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1.
J Struct Biol ; 215(3): 108006, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37507029

RESUMO

Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.


Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , Proteínas de Saccharomyces cerevisiae , Humanos , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
2.
Proteins ; 90(3): 889-897, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34796993

RESUMO

Eukaryotic translation initiates upon recruitment of the EIF2-GTP·Met-tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, ß, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α-ε subunits). During stress-induced conditions, phosphorylation of the α-subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ-subunit. We show that the IPA mRNA isoform is translated into a C-terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer-specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co-exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress-induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Bases de Dados de Ácidos Nucleicos , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/genética , Humanos , Células MCF-7 , Modelos Moleculares , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Isoformas de Proteínas , Relação Estrutura-Atividade
3.
Biomed Res ; 42(3): 95-102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34092754

RESUMO

Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.


Assuntos
Apoptose , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Antineoplásicos/farmacologia , Morte Celular , Clonagem Molecular , Códon de Terminação , Fator de Iniciação 2 em Eucariotos/química , Proteínas Ativadoras de GTPase/química , Células HCT116 , Humanos , Fosforilação , Domínios Proteicos , Transfecção
4.
PLoS Comput Biol ; 16(5): e1007864, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453748

RESUMO

Interactions between disordered proteins involve a wide range of changes in the structure and dynamics of the partners involved. These changes can be classified in terms of binding modes, which include disorder-to-order (DO) transitions, when proteins fold upon binding, as well as disorder-to-disorder (DD) transitions, when the conformational heterogeneity is maintained in the bound states. Furthermore, systematic studies of these interactions are revealing that proteins may exhibit different binding modes with different partners. Proteins that exhibit this context-dependent binding can be referred to as fuzzy proteins. Here we investigate amino acid code for fuzzy binding in terms of the entropy of the probability distribution of transitions towards decreasing order. We implement these entropy calculations into the FuzPred (http://protdyn-fuzpred.org) algorithm to predict the range of context-dependent binding modes of proteins from their amino acid sequences. As we illustrate through a variety of examples, this method identifies those binding sites that are sensitive to the cellular context or post-translational modifications, and may serve as regulatory points of cellular pathways.


Assuntos
Sítios de Ligação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/química , Algoritmos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Fator de Iniciação 2 em Eucariotos/química , Lógica Fuzzy , Humanos , Proteínas Intrinsicamente Desordenadas/química , Probabilidade , Domínios Proteicos , Dobramento de Proteína , Curva ROC , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteína Supressora de Tumor p53/química , eIF-2 Quinase/química
5.
Int J Mol Sci ; 21(6)2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32192132

RESUMO

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,ß,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Evolução Molecular , Técnicas de Silenciamento de Genes , Humanos , Mamíferos , Camundongos Knockout , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Transdução de Sinais , Estresse Fisiológico , Relação Estrutura-Atividade , Sítio de Iniciação de Transcrição , Leveduras/genética , Leveduras/metabolismo
6.
Cell Death Dis ; 10(6): 418, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138785

RESUMO

Hepatocellular carcinoma (HCC) is the one of the most common cancers worldwide. Because the side effects of current treatments are severe, new effective therapeutic strategies are urgently required. Pterostilbene (PT), a natural analogue of resveratrol, has diverse pharmacologic activities, including antioxidative, anti-inflammatory and antiproliferative activities. Here we demonstrated that PT inhibits HCC cell growth without the induction of apoptosis in an endoplasmic reticulum (ER) stress- and autophagy-dependent manner. Mechanistic studies indicated that the combination of salubrinal and PT modulates ER stress-related autophagy through the phospho-eukaryotic initiation factor 2α/activating transcription factor-4/LC3 pathway, leading to a further inhibition of eIF2α dephosphorylation and the potentiation of cell death. An in vivo xenograft analysis revealed that PT significantly reduced tumour growth in mice with a SK-Hep-1 tumour xenograft. Taken together, our results yield novel insights into the pivotal roles of PT in ER stress- and autophagy-dependent cell death in HCC cells.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Estilbenos/farmacologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estilbenos/química , Estilbenos/uso terapêutico , Tioureia/análogos & derivados , Tioureia/farmacologia , Tioureia/uso terapêutico , Transplante Heterólogo
7.
EBioMedicine ; 42: 470-480, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30878599

RESUMO

BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.


