RESUMEN
MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.
Asunto(s)
Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Cristalografía por Rayos X , Humanos , Análisis de los Mínimos Cuadrados , Espectroscopía de Resonancia Magnética , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Ribonucleasa III/metabolismo , TemperaturaRESUMEN
Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.
Asunto(s)
Sistema de Lectura Ribosómico , ARN Viral/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Secuencia de Bases , Codón , Secuencia Conservada , Dimerización , Secuencias Invertidas Repetidas , Cinética , Magnesio/química , Viabilidad Microbiana , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , TemperaturaRESUMEN
The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.
Asunto(s)
Regiones no Traducidas 3' , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Modelos Biológicos , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Estabilidad del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Aptámeros de Nucleótidos , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , Leucemia/genética , Leucemia/mortalidad , Oligodesoxirribonucleótidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , Estabilidad del ARN/efectos de los fármacos , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , NucleolinaRESUMEN
Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington's disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues. The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins. Anfinsen's paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP-nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding-misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.
Asunto(s)
Priones/química , Priones/metabolismo , Pliegue de Proteína , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Agua/química , Humanos , Ligandos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismoRESUMEN
Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.
Asunto(s)
Antígenos de Superficie/metabolismo , Carcinoma de Células Escamosas/patología , Leucemia/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas de Unión al ARN/metabolismo , Carcinoma de Células Escamosas/genética , Centrifugación por Gradiente de Densidad , Proteínas ELAV , Proteína 1 Similar a ELAV , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HL-60 , Humanos , Inmunoprecipitación , Leucemia/genética , Fosfoproteínas/metabolismo , Polirribosomas/metabolismo , Unión Proteica , Proto-Oncogenes Mas , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico/genética , NucleolinaRESUMEN
The tumor suppressor protein p53 is a nuclear protein that serves as an important transcription factor. The region responsible for sequence-specific DNA interaction is located in its core domain (p53C). Although full-length p53 binds to DNA as a tetramer, p53C binds as a monomer since it lacks the oligomerization domain. It has been previously demonstrated that two core domains have a dimerization interface and undergo conformational change when bound to DNA. Here we demonstrate that the interaction with a consensus DNA sequence provides the core domain of p53 with enhanced conformational stability at physiological salt concentrations (0.15 M). This stability could be either increased or abolished at low (0.01 M) or high (0.3 M) salt concentrations, respectively. In addition, interaction with the cognate sequence prevents aggregation of p53C into an amyloid-like structure, whereas binding to a nonconsensus DNA sequence has no effect on p53C stability, even at low ionic strength. Strikingly, sequence-specific DNA binding also resulted in a large stabilization of full-length p53, whereas nonspecific sequence binding led to no stabilization. The effects of cognate DNA could be mimicked by high concentrations of osmolytes such as glycerol, which implies that the stabilization is caused by the exclusion of water. Taken together, our results show an enhancement in protein stability driven by specific DNA recognition. When cognate DNA was added to misfolded protein obtained after a pressurization cycle, the original conformation was mostly recovered. Our results may aid the development of therapeutic approaches to prevent misfolded species of p53.
Asunto(s)
ADN/farmacología , Pliegue de Proteína/efectos de los fármacos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Naftalenosulfonatos de Anilina/química , Secuencia de Consenso , ADN/metabolismo , Colorantes Fluorescentes/química , Glicerol/química , Humanos , Presión Hidrostática , Luz , Concentración Osmolar , Polidesoxirribonucleótidos/química , Polidesoxirribonucleótidos/metabolismo , Unión Proteica/genética , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Dispersión de Radiación , Espectrometría de Fluorescencia , Proteína p53 Supresora de Tumor/genética , Agua/químicaRESUMEN
The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.
Asunto(s)
Guanidina/química , Proteína p53 Supresora de Tumor/química , Dimerización , Cinética , Complejos Multiproteicos/química , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Foot-and-mouth disease virus (FMDV) is the causative agent of the foot-and-mouth disease (FMD). Alternative FMD vaccines have been pursued due to important disadvantages of the one currently in use. High hydrostatic pressure (HP) has been observed to inactivate some viruses. Here, we investigated the effects of HP on FMDV O1 Campos-Vallée (CVa) infectivity. A treatment consisting of 2.5 kbar at -15 degrees C and 1M urea, completely abolished FMDV infectivity, maintaining the integrity of its capsid structure. Moreover, its ability to elicit neutralizing antibody production in rabbits was preserved. Taken together, our results suggest that HP could be a safe, simple, cheap and reproducible way for viral vaccine production.
Asunto(s)
Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Presión Hidrostática , Vacunación/métodos , Vacunas Virales/inmunología , Inactivación de Virus , Animales , Anticuerpos Antivirales/sangre , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Virus de la Fiebre Aftosa/ultraestructura , Pruebas de Neutralización , Conejos , Temperatura , Urea , Replicación ViralRESUMEN
The wild-type p53 protein can be driven into a conformation corresponding to that adopted by structural mutant forms by heterodimerization with a mutant subunit. To seek partially folded states of the wild-type p53 core domain (p53C) we used high hydrostatic pressure (HP) and subzero temperatures. Aggregation of the protein was observed in parallel with its pressure denaturation at 25 and 37 degrees C. However, when HP experiments were performed at 4 degrees C, the extent of denaturation and aggregation was significantly less pronounced. On the other hand, subzero temperatures under pressure led to cold denaturation and yielded a non-aggregated, alternative conformation of p53C. Nuclear magnetic resonance (1H15N-NMR) data showed that the alternative p53C conformation resembled that of the hot-spot oncogenic mutant R248Q. This alternative state was as susceptible to denaturation and aggregation as the mutant R248Q when subjected to HP at 25 degrees C. Together these data demonstrate that wild-type p53C adopts an alternative conformation with a mutant-like stability, consistent with the dominant-negative effect caused by many mutants. This alternative conformation is likely related to inactive forms that appear in vivo, usually driven by interaction with mutant proteins. Therefore, it can be a valuable target in the search for ways to interfere with protein misfolding and hence to prevent tumor development.
Asunto(s)
Imitación Molecular , Mutación Puntual/genética , Conformación Proteica , Proteína p53 Supresora de Tumor/química , Sitios de Unión , Cromatografía en Gel , Cartilla de ADN/química , Fluorescencia , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plásmidos , Reacción en Cadena de la Polimerasa , Presión , Estructura Terciaria de Proteína , Temperatura , Termodinámica , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.