RESUMO
BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.
Assuntos
Dano ao DNA/genética , Disceratose Congênita/genética , Fibroblastos/metabolismo , Ribossomos/fisiologia , Telômero/genética , Proteína Supressora de Tumor p53/genética , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Senescência Celular/fisiologia , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Fibroblastos/citologia , Perfilação da Expressão Gênica , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismoRESUMO
The human SBDS gene and its yeast ortholog SDO1 encode essential proteins that are involved in ribosome biosynthesis. SDO1 has been implicated in recycling of the ribosomal biogenesis factor Tif6p from pre-66S particles as well as in translation activation of 60S ribosomes. The SBDS protein is highly conserved, containing approximately 250 amino acid residues in animals, fungi and Archaea, while SBDS orthologs of plants and a group of protists contain an extended C-terminal region. In this work, we describe the characterization of the Trypanosoma cruzi SBDS ortholog (TcSBDS). TcSBDS co-fractionates with polysomes in sucrose density gradients, which is consistent with a role in ribosome biosynthesis. We show that TcSBDS contains a C-terminal extension of 200 amino acids that displays the features of intrinsically disordered proteins as determined by proteolytic, circular dichroism and NMR analyses. Interestingly, the C-terminal extension is responsible for TcSBDS-RNA interaction activity in electrophoretic mobility shift assays. This finding suggests that Trypanosomatidae and possibly also other organisms containing SBDS with extended C-terminal regions have evolved an additional function for SBDS in ribosome biogenesis.
Assuntos
Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Dados de Sequência Molecular , Polirribossomos/fisiologia , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
There is a great variety of resistance mechanisms observed in bacteria. Several mechanisms can operate simultaneously against a single antibiotic, and there are no species-specific mechanisms described so far. In many cases, resistance is mediated by mobile genetic elements (plasmids, phages and transposons), spreading among different bacterial genera, carrying in many instances multiresistant determinants. Administration of one type of antimicrobial agent can select resistance to other groups of antimicrobials. In summary, more details are found every day on these molecular processes. All this constitute an important tool for the design of new antimicrobials with broad specificity and higher efficacy in an attempt to control bacterial resistance. More important yet, this knowledge has a necessary implication which translates into a rational and adequate use of antimicrobial agents in the fight against bacterial infections.
Assuntos
Resistência Microbiana a Medicamentos/fisiologia , Antibacterianos/metabolismo , Permeabilidade da Membrana Celular , Parede Celular/metabolismo , Resistência Microbiana a Medicamentos/genética , Hidrólise , Ribossomos/fisiologia , beta-Lactamases , beta-LactamasRESUMO
A yeast ribosomal subunit association factor (AF) has been purified from a high-salt ribosomal wash. The purified enzyme is a thermostable protein that associates ribosomal subunits at low Mg2+ concentration without requiring energy. It appears to be an aggregate of trimers or dimers (molecular mass 125 or 79 kDa) which on sodium dodecyl sulfate gels shows the presence of a major protein band whose estimated molecular mass is 43 kDa. Evidence also indicates the existence of a 50-kDa polypeptide which seems to be unstable since with freezing and thawing it gives rise to the 43-kDa polypeptide. It was shown that the labelled factor interacts with 80S ribosomes and with 40S ribosomal subunits. The purified polypeptide reacts with antibodies directed against EF-1 alpha, this last protein recognizing the antibodies raised against AF. Likewise, both EF-1 alpha and AF associate ribosomal subunits in the same way. When EF-1 is heated, it not only maintains its association activity, but also behaves like a 43-kDa polypeptide in an SDS electrophoresis run. These observations strongly suggest that AF originates from EF-1 alpha, which implies that the well-known elongation factor may also play a role in the initiation step of protein synthesis.
Assuntos
Proteínas Fúngicas/biossíntese , Fatores de Alongamento de Peptídeos/fisiologia , Ribossomos/fisiologia , Saccharomyces cerevisiae/fisiologia , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fator 1 de Elongação de Peptídeos , Ribonucleoproteínas/fisiologiaRESUMO
Se estudian los perfiles ribosomales en un miometrio normal en menopausia, y tres miometrios normales al final del embarazo. Se estudian tambien estos perfiles ribosomales en tres miometrios patologicos en adenomiosis, en miomatosis y en sarcomatosis. La mayor actividad en sintesis proteica miometrial fue revelada en orden decreciente por la adenomiosis, la sarcomatosis, la miomatosis, el embarazo a termino y finalmente la menopausia.
Assuntos
Humanos , Células Eucarióticas/análise , Células Eucarióticas/citologia , Células Eucarióticas/patologia , Miométrio , Ribossomos/análise , Ribossomos/fisiologiaRESUMO
The mechanism by which insulin controls protein metabolism is not fully understood. Insulin stimulates protein synthesis; it also enhances transport of some amino acids, but the latter action does not appear to be sufficient explanation of the increase in synthesis. The various actions seem to be independent of the effects of glucose metabolism. In diabetic muscle there are a fewer than normal polysomes, and insulin rapidly enhances attachment of monomers to messenger-RNA. Insulin also increases the effectiveness of cell sap in catalyzing protein synthesis by ribosomal systems. The way in which the hormone may affect either initiation or peptide synthesis is not known. Experiments are reported bearing on whether availability of amino acids could be a mechanism by which effects of insulin are mediated. Activity of liver and muscle soluble fractions declines on fasting and, for the latter tissue, possibly also on a low protein diet. Sap from fasting animals allows a much smaller response of isolated ribosomes to added amino acids. Availability of glutamate in amino acid mixtures may be of special importance. However, insulin can influence the activity of the sap fraction of diaphram muscle during incubation without the presence of amino acids in the medium. Understanding of what mechanisms are involved will depend on resolution of the critical sap factors (Summary)