and cell adaptation. This is the first report showing that this small heat shock protein could elicit the human immune response.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP30/genética , Penicillium/genética , Sequência de Aminoácidos , Northern Blotting , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Glutationa Transferase/genética , Proteínas de Choque Térmico HSP30/biossíntese , Proteínas de Choque Térmico HSP30/metabolismo , Dados de Sequência Molecular , Penicillium/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , TemperaturaRESUMO
Small heat shock proteins (sHSPs) are chaperones that are crucial in the heat shock response but also have important non-stress roles within the cell. HSP70 in Trichophyton rubrum is already detected and carefully characterised; however, no study was carried out for HSP30 in this pathogenic fungus. In the present study, T. rubrum was obtained from patients with dermatophytosis and cultured in appropriate conditions. High-molecular-weight DNA was extracted using standard extraction methods. Pairs of 21 nt primers were designed from highly conserved regions of the similar genes in other eukaryotic cells. Mentioned primers were utilised in PCR using isolated genomic DNA and extracted RNA templates of T. rubrum. The PCR fragments were then sequenced and 415 nucleotides of HSP30 in this pathogenic fungus were detected; the open reading frame had 156 nucleotides and was coding 51 amino acids. This gene (called TrHSP30) is registered in GenBank at National Center for Biotechnology Information (NIH, USA) database. Detection of TrHSP30 gene may open the way to determination of its possible role in the pathogenesis of dermatophyte infections due to T. rubrum.