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Mol Cell ; 81(1): 88-103.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33220178


The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Regulação Alostérica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HeLa , Humanos , Fosforilação
Science ; 359(6383): 1533-1536, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29599245


The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the ß and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Acetamidas/farmacologia , Animais , Microscopia Crioeletrônica , Cicloexilaminas/farmacologia , Fator de Iniciação 2B em Eucariotos/genética , Células HeLa , Humanos , Camundongos , Mutagênese , Fosforilação , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Estresse Fisiológico/efeitos dos fármacos
Pathol Oncol Res ; 20(3): 707-17, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24599561


All-trans-retinoic acid (atRA), the oxidized form of vitamin A (retinol), regulates a wide variety of biological processes, such as cell proliferation and differentiation. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs, RALDHs) as well as aldo-keto reductases (AKRs) catalyze atRA production. The reduced atRA biosynthesis has been observed in several human tumors, including colorectal cancer. However, subsets of atRA-synthesizing enzymes have not been determined in colorectal tumors. We investigated the expression patterns of genes involved in atRA biosynthesis in normal human colorectal tissues, primary carcinomas and cancer cell lines by RT-PCR. These genes were identified using transcriptomic data analysis (expressed sequence tags, RNA-sequencing, microarrays). Our results indicate that each step of the atRA biosynthesis pathway is dysregulated in colorectal cancer. Frequent and significant decreases in the mRNA levels of the ADH1B, ADH1C, RDHL, RDH5 and AKR1B10 genes were observed in a majority of colorectal carcinomas. The expression levels of the RALDH1 gene were reduced, and the expression levels of the cytochrome CYP26A1 gene increased. The human colon cancer cell lines showed a similar pattern of changes in the mRNA levels of these genes. A dramatic reduction in the expression of genes encoding the predominant retinol-oxidizing enzymes could impair atRA production. The most abundant of these genes, ADH1B and ADH1C, display decreased expression during progression from adenoma to early and more advanced stage of colorectal carcinomas. The diminished atRA biosynthesis may lead to alteration of cell growth and differentiation in the colon and rectum, thus contributing to the progression of colorectal cancer.

Adenoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Bases de Dados Factuais , Perfilação da Expressão Gênica , Tretinoína/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , Adenoma/patologia , Álcool Desidrogenase/genética , Oxirredutases do Álcool/genética , Aldeído Redutase/genética , Aldo-Ceto Redutases , Estudos de Casos e Controles , Colo/metabolismo , Neoplasias Colorretais/patologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reto/metabolismo
J Biol Chem ; 287(53): 44338-44, 2012 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-23148209


Loss-of-function mutations in EIF2AK3, encoding the pancreatic endoplasmic reticulum (ER) kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However, to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK modulation of protein synthesis rates. Here, we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.

Retículo Endoplasmático/enzimologia , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , eIF-2 Quinase/antagonistas & inibidores , Animais , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Glucose/metabolismo , Técnicas In Vitro , Insulina/química , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Biossíntese de Proteínas/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ratos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo