RESUMO
Dysregulation of mRNA translation is involved in the onset and progression of different types of cancer. To gain insight into novel genetic strategies to avoid this malady, we reviewed the available genomic, transcriptomic, and proteomic data about the translational machinery from the naked-mole rat (NMR) Heterocephalus glaber, a new model of study that exhibits high resistance to cancer. The principal features that might confer cancer resistance are 28S rRNA fragmentation, RPL26 and eIF4G overexpression, global downregulation of mTOR pathway, specific amino acid residues in RAPTOR (P908) and RICTOR (V1695), and the absence of 4E-BP3. These features are not only associated with cancer but also might couple longevity and adaptation to hypoxia. We propose that the regulation of translation is among the strategies endowing NMR cancer resistance.
Assuntos
Resistência à Doença/genética , Ratos-Toupeira/genética , Neoplasias/genética , Transcriptoma/genética , Animais , Fator de Iniciação Eucariótico 4G/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Longevidade/genética , Neoplasias/patologia , RNA Ribossômico 28S/genética , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Regulatória Associada a mTOR/genética , Hipóxia Tumoral/genéticaRESUMO
Kinetoplastida, a class of early-diverging eukaryotes that includes pathogenic Trypanosoma and Leishmania species, display key differences in their translation machinery compared with multicellular eukaryotes. One of these differences involves a larger number of genes encoding eIF4E and eIF4G homologs and the interaction pattern between the translation initiation factors. eIF4G is a scaffold protein which interacts with the mRNA cap-binding factor eIF4E, the poly(A)-binding protein, the RNA helicase eIF4A and the eIF3 complex. It contains the so-called middle domain of eIF4G (MIF4G), a multipurpose adaptor involved in different protein-protein and protein-RNA complexes. Here, the crystal structure of the MIF4G domain of T. cruzi EIF4G5 is described at 2.4â Å resolution, which is the first three-dimensional structure of a trypanosomatid MIF4G domain to be reported. Structural comparison with IF4G homologs from other eukaryotes and other MIF4G-containing proteins reveals differences that may account for the specific interaction mechanisms of MIF4G despite its highly conserved overall fold.
Assuntos
Proteínas de Bactérias/química , Fator de Iniciação Eucariótico 4G/química , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Trypanosoma cruzi/genéticaRESUMO
Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biossíntese de Proteínas/genética , Multimerização Proteica/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Chlorocebus aethiops , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Mutação Puntual/genética , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Viral/genética , Rotavirus/genética , Infecções por Rotavirus/virologia , Alinhamento de Sequência , Replicação Viral/genéticaRESUMO
In higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Site-directed mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation.
Assuntos
Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Leishmania major/genética , Iniciação Traducional da Cadeia Peptídica , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Sítios de Ligação , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/química , Fator de Iniciação Eucariótico 4G/metabolismo , Regulação da Expressão Gênica , Leishmania major/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/metabolismoRESUMO
Dietary soy is thought to be cancer-preventive; however, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein or combined soy isoflavones (genistein, daidzein, and glycitein) increased primary mammary tumor growth and metastasis. Cancer-promoting molecules, including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E, were up-regulated in mammary tumors from mice that received dietary daidzein. Herein, we show that increased eIF expression in tumor extracts of mice after daidzein diets is associated with protein expression of mRNAs with internal ribosome entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. Results with metastatic cancer cell lines show that some of the effects of daidzein in vivo can be recapitulated by the daidzein metabolite equol. In vitro, equol, but not daidzein, up-regulated eIF4G without affecting eIF4E or its regulator, 4E-binding protein (4E-BP), levels. Equol also increased metastatic cancer cell viability. Equol specifically increased the protein expression of IRES containing cell survival and proliferation-promoting molecules and up-regulated gene and protein expression of the transcription factor c-Myc. Moreover, equol increased the polysomal association of mRNAs for p 120 catenin and eIF4G. The elevated eIF4G in response to equol was not associated with eIF4E or 4E-binding protein in 5' cap co-capture assays or co-immunoprecipitations. In dual luciferase assays, IRES-dependent protein synthesis was increased by equol. Therefore, up-regulation of eIF4G by equol may result in increased translation of pro-cancer mRNAs with IRESs and, thus, promote cancer malignancy.
Assuntos
Neoplasias da Mama/metabolismo , Equol/efeitos adversos , Fator de Iniciação Eucariótico 4G/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glycine max/química , Fitoestrógenos/efeitos adversos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Suplementos Nutricionais/efeitos adversos , Equol/química , Equol/farmacologia , Fator de Iniciação 4E em Eucariotos/biossíntese , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Isoflavonas/efeitos adversos , Isoflavonas/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fitoestrógenos/química , Fitoestrógenos/farmacologia , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transplante Heterólogo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
OBJECTIVE: The aim of the present study was to identify differentially expressed genes that might be associated with the phenotype of superficial and invasive bladder cancer. METHODS: Differential display reverse transcriptase PCR (DDRT-PCR) was used to compare the expression pattern between normal bladder tissue and 4 groups of transitional cell carcinomas of the bladder regarding clinical stage and grade. RESULTS: We were able to identify 72 different transcripts, of which 57 (79%) showed homology to known genes, 12 (17%) to hypothetical proteins and 3 (4%) to human expressed sequence tags. Among the differentially expressed genes, SFRP1,CEP63 and EIF4G2 were further validated by quantitative RT-PCR in a series of 50 transitional cell carcinomas. Overall, the transcripts of these three genes were shown to be downregulated in the bladder tumors analyzed. In accordance with the DDRT-PCR results, the SFRP1 transcripts were shown to be downregulated in 90% (45/50) of the bladder tumors as compared with the normal bladder tissue. Although EIF4G2 and CEP63 transcripts exhibited three different expression patterns, downregulation was found in about 50% of the cases analyzed. In addition, downregulation of both CEP63 and EIF4G2 gene transcription was associated with invasive tumors. CONCLUSION: The use of DDRT-PCR analysis to compare expression patterns among different subgroups of bladder tumors allowed us to identify a significant number of genes implicated in different cellular pathways that, when up- or downregulated, might play a role in the tumorigenic process of the bladder.