RESUMO
The levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are controlled by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element (ERSE), the consensus sequence of which is CCAAT-N(9)-CCACG. We recently proposed that ER stress response factor (ERSF) binding to ERSE is a heterologous protein complex consisting of the constitutive component NF-Y (CBF) binding to CCAAT and an inducible component binding to CCACG and identified the basic leucine zipper-type transcription factors ATF6alpha and ATF6beta as inducible components of ERSF. ATF6alpha and ATF6beta produced by ER stress-induced proteolysis bind to CCACG only when CCAAT is bound to NF-Y, a heterotrimer consisting of NF-YA, NF-YB, and NF-YC. Interestingly, the NF-Y and ATF6 binding sites must be separated by a spacer of 9 bp. We describe here the basis for this strict requirement by demonstrating that both ATF6alpha and ATF6beta physically interact with NF-Y trimer via direct binding to the NF-YC subunit. ATF6alpha and ATF6beta bind to the ERSE as a homo- or heterodimer. Furthermore, we showed that ERSF including NF-Y and ATF6alpha and/or beta and capable of binding to ERSE is indeed formed when the cellular UPR is activated. We concluded that ATF6 homo- or heterodimers recognize and bind directly to both the DNA and adjacent protein NF-Y and that this complex formation process is essential for transcriptional induction of ER chaperones.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação/genética , Fator de Ligação a CCAAT/química , Sequência Consenso , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/química , Dimerização , Fatores de Ligação G-Box , Células HeLa , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Mutação , Estresse Oxidativo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Fatores de Transcrição/químicaRESUMO
Transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) is induced by accumulation of unfolded proteins in the ER. This intracellular signaling, known as the unfolded protein response (UPR), is mediated by the cis-acting ER stress response element (ERSE) in mammals. In addition to ER chaperones, the mammalian transcription factor CHOP (also called GADD153) is induced by ER stress. We report here that the transcription factor XBP-1 (also called TREB5) is also induced by ER stress and that induction of CHOP and XBP-1 is mediated by ERSE. The ERSE consensus sequence is CCAAT-N(9)-CCACG. As the general transcription factor NF-Y (also known as CBF) binds to CCAAT, CCACG is considered to provide specificity in the mammalian UPR. We recently found that the basic leucine zipper protein ATF6 isolated as a CCACG-binding protein is synthesized as a transmembrane protein in the ER, and ER stress-induced proteolysis produces a soluble form of ATF6 that translocates into the nucleus. We report here that overexpression of soluble ATF6 activates transcription of the CHOP and XBP-1 genes as well as of ER chaperone genes constitutively, whereas overexpression of a dominant negative mutant of ATF6 blocks the induction by ER stress. Furthermore, we demonstrated that soluble ATF6 binds directly to CCACG only when CCAAT exactly 9 bp upstream of CCACG is bound to NF-Y. Based on these and other findings, we concluded that specific and direct interactions between ATF6 and ERSE are critical for transcriptional induction not only of ER chaperones but also of CHOP and XBP-1.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico , Proteínas Nucleares/genética , Dobramento de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Transporte/genética , Extratos Celulares , Núcleo Celular/metabolismo , Retículo Endoplasmático , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica , Células HeLa , Humanos , Mamíferos , Chaperonas Moleculares/genética , Mutagênese , Fatores de Transcrição de Fator Regulador X , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição CHOP , Transcrição Gênica , Proteína 1 de Ligação a X-BoxRESUMO
An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions ("ER stress"). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin-proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little of no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Proteínas/metabolismo , Splicing de RNA , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Genes Fúngicos , Variação Genética , Zíper de Leucina/genética , Mutação , Biossíntese de Proteínas , Dobramento de Proteína , Proteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Fatores de Transcrição/efeitos dos fármacos , Tunicamicina/farmacologia , Leveduras/genéticaRESUMO
The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Endopeptidases/metabolismo , Retículo Endoplasmático/genética , Chaperona BiP do Retículo Endoplasmático , Imunofluorescência , Glicosilação , Células HeLa , Humanos , Peso Molecular , Proteínas Nucleares/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Solubilidade , Tapsigargina/farmacologia , Transfecção , Tunicamicina/farmacologiaRESUMO
Current models of both heat induction and the chaperone-mediated feedback control of the sigma32 regulon in Escherichia coli have been further substantiated, and the extent of conservation among Gram-negative bacteria has been assessed. Analyses of the 'CIRCE' and other regulons or operons in Gram-positive and Gram-negative bacteria have provided new insights into their significance and regulatory mechanisms.