Assuntos
Fator de Iniciação 2 em Eucariotos/genética , Genes Ligados ao Cromossomo X , Glucose/metabolismo , Hipopituitarismo/etiologia , Hipopituitarismo/metabolismo , Fenótipo , Substituição de Aminoácidos , Apoptose , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Pré-Escolar , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Hipopituitarismo/diagnóstico , Hibridização In Situ , Lactente , Imageamento por Ressonância Magnética , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Biossíntese de Proteínas
8.
Mol Biol Rep ; 46(1): 1275-1284, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30694453

RESUMO

Recent in vitro studies have shown that vitamin C (Vit C) with pro-oxidative properties causes cytotoxicity of breast cancer cells by selective oxidative stress. However, the effect of Vit C in itself at different concentration levels on MCF-7 breast cancer cell line after 24 h, has not yet been described. We aimed to examine the effect of Vit C on the viability and signalling response of MCF-7/WT (MCF-7 wild-type) cells that were exposed to various concentrations (0.125-4 mM) of Vit C during 24 h. The cytotoxic effect of Vit C on MCF-7/VitC (MCF-7/WT after added 2 mM Vit C) was observed, resulting in a decrease of cell index after 12 h. Also, the cytotoxicity of Vit C (2 mM) after 24 h was confirmed by flow cytometry, i.e., increase of dead, late apoptotic, and depolarized dead MCF-7/VitC cells compared to MCF-7/WT cells. Moreover, changes in proteomic profile of MCF-7/VitC cells compared to the control group were investigated via label-free quantitative mass spectrometry and post-translational modification. Using bioinformatics assessment (i.e., iPathwayGuide and SPIA R packages), a significantly impacted pathway in MCF-7/VitC was identified, namely the protein processing in endoplasmic reticulum. The semi-quantitative change (log2fold change = 4.5) and autophosphorylation at Thr-446 of protein kinase (PKR) (involved in this pathway) indicates that PKR protein could be responsible for the unfolded protein response and inhibition of the cell translation during endoplasmic reticulum stress, and eventually, for cell apoptosis. These results suggest that increased activity of PKR (Thr-446 autophosphorylation) related to cytotoxic effect of Vit C (2 mM) may cause the MCF-7 cells death.


Assuntos
Ácido Ascórbico/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , eIF-2 Quinase/química , eIF-2 Quinase/metabolismo
9.
J Transl Med ; 17(1): 20, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30634982

RESUMO

BACKGROUND: Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported. METHODS: We evaluated levels and localization of eIF2α/peIF2α in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscopy analyses. Furthermore, we performed a sequence analyses and structure and dynamics studies of eIF2α protein to reveal the role of eIF2α and its correlations in different pathways involved in the invasive phase of melanoma. RESULTS: We found peIF2α both in cytoplasm and nucleus. Nuclear localization was more represented in V600BRAF melanoma cell lines. Our studies on eIF2α protein sequence indicated the presence of a predicted bipartite NLS as well as a nuclear export signal NES and an S1 domain, typical of RNA interacting proteins. Furthermore, we found high levels of transcription factor EB (TFEB), a component of the MiT/TFE family, and low ß-catenin levels in V600BRAF cells. CONCLUSIONS: Based on our results, we suggest that peIF2α nuclear localization can be crucial in ER stress response and in driving the metastatic spread of melanoma, through lysosomal signaling and Wnt/ß-catenin pathway. In conclusion, this is the first evidence of nuclear localization of peIF2α, representing a possible target for future therapeutic approaches for metastatic melanoma.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Melanoma/metabolismo , Biossíntese de Proteínas , Neoplasias Cutâneas/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/química , Humanos , Fosforilação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , beta Catenina/metabolismo , Melanoma Maligno Cutâneo
10.
Free Radic Res ; 52(11-12): 1359-1370, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30693837

RESUMO

Ultraviolet A (UVA) irradiation is a potential environmental stressor, which contributes to inflammation, photoaging, and carcinogenesis. UVA causes endoplasmic reticulum stress, hence phosphorylates the α subunit of eIF2. Meanwhile, UVA also induces expression of haem oxygenase-1 (HO-1) and nuclear factor erythroid-derived two related factor 2 (Nrf2) in human skin cells. In mouse JB6 cell, we found high dose UVA could change cell morphology, cause cell viability loss. UVA irradiation activated phosphorylation of eIF2α and Nrf2-HO-1 pathway in a dose-dependent manner. Besides, modulation of eIF2α phosphorylation status could alter expression pattern of Nrf2-HO-1 signalling. Salubrinal, a selective inhibitor of eIF2α dephosphorylation, increased the S phase in cell cycle of JB6 cells after UVA irradiation, suggesting phosphorylation status of eIF2α may affect cellular homeostasis under UVA irradiation. The study directed to further acknowledge about the relationship of UVA-induced eIF2α phosphorylation and Nrf2-HO-1 pathway, which may play a role in phototherapy and photo protection.


Assuntos
Células Epidérmicas/metabolismo , Células Epidérmicas/efeitos da radiação , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Heme Oxigenase-1/biossíntese , Raios Ultravioleta , Animais , Sobrevivência Celular , Células Cultivadas , Perfilação da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Camundongos , Fosforilação , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo
11.
Nucleic Acids Res ; 45(20): 11962-11979, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29036434

RESUMO

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric GTPase, which plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP•Met-tRNAi ternary complex (TC), which is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, which triggers the integrated stress response (ISR). Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms of eIF2B action and regulation remain unknown. Here we employ a combination of NMR, fluorescence spectroscopy, site-directed mutagenesis, and thermodynamics to elucidate the mechanisms of eIF2B action and its regulation by phosphorylation of the substrate eIF2. We present: (i) a novel mechanism for the inhibition of eIF2B activity, whereby eIF2α phosphorylation destabilizes an autoregulatory intramolecular interaction within eIF2α; and (ii) the first structural model for the complex of eIF2B with its substrate, eIF2-GDP, reaction intermediates, apo-eIF2 and eIF2-GTP, and product, TC, with direct implications for the eIF2B catalytic mechanism.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Espectrometria de Fluorescência , Termodinâmica
12.
Mol Cell ; 67(3): 447-456.e7, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28732596

RESUMO

After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Complexos Multiproteicos , Mutação , Conformação de Ácido Nucleico , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Relação Estrutura-Atividade , Transfecção
13.
Nucleic Acids Res ; 43(17): 8392-404, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26264663

RESUMO

A plethora of stresses trigger a rapid downregulation of protein synthesis. However, a fraction of mRNAs continue to be recruited onto polysomes and their protein products play a key role in deciding cell fate. These transcripts are characterized by the presence of uORFs within their 5' TL coupling protein expression to reinitiation. The translational brake arises due to the activation of a family of kinases targeting the α subunit of the trimolecular eIF2(αßγ) initiation factor. Phosphorylation of eIF2αSer51 inhibits ternary complex regeneration reducing the pool of 43S ribosomes. It is popular to mimic this event, and hence the integrated stress response (ISR), by the expression of the phosphomimetic eIF2αS51D. However, we report that whereas the ISR is reproduced by eIF2αS51D expression in human HEK293T cells this is not the case in N2a mouse neuroblastoma cells. With regards to translational downregulation, this arises due to the failure of the phosphomimetic protein to assemble an eIF2 complex with endogenous eIF2ß/γ. This can be compensated for by the transient co-expression of all three subunits. Curiously, these conditions do not modulate reinitiation and consequently fail to trigger the ISR. This is the first demonstration that the inhibitory and reinitiation functions of eIF2αS/D can be separated.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Estresse Fisiológico/genética , Animais , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/química , Células HEK293 , Humanos , Camundongos , Fosforilação , Subunidades Proteicas/metabolismo
14.
Structure ; 23(9): 1596-1608, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26211610

RESUMO

Eukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2γ. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg(2+), and conserved residues contacting ATP-Mg(2+) are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2αγ onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2γ to eIF2α.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Fator de Iniciação 2 em Eucariotos/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
15.
Fish Shellfish Immunol ; 40(2): 609-15, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25149588

RESUMO

The alpha subunit of Eukaryotic Initiation Factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses, which including virus infection. To investigate whether WSSV infection can activate the PERK-eIF2α pathway, the eIF2α in shrimp Litopenaeus vannamei, designed as LveIF2α, was analyzed. The LveIF2α, a 332-amino acid polypeptide, shares a high degree of similarity with eIF2α from other species, having two eIF2α protein signatures at the 13-88 aa and 192-243 aa. The WSSV challenge experiment showed that the protein level of the total LveIF2α was decreased after infection, while the phosphorylation of LveIF2α has no significant change, which indicated that the phosphorylation ratio of LveIF2α was increased after infection. Furthermore, inhibitor treatment led to a significant decrease of WSSV loads. Moreover, the Binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) stress sensor, and PERK were also investigated during virus infection and it was shown that they were both up-regulated. Taken together, these results suggested that WSSV infection can induce ER stress and activated the unfolded protein response (UPR), and the PERK-eIF2α pathway is important for innate immune during WSSV infection in shrimp.


