An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.
Quantitative description of protein interactions is crucial to understand and model molecular systems regulating various cellular activities. Here, we developed a novel peptide-concatenated standard (PCS) strategy for accurate mass spectrometric quantification of component stoichiometry of multiprotein complexes. In this strategy, tryptic peptides suitable for quantification are selected with their natural flanking sequences from each component of multiprotein complex and concatenated into a single synthetic protein called PCS. The concatenation guarantees equimolarity among the peptides added to the sample to obviate the need for preparation of accurately known amounts of individual peptides. The flanking sequences would equalize the excision efficiency of each peptide between the PCS and the target protein to improve the accuracy of quantification. To validate this strategy, we quantified the budding yeast eIF2Bgamma, the gamma subunit of eukaryotic initiation factor 2B, using a PCS composed of tryptic peptides from eIF2Bgamma with their flanking sequences. An identical sample-to-standard signal ratio was obtained within 5% measured error for these peptides, including the one prone to incomplete digestion, thereby proving the principle of PCS strategy. We applied the strategy to reveal the stoichiometry of the eIF2B-eIF2 complex using a PCS covering the 5 eIF2B and 3 eIF2 components. While the complex contained equimolar amounts of the eIF2B subunits, the ratio of each eIF2 subunit to eIF2B was 30-40%. The PCS strategy would provide a versatile method to quantitatively analyze compositional alteration of multiprotein complexes or dynamics of protein-protein interactions in response to various stimuli.
The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.
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