Assuntos
Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico/fisiologia , Fator sigma/genética , Sequência Conservada , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Proteínas de Choque Térmico/genética , Modelos Genéticos , Regulon , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli , Fator F/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Reagentes de Ligações Cruzadas , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator F/efeitos dos fármacos , Dosagem de Genes , Chaperonas Moleculares/fisiologia , Mutagênese Insercional , Ligação Proteica/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Análise de Sequência de DNARESUMO
The E. coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32. We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels. Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Sítios de Ligação/genética , Sequência Conservada , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.
Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/efeitos dos fármacos , Etanol/farmacologia , Mutação , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Mapeamento Cromossômico , Cromossomos Bacterianos , Colífagos , Vírus de DNA , Ácido Desoxicólico/farmacologia , Resistência Microbiana a Medicamentos , Escherichia coli/crescimento & desenvolvimento , Microscopia Eletrônica , Vírus de RNA , Rifampina/farmacologia , Temperatura , Fatores de TempoRESUMO
We report the isolation and characterization of a gene (designated mcp60) encoding the mitochondrial (mt) 60-kDa heat-shock protein (HSP60) in the fission yeast Schizosaccharomyces pombe. The deduced amino-acid sequence (582 aa) of this gene is highly similar to the known mt HSP60 from diverse organisms. When its sequence was related to the known functional domains of bacterial HSP60 (GroEL), the similarity was particularly high for the intermediate domains that connect the apical domain with the equatorial domain. The mRNA level of mcp60 increased several-fold upon temperature upshift (from 25 to 35 degrees C), while gradually decreased during sporulation. Gene disruption experiments revealed that mcp60 is essential for cell viability at all temperatures.
Assuntos
Chaperonina 60/genética , Mitocôndrias/química , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/química , Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Schizosaccharomyces/enzimologia , Homologia de Sequência de AminoácidosRESUMO
Although heat shock factor 2 (HSF2) binds to heat shock element (HSE) constitutively during differentiation, development and spermatogenesis, little is known about the nature and mechanism of transcriptional control of heat shock genes by HSF2. We screened a human testis cDNA library for proteins that can associate with HSF2 by the yeast two-hybrid system, and isolated clones encoding a novel protein, designated HSF2 binding protein (HSF2BP), that associates with HSF2 in vitro and in vivo and is specifically expressed in testis. The interaction seemed to occur between the trimerization domain of HSF2 and the amino terminal hydrophilic region of HSF2BP that comprises two leucine zipper motifs. HSF2BP may therefore be involved in modulating HSF2 activation in testis.
Assuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas/genética , Proteínas/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Biblioteca Gênica , Proteínas de Choque Térmico/química , Humanos , Técnicas In Vitro , Zíper de Leucina/genética , Masculino , Dados de Sequência Molecular , Ligação Proteica , Proteínas/química , Saccharomyces cerevisiae/genética , Distribuição Tecidual , Fatores de Transcrição/química , TransfecçãoRESUMO
A DNA fragment from Pseudomonas aeruginosa containing the rpoH gene encoding the heat-shock sigma factor sigma 32 has been cloned and sequenced. The gene is expressed in Escherichia coli and complements an rpoH- strain. An open reading frame encoding 284 amino acids shows 61% identity and 78% similarity to the RpoH protein of Escherichia coli.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Pseudomonas aeruginosa/genética , Fator sigma/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Citrobacter freundii/genética , Clonagem Molecular , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The chaperonin containing TCP-1 (CCT) is a eukaryotic molecular chaperone consisting of eight subunit species and assists in the folding of cytosolic proteins. We show here that all eight mouse CCT subunit genes contain sequences called heat shock elements for binding heat shock transcription factors (HSFs) by electrophoretic mobility shift assays and that these genes are transcriptionally activated by HSFs in reporter gene assays using HeLa cells transiently overexpressing HSFs. These results suggest that HSF1 and/or HSF2 play a role in Cct gene expression.
