RESUMO
BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).
Assuntos
Calreticulina/genética , Mutação , Síndromes Mielodisplásicas/genética , Mielofibrose Primária/genética , Trombocitemia Essencial/genética , Sequência de Aminoácidos , Doenças da Medula Óssea/genética , Calreticulina/análise , Éxons , Humanos , Janus Quinase 2/genética , Leucemia Mieloide/genética , Dados de Sequência Molecular , Neoplasias/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.
Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/química , Proteínas de Choque Térmico , Proteínas de Membrana , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Cricetinae , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Camundongos , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Peso Molecular , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Termodinâmica , Transfecção , eIF-2 Quinase/química , eIF-2 Quinase/isolamento & purificaçãoRESUMO
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas de Choque Térmico , Fatores de Iniciação de Peptídeos/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição , Animais , Antígenos de Diferenciação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células CHO , Proteínas de Transporte/metabolismo , Domínio Catalítico , Proteínas de Ciclo Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática , Retroalimentação , Regulação da Expressão Gênica , Camundongos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fator de Iniciação 2 em Procariotos , Ligação Proteica , Biossíntese de Proteínas , Desnaturação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.
Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Proteínas de Membrana , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Endorribonucleases/genética , Ativação Enzimática , Marcação de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Complexos Multienzimáticos/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas/química , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fator 2 Associado a Receptor de TNF , Tapsigargina/farmacologia , Técnicas do Sistema de Duplo-Híbrido , eIF-2 Quinase/metabolismoRESUMO
Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene. This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator. However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites. To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences. We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site. Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding. The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms. This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb. Rev-Erb also appeared to bind to the selected site as a monomer. Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand. Thus, Rev-Erb is a transcriptional activator whose properties differ dramatically from those of classical nuclear hormone receptors, including the TR encoded on the opposite strand of the same genomic locus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Produtos do Gene rev/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Ligação Competitiva , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Coriocarcinoma , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Oligodesoxirribonucleotídeos/metabolismo , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Reticulócitos/metabolismo , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores dos Hormônios Tireóideos , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Transcrição Gênica , Ativação Transcricional , Sequência de Bases , Sequência Consenso , Proteínas Fúngicas/genética , Humanos , Dados de Sequência Molecular , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Sequências Repetitivas de Ácido Nucleico , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Análise de Sequência , Relação Estrutura-Atividade , Tretinoína/farmacologiaRESUMO
Binding of the thyroid hormone receptor (TR) to thyroid hormone-responsive elements (TREs) is crucial for regulation of gene expression by thyroid hormone. The TR binds to each half-site of a palindromic TRE separately, as a monomer, or simultaneously, as a homodimer. In addition, the TR monomer interacts with a 42-kDa protein that may be responsible for an increase in the apparent size and stability of the TR-TRE complex after incubation with liver nuclear extract. The multiple DNA-binding forms of the TR contact the TRE differently but compete for binding in a dynamic equilibrium which is highly dependent on the relative concentrations of TR and nuclear protein. Thus, protein-protein interactions are likely to determine the context in which the TR binds to target genes and regulates the transcriptional response to thyroid hormone.
Assuntos
DNA/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Escherichia coli/metabolismo , Cinética , Extratos Hepáticos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação/genética , Ratos , Receptores dos Hormônios Tireóideos/genética , Proteínas Recombinantes/biossíntese , Sequências Repetitivas de Ácido NucleicoRESUMO
Ligand-independent transcriptional repression is an important function of nuclear hormone receptors. An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR). N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR. Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb. Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR. The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors. N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors. These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Sequência Consenso , Sequência Conservada , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Escherichia coli , Glutationa Transferase/biossíntese , Humanos , Rim , Dados de Sequência Molecular , Biossíntese de Proteínas , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores do Ácido Retinoico/química , Receptores dos Hormônios Tireóideos/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Repressoras/biossíntese , Proteínas Repressoras/química , Receptor alfa de Ácido Retinoico , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Mutation of the orphan nuclear receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have found that RORalpha activates transcription from only a subset of sites to which it binds strongly as a monomer. RORalpha also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of RORalpha, we fused its C terminus to a heterologous DNA-binding domain. Mutational analysis revealed that RORalpha contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of RORalpha polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of RORalpha inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by RORalpha is complex and likely to be regulated in a cell type- and target gene-specific manner.
