Screening And Identification Of Target Proteins Of Exogenous IbeB In Embryonic Stem Cells

IntroductionResearchers have found some bacterial protein effectors when pathogenic bacteria interact with host cells. These protein effectors are secreted by bacteria, some of which can be transmitted to the host cells'cytoplasm or nucleus. These proteins not only involve in the process of bacterial invasion to host cells, but also affect some important biological functions of the host cells. For example, outer membrane protein InlB of Listeria monocytogenes affects liver regeneration by hepatocyte growth factor receptor (HGF-R