Assuntos
Chaperoninas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Chaperonina com TCP-1 , Chaperoninas/genética , Proteínas de Ligação a DNA/genética , Genes Reporter , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Células K562 , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ligação Proteica , Temperatura , Fatores de Transcrição/genéticaRESUMO
When cells of E coli are transferred from 30 to 42 degrees C, the cellular level of sigma 32 (rpoH gene product) increases transiently, resulting in increased transcription of a set of heat shock genes. Both increased synthesis and increased stability of sigma 32 contribute to transient accumulation of sigma 32. Evidence suggests that synthesis of sigma 32 is enhanced primarily at the level of rpoH translation. We have constructed and examined the expression of deletion derivatives of rpoH-lacZ gene fusion at 30 degrees C and after shift to 42 degrees C. It was revealed that two cis-acting sequences within the rpoH coding region are involved in thermal regulation of fusion protein synthesis. One region immediately downstream of the initiation codon is required for high level expression, whereas the other internal region is involved in repression at low temperature. Thus, these regions act as positive and negative cis-elements in thermal regulation of rpoH translation. The rpoH mRNA secondary structure model suggesting an interplay between the two regions has been proposed to account for the temperature-induced sigma 32 synthesis as a primary cellular response to the heat shock stress.
Assuntos
Escherichia coli/genética , Proteínas de Choque Térmico/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Fator sigma/genética , Fatores de Transcrição , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas Recombinantes de Fusão/biossíntese , Fator sigma/biossíntese , Transcrição GênicaRESUMO
OBJECTIVE: The present study was carried out to examine the involvement of dopamine in the pressure-natriuresis phenomenon which has been postulated as a major regulator of extracellular fluid volume and thereby arterial pressure. DESIGN: Dopaminergic modulation of the pressure-natriuresis response was studied in the innervated and denervated rat kidney, to allow a distinction between the effects of neural and extraneural dopamine. METHODS: The pressure-natriuresis response was studied in anesthetized Sprague-Dawley rats, in which neural and hormonal influences on the kidney were fixed by denervating the kidney and by intravenous infusion of aldosterone, hydrocortisone, vasopressin and norepinephrine. The innervation to the kidney remained intact in some experiments with the selective dopamine-1 antagonist SCH 23390. Urinary excretion of dopamine during the pressure-natriuresis response was also examined in the innervated and denervated rat kidney. RESULTS: Although infusion of dopamine at a dose of 2 micrograms/kg per min had no effect on the pressure-natriuresis response in rats in which neural and hormonal influences on the kidney were fixed, the slopes of the relations between urine flow, sodium excretion and mean arterial pressure in rats given 10 micrograms/kg per min dopamine were significantly greater than those found in the control rats. Renal plasma flow increased significantly in the dopamine-treated rats whilst glomerular filtration rate did not differ between the control and dopamine-treated rats. The dopamine-induced increase in the slope of pressure-natriuresis relationship and renal plasma flow were completely blocked by 0.5 micrograms/kg per min SCH 23390. However, infusion of SCH 23390 alone at 0.5 micrograms/kg per min did not significantly alter the pressure-natriuresis response in rats with either denervated or innervated kidney. In addition, urinary excretion of dopamine derived from neither neural nor extraneural origins was altered in parallel with variations in mean arterial pressure. CONCLUSION: These results suggest that exogenous administration of dopamine may affect the pressure-natriuresis response by altering the magnitude of arterial pressure-induced changes in tubular sodium reabsorption, via an action of dopamine-1 receptors. However, endogenous dopamine does not appear to be capable of modulating the pressure-natriuresis response.
Assuntos
Pressão Sanguínea/fisiologia , Dopamina/fisiologia , Rim/fisiologia , Natriurese/fisiologia , Animais , Benzazepinas/farmacologia , Denervação , Dopamina/farmacologia , Espaço Extracelular/fisiologia , Hipertensão/fisiopatologia , Rim/inervação , Masculino , Ratos , Ratos Endogâmicos , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores Dopaminérgicos/fisiologia , Circulação Renal/fisiologiaRESUMO
Substituted 2-sulfinylimidazoles were synthesized and investigated as potential inhibitors of gastric H+/K(+)-ATPase. The 4,5-unsubstituted imidazole series 6-11 and the 1,4,5,6-tetrahydrocyclopent[d]imidazole series 12 were found to be potent inhibitors of the acid secretory enzyme H+/K(+)-ATPase. Structure-activity relationships indicate that the substitution of 2-pyridyl groups at the 1-position of the imidazole moiety combined with (2-aminobenzyl)-sulfinyl groups at the 2-position leads to highly active compounds with a favorable chemical stability. Other substitution patterns in the imidazole moiety result in reducing biological activities. 2-[(2-Aminobenzyl) sulfinyl]-1-[2-(3-methylpyridyl)]-1,4,5,6-tetrahydrocyclopent++ ++ ++ [d]-imidazole (12h, T-776) was selected for further development as a potential clinical candidate. Extensive study on the acid degradation of 12h indicates a mechanism of action different from that of omeprazole, the first H+/K(+)-ATPase inhibitor introduced to the market.
Assuntos
Inibidores Enzimáticos/farmacologia , Mucosa Gástrica/enzimologia , Imidazóis/farmacologia , Inibidores da Bomba de Prótons , Animais , Inibidores Enzimáticos/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Imidazóis/química , Masculino , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
In procaryotes such as Escherichia coli, transcriptional activation of heat shock genes in response to elevated temperature is caused primarily by transient increase in the amount of sigma 32 (rpoH gene product) specifically required for transcription from the heat shock promoters. The increase in sigma 32 level results from increased translation of rpoH mRNA and from stabilization of sigma 32 which is ordinarily very unstable. Some of the factors and cis-acting elements that constitute the complex regulatory circuits have been identified and characterized, but detailed mechanisms as well as nature of sensors and signals remain to be elucidated. Whereas this "classical" heat shock regulon (sigma 32 regulon) provides major protective functions against thermal stress, a second heat shock regulon mediated by sigma E (sigma 24) encodes functions apparently required under more extreme conditions, and is activated by responding to extracytoplasmic signals. These regulons mediated by minor sigma factors (sigma 32 in particular) appear to be conserved in most gram-negative bacteria, but not in gram-positive bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma/metabolismo , Chaperoninas/biossíntese , Neoplasias do Colo/metabolismo , Neoplasias Hepáticas/metabolismo , Chaperonina com TCP-1 , Colo/metabolismo , Citosol/metabolismo , Humanos , Fígado/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjögren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/sangue , Chaperonina 60/imunologia , Chaperoninas/imunologia , Doenças Reumáticas/sangue , Adulto , Artrite Reumatoide/sangue , Chaperonina com TCP-1 , Reações Cruzadas , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Masculino , Pessoa de Meia-Idade , Doença Mista do Tecido Conjuntivo/sangue , Família Multigênica , Síndrome de Sjogren/sangueRESUMO
150 kDa oxygen-regulated protein (ORP150) is one of the endoplasmic reticulum (ER)-resident stress proteins. We have cloned and sequenced the entire human ORP150 gene covering over 15-kb. Analyses of transcription initiation sites and transcriptional regulatory sequences revealed that at least three distinct mRNA species were produced by alternative promoters: two of them starting from alternative exon 1 (1A or 1B), and the third one starting from exon 2, six nucleotides upstream of the first AUG initiation codon. Among them, the transcript that begins with exon 1B was preferentially induced by hypoxia or tunicamycin treatment. A cis-acting segment involved in the stress-dependent induction was found at the 5'-end of exon 1A, which could account for the selective induction of the transcription from exon 1B. Furthermore, in vitro analyses of translation of the third mRNA suggested the constitutive expression of the cytosolic ORP150 due to the lack of the signal peptide resulting from differential translation initiation.
Assuntos
Genes Reguladores/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , RNA Mensageiro/biossíntese , Estresse Fisiológico/genética , Sequência de Bases , Northern Blotting , Hipóxia Celular , Eletroforese em Gel de Ágar , Biblioteca Genômica , Proteínas de Choque Térmico HSP70 , Humanos , Immunoblotting , Luciferases/metabolismo , Dados de Sequência Molecular , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteínas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Células Tumorais CultivadasRESUMO
Adenosinetriphosphatase (ATPase) [EC 3.6.1,3] activity has been found to exist in most preparations of DNA-dependent RNA polymerase [EC 2.7.7.6] obtained from Escherichia coli by a number of purification procedures so far established. Electrophoretic analysis on polyacrylamide gels demonstrated that ATP hydrolysis and RNA synthesis were catalyzed by two distinct enzyme proteins. It appears that the two enzymes are associated or have similar molecular properties. Separation of the two enzymes, the object of the present work, was achieved by three independent methods: ion exchange chromatography on a phosphocellulose column, electrophoresis in glycerol gradients, or high-salt glycerol gradient centrifugation.