Assuntos
Cerebelo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Receptores Citoplasmáticos e Nucleares/fisiologia , Transativadores/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Substâncias Macromoleculares , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Correpressor 2 de Receptor Nuclear , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/química , Transcrição Gênica/efeitos dos fármacosRESUMO
We have isolated complementary DNA clones encoding a novel orphan member of the nuclear receptor superfamily, termed BD73. This protein shows strong amino acid sequence similarity to the previously described Rev-ErbA alpha. Unlike Rev-Erb, in which the opposite strand of the C-terminal coding region encodes the C-terminal portion of a variant thyroid hormone receptor isoform, the opposite strand of the C-terminal coding region of BD73 does not have any extensive open reading frames. BD73 messenger RNA is expressed in a wide variety of tissues and cell lines. In quiescent HepG2 cells, BD73 messenger RNA levels are strongly induced by planar aromatic antioxidants. Like Rev-Erb, BD73 binds as a monomer to a DNA sequence which consists of a specific A/T-rich sequence upstream of the consensus hexameric half-site specified by the P box of the DNA-binding domain. Amino acid sequence comparisons suggest that the A box sequence, which has been suggested to mediate monomer binding by other superfamily members, lies closer to the DNA-binding domain in BD73 and Rev-Erb than in other receptors. Under the conditions examined, neither BD73 nor Rev-Erb activated reporters containing multiple copies of their common binding site. Thus, these two orphans may require an as yet unidentified ligand or other signal for such activation. Together, BD73 and Rev-Erb define a subgroup of orphan receptors that bind as monomers to a half-site flanked by a specific and extended A/T-rich sequence.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Sequência de Aminoácidos , Antioxidantes/farmacologia , Sequência de Bases , Sítios de Ligação , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Família Multigênica , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Proteínas/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/química , Receptores dos Hormônios Tireóideos/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative disorders that are currently incurable.
Assuntos
Acetamidas/uso terapêutico , Cicloexilaminas/uso terapêutico , Doenças Neurodegenerativas/prevenção & controle , Pâncreas/metabolismo , Acetamidas/efeitos adversos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Cicloexilaminas/efeitos adversos , Immunoblotting , Camundongos , Pâncreas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Espectrometria de Massas em TandemRESUMO
The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR.
Assuntos
Aminoácidos/deficiência , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Antibacterianos/farmacologia , Northern Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Células HeLa , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Leucina/metabolismo , Fatores de Tempo , Fator de Transcrição CHOP , Tunicamicina/farmacologiaRESUMO
We used an antiserum to a hydrophobic 6 KD surfactant-associated protein to localize this protein in human lung tissue. This antiserum does not crossreact with the 35 KD surfactant-associated protein. By light microscopy using the indirect immunoperoxidase technique, the protein appears to be localized within Type II alveolar epithelial cells. Staining is also detectable in alveolar macrophages and occasionally within the lumina of alveoli and bronchioles. No staining was detected within the alveolar septa or in association with blood vessels. An identical distribution is seen for the 35 KD surfactant-associated protein using an antiserum specific for that protein.
Assuntos
Técnicas Imunoenzimáticas , Pulmão/análise , Proteínas/análise , Surfactantes Pulmonares/análise , Brônquios/análise , Eletroforese em Gel de Poliacrilamida , Epitélio/análise , Humanos , Soros Imunes/imunologia , Imunoensaio , Imuno-Histoquímica , Proteínas/imunologia , Alvéolos Pulmonares/análise , Distribuição TecidualRESUMO
Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2alpha (eIF2alpha) in response to a stress signal from the endoplasmic reticulum (ER). Activation of this process leads to reduced rates of initiation of protein translation during ER stress. Here we describe the cloning of perk, a gene encoding a type I transmembrane ER-resident protein. PERK has a lumenal domain that is similar to the ER-stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2alpha kinases, PKR and HRI. ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2alpha on serine residue 51, inhibiting translation of messenger RNA into protein. These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress.