Assuntos
Proteínas de Artrópodes/genética , Fator de Iniciação 2 em Eucariotos/genética , Regulação da Expressão Gênica , Imunidade Inata , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia
16.
J Biomol Screen ; 19(7): 1024-34, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24598103

RESUMO

PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the K(m) of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer-based assay that yielded a robust Z' factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/química , Animais , Simulação por Computador , Desenho de Fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/química , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Camundongos , Fenótipo , Fosforilação , Transdução de Sinais
17.
Mol Cancer ; 12(1): 135, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24215632

RESUMO

BACKGROUND: 6-Shogaol is a promising antitumor agent isolated from dietary ginger (Zingiber officinale). However, little is known about the efficacy of 6-shogaol on leukemia cells. Here we investigated the underlying mechanism of 6-shogaol induced apoptosis in human leukemia cells in vitro and in vivo. METHODS: Three leukemia cell lines and primary leukemia cells were used to investigate the apoptosis effect of 6-shogaol. A shotgun approach based on label-free proteome with LC-CHIP Q-TOF MS/MS was employed to identify the cellular targets of 6-shogaol and the differentially expressed proteins were analyzed by bioinformatics protocols. RESULTS: The present study indicated that 6-shogaol selectively induced apoptosis in transformed and primary leukemia cells but not in normal cells. Eukaryotic translation initiation factor 2 alpha (eIF2α), a key regulator in apoptosis signaling pathway, was significantly affected in both Jurkat and U937 proteome profiles. The docking results suggested that 6-shogaol might bind well to eIF2α at Ser51 of the N-terminal domain. Immunoblotting data indicated that 6-shogaol induced apoptosis through a process involving dephosphorylation of eIF2α and caspase activation-dependent cleavage of eIF2α. Furthermore, 6-shogaol markedly inhibited tumor growth and induced apoptosis in U937 xenograft mouse model. CONCLUSION: The potent anti-leukemia activity of 6-shogaol found both in vitro and in vivo in our study make this compound a potential anti-tumor agent for hematologic malignancies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Catecóis/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Leucemia/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/química , Caspase 3/metabolismo , Caspase 7/metabolismo , Catecóis/química , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/química , Células HL-60 , Humanos , Células Jurkat , Leucemia/enzimologia , Leucemia/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Simulação de Acoplamento Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Struct Mol Biol ; 19(8): 744-53, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22751017

RESUMO

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Sítios de Ligação , Fator de Iniciação 2 em Eucariotos/química , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
19.
Bioorg Med Chem Lett ; 22(1): 402-9, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22153346

RESUMO

Symmetrical N,N'-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3-(trifluoromethyl)phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNA(i)(Met) ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N'-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.


Assuntos
Antineoplásicos/farmacologia , Química Farmacêutica/métodos , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células , Desenho de Fármacos , Fator de Iniciação 2 em Eucariotos/química , Genes Reporter , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Fosforilação , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/química , Relação Estrutura-Atividade , Fator de Transcrição CHOP/química , Transfecção , Ureia/química
20.
Nat Struct Mol Biol ; 18(11): 1227-34, 2011 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-22002225

RESUMO

In contrast to prokaryotic elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds methionyl initiator transfer RNA (Met-tRNA(i)(Met)) to the P-site of the 40S ribosomal subunit. The results of directed hydroxyl radical probing experiments to map binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNA(i)(Met) revealed that eIF2γ primarily contacts the acceptor stem of Met-tRNA(i)(Met) and identified a key binding interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Whereas the analogous domain III of EF-Tu contacts the T stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, not Met-tRNA(i)(Met). Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Guanosina Trifosfato/metabolismo , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cisteína/genética , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Guanosina Trifosfato/química , Radical Hidroxila/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Ribossômico 18S/química , RNA Ribossômico 18S/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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