RESUMO
Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.
Assuntos
Proteômica , Espermidina , Humanos , Espermidina/metabolismo , Fatores de Iniciação de Peptídeos/genética , Diferenciação Celular , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at â¼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.
Assuntos
Ribossomos , Saccharomyces cerevisiae , Códon de Iniciação/metabolismo , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de ProteínasRESUMO
Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs. Recent studies in bacteria and eukaryotes have revealed the functions of evolutionarily conserved uORF-encoded peptides. Some of these uORF-encoded nascent peptides enable responses to specific metabolites to modulate the translation of their mRNAs by stalling ribosomes and through ribosome stalling may also modulate the level of their mRNAs. In this review, we highlight several examples of conserved uORF nascent peptides that stall ribosomes to regulate gene expression in response to specific metabolites in bacteria, fungi, mammals, and plants.
Assuntos
Fases de Leitura Aberta/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , Ribossomos/genéticaRESUMO
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 ProteínasRESUMO
Polyamines are organic polycations that bind to a variety of cellular molecules, including nucleic acids. Within cells, polyamines contribute to both the efficiency and fidelity of protein synthesis. In addition to directly acting on the translation apparatus to stimulate protein synthesis, the polyamine spermidine serves as a precursor for the essential post-translational modification of the eukaryotic translation factor 5A (eIF5A), which is required for synthesis of proteins containing problematic amino acid sequence motifs, including polyproline tracts, and for termination of translation. The impact of polyamines on translation is highlighted by autoregulation of the translation of mRNAs encoding key metabolic and regulatory proteins in the polyamine biosynthesis pathway, including S-adenosylmethionine decarboxylase (AdoMetDC), antizyme (OAZ), and antizyme inhibitor 1 (AZIN1). Here, we highlight the roles of polyamines in general translation and also in the translational regulation of polyamine biosynthesis.
Assuntos
Poliaminas/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Animais , Humanos , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.
Assuntos
Proteínas de Transporte/biossíntese , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Poliaminas/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Códon de Iniciação , Sequência Conservada , Células HEK293 , Humanos , Camundongos , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.
Assuntos
Aminoácidos/metabolismo , Lisina/análogos & derivados , Biossíntese Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Poliaminas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Lisina/metabolismo , Conformação de Ácido Nucleico , Fatores de Iniciação de Peptídeos/química , Peptídeos/metabolismo , Prolina/análogos & derivados , Prolina/química , Prolina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , RNA de Transferência de Prolina/química , RNA de Transferência de Prolina/metabolismo , Proteínas de Ligação a RNA/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3' UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.
Assuntos
Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas , Motivos de Aminoácidos , Códon de Terminação , Perfilação da Expressão Gênica/métodos , Hidrólise , Cinética , Fatores de Iniciação de Peptídeos/genética , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.
Assuntos
Peptídeos/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Ribossomos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Modelos Moleculares , Peptídeos/química , Prolina/química , Ligação Proteica , RNA de Transferência de Prolina/metabolismo , Ribossomos/metabolismoRESUMO
In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.
Assuntos
Regulação Fúngica da Expressão Gênica , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia PeptídicaRESUMO
Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals a previously unseen conformation of an eIF5A-ribosome complex and highlights a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A-ribosome association.
Assuntos
Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a ß-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.
Assuntos
Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/química , Peptídeos/metabolismo , Proteínas de Ligação a RNA/química , Ribossomos/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
Assuntos
Motivos de Aminoácidos , Fatores de Iniciação de Peptídeos/fisiologia , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/fisiologia , Modelos Biológicos , Modelos Moleculares , Estrutura Terciária de Proteína , Ribossomos/metabolismo , Ribossomos/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium, the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasite's erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.
Assuntos
Plasmodium falciparum/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Animais , Anopheles , Códon , DNA/genética , Proteínas Fúngicas/química , Células Hep G2 , Humanos , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Serina/químicaRESUMO
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/metabolismoRESUMO
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at serine 51 inhibits protein synthesis in cells subjected to various forms of stress including virus infection. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon (IFN) action. We have investigated a possible functional relationship between the E6 oncoprotein and eIF2alpha phosphorylation by an inducible-dimerization form of the IFN-inducible protein kinase PKR. Herein, we demonstrate that HPV type 18 E6 protein synthesis is rapidly repressed upon eIF2alpha phosphorylation caused by the conditional activation of the kinase. The remainder of E6, however, can rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis. E6 physically associates with GADD34/PP1 holophosphatase complex, which mediates translational recovery, and facilitates eIF2alpha dephosphorylation. Inhibition of eIF2alpha phosphorylation by E6 mitigates eIF2alpha-dependent responses to transcription and translation of proapoptotic genes. These findings demonstrate, for the first time, a role of the oncogenic E6 in apoptotic signaling induced by PKR and eIF2alpha phosphorylation. The functional interaction between E6 and the eIF2alpha phosphorylation pathway may have important implications for HPV infection and associated pathogenesis.
Assuntos
Morte Celular/fisiologia , Proteínas de Ligação a DNA , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Proteínas Oncogênicas Virais/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Aminocumarinas , Antígenos de Diferenciação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cumarínicos/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Interferon-alfa/metabolismo , Proteínas Oncogênicas Virais/genética , Papillomaviridae/metabolismo , Infecções por Papillomavirus , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1 , Subunidades Proteicas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica , Proteína X Associada a bcl-2 , eIF-2 Quinase/metabolismoRESUMO
Phosphorylation of the translation initiation factor eIF2 on Ser51 of its alpha subunit is a key event for regulation of protein synthesis in all eukaryotes. M156R, the product of the myxoma virus M156R open reading frame, has sequence similarity to eIF2alpha as well as to a family of viral proteins that bind to the interferon-induced protein kinase PKR and inhibit phosphorylation of eIF2alpha. In this study, we demonstrate that, like eIF2alpha. M156R is an efficient substrate for phosphorylation by PKR and can compete with eIF2alpha. To gain insights into the substrate specificity of the eIF2alpha kinases, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel beta-barrel with two of the strands connected by a loop and an alpha-helix. The similarity between M156R and the beta-barrel structure in the N terminus of eIF2alpha suggests that the viral homologs mimic eIF2alpha structure in order to compete for binding to PKR. A homology-modeled structure of the well-studied vaccinia virus K3L was generated on the basis of alignment with M156R. Comparison of the structures of the K3L model, M156R, and human eIF2alpha indicated that residues important for binding to PKR are located at conserved positions on the surface of the beta-barrel and in the mobile loop, identifying the putative PKR recognition motif.