Assuntos
Retículo Endoplasmático/enzimologia , Biossíntese de Proteínas , Dobramento de Proteína , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , eIF-2 Quinase/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Clonagem Molecular , Escherichia coli , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Humanos , Membranas Intracelulares/enzimologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , eIF-2 Quinase/química , eIF-2 Quinase/fisiologiaRESUMO
The effects of postnatal dexamethasone treatment in vivo on the synthesis of surfactant protein A (SP-A) were examined at the protein and RNA levels. Rats ranging from 1 day old to adult were injected with 200 micrograms of dexamethasone/kg body wt or with vehicle alone and were killed 24 h after injection. One portion of the lung was metabolically labeled with [35S]methionine, the proteins immunoprecipitated using an antiserum to SP-A, and analyzed electrophoretically. Both newly synthesized intracellular and secreted SP-A levels were increased by dexamethasone, reaching averages of 2.3 and 4.5 times control values, respectively. Another portion of the lung tissue was used for RNA analysis. SP-A mRNA levels were also elevated an average of 1.4 times control values by hormone treatment. Dose-response experiments using 16-day-old pups showed that both total SP-A, as measured by enzyme-linked immunosorbent assay, and total SP-A mRNA levels were elevated with dexamethasone treatment, reaching maximal stimulation at 2 mg. We conclude that postnatal dexamethasone treatment in vivo results in increased levels of both newly synthesized SP-A and SP-A mRNA, suggesting that pretranslational events may in part contribute to this process.
Assuntos
Dexametasona/farmacologia , Pulmão/crescimento & desenvolvimento , Proteolipídeos/biossíntese , Surfactantes Pulmonares/biossíntese , Animais , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Peso Molecular , Proteolipídeos/genética , Proteolipídeos/isolamento & purificação , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
Most of the criteria for competence in current use emphasize cognitive rather than affective dimensions. Our clinical experience indicates that affective disorders may impair competence in a detectable and identifiable way. In particular, patients with major affective disorders can retain the cognitive capacity to understand the risks and benefits of a medication, yet fail to appreciate its benefits. A case study of a pathologic grief reaction is introduced to illustrate how cognitive and affective impairments may coexist and require separate remedial strategies for restoration. Further empirical work on the role of affective disorder in impairing competence is warranted and planned.
Assuntos
Prova Pericial/legislação & jurisprudência , Consentimento Livre e Esclarecido/legislação & jurisprudência , Pessoas Mentalmente Doentes , Transtornos do Humor/diagnóstico , Transtornos do Humor/psicologia , Direito a Morrer/legislação & jurisprudência , Recusa do Paciente ao Tratamento , Idoso , Compreensão , Transtorno Depressivo/diagnóstico , Transtorno Depressivo/psicologia , Humanos , Masculino , Equipe de Assistência ao Paciente , Medição de Risco , Papel do DoenteRESUMO
Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.
Assuntos
Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Dobramento de Proteína , eIF-2 Quinase/metabolismo , Adaptação Fisiológica , Animais , Sobrevivência Celular , Cicloeximida/farmacologia , Camundongos , Mutação , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Tunicamicina/farmacologia , eIF-2 Quinase/genéticaRESUMO
The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Pâncreas/enzimologia , Biossíntese de Proteínas/fisiologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/ultraestrutura , Feminino , Glucagon/análise , Hiperglicemia/metabolismo , Marcação In Situ das Extremidades Cortadas , Insulina/análise , Insulina/sangue , Insulina/genética , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Pâncreas/citologia , Pâncreas/metabolismo , Fenótipo , Fosforilação , Proinsulina/metabolismo , RNA Mensageiro/análise , Vesículas Secretórias/metabolismoRESUMO
Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis. Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells. When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153). This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis. Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals.