RESUMEN
The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.
Asunto(s)
Metaloendopeptidasas/metabolismo , Invasividad Neoplásica , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Factor de Crecimiento Epidérmico/metabolismo , Histidina , Espectrometría de Masas/métodos , Ratones , Mutación Missense , Unión Proteica , ProteómicaRESUMEN
Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3' UTR. These results suggest that the DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3' UTR is required for DENV replication.
Asunto(s)
Regiones no Traducidas 3'/genética , ARN Helicasas DEAD-box/metabolismo , Virus del Dengue/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Viral/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Células HeLa , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Espectrometría de Masas/métodos , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ensamble de Virus , Replicación ViralRESUMEN
The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.
Asunto(s)
Mutación Missense , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Fosfohidrolasa PTEN/metabolismo , Aminoácidos/farmacología , Línea Celular Tumoral , Humanos , Marcaje Isotópico/métodos , Proteínas de Neoplasias/genética , Neoplasias/genética , Fosfohidrolasa PTEN/genéticaRESUMEN
Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.