RESUMEN
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.
Asunto(s)
Proteínas/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Humanos , Inmunoprecipitación , Unión ProteicaRESUMEN
We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 microg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 microg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate.
Asunto(s)
Biomarcadores de Tumor/análisis , Reactores Biológicos , Proteínas/análisis , Proteómica/métodos , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatografía en Gel/métodos , Humanos , Proteínas/química , Proteómica/instrumentaciónRESUMEN
Progesterone-induced oocyte maturation is thought to involve the inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. Our previous studies demonstrated that injection of inhibitors of G protein betagamma complex induces hormone-independent oocyte maturation. In contrast, over-expression of Xenopus Gbeta1 (xGbeta1), alone or together with bovine Ggamma2, elevates oocyte cAMP and inhibits progesterone-induced oocyte maturation. To further investigate the mechanism of Gbetagamma-induced oocyte maturation, we generated a mutant xGbeta1, substituting Asp-228 for Gly (D228G). An equivalent mutation in the mammalian Gbeta1 results in the loss of its ability to activate adenylyl cyclases. Indeed, co-injection of xGbeta1D228G with Ggamma2 failed to increase oocyte cAMP or inhibit progesterone-induced oocyte maturation. To directly demonstrate that oocytes contained a Gbetagamma-regulated adenylyl cyclase, we analyzed cAMP formation in vitro by using oocyte membrane preparations. Purified brain Gbetagamma complexes significantly activated membrane-bound adenylyl cyclase activities. Multiple adenylyl cyclase isoforms were identified in frog oocytes by PCR using degenerate primers corresponding to highly conserved catalytic amino acid sequences. Among these we identified a partial Xenopus adenylyl cyclase 7 (xAC7) that was 65% identical in amino acid sequence to human AC7. A dominant-negative mutant of xAC7 induced hormone-independent oocyte maturation and accelerated progesterone-induced oocyte maturation. Theses findings suggest that xAC7 is a major component of the G2 arrest mechanism in Xenopus oocytes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Fase G2/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Oocitos/crecimiento & desarrollo , Adenilil Ciclasas/genética , Adenilil Ciclasas/aislamiento & purificación , Adenilil Ciclasas/metabolismo , Sustitución de Aminoácidos/genética , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Secuencia Conservada/genética , AMP Cíclico/biosíntesis , ADN Complementario/análisis , ADN Complementario/genética , Femenino , Fase G2/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Datos de Secuencia Molecular , Mutación/genética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Progesterona/metabolismo , Progesterona/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Fracciones Subcelulares , Regulación hacia Arriba/genética , Xenopus laevisRESUMEN
Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.