Regulation of Xenopus oocyte meiosis arrest by G protein βγ subunits

AbstractBackground: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the α subunits of classical Gi-type G proteins.Results: To investigate the possibility that G protein βγ subunits, rather than α subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein βγ scavengers (Gtα and βARK-CCAAX) to sequester free Gβγ dimers. We demonstrated that the injection of mRNA encoding either of these Gβγ scavengers induced oocyte maturation. The Gβγ scavengers bound an endogenous, membrane-associated Gβ subunit, indistinguishable from Xenopus Gβ1 derived from mRNA injection. The injection of Xenopus Gβ1 mRNA, together with bovine Gγ2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation.Conclusion: An endogenous G protein βγ dimer, likely including Xenopus Gβ1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gβγ scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gβγ

Large-scale mapping of human protein–protein interactions by mass spectrometry

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

Multiplexed proteomic reactor for the processing of proteomic samples

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 μg 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 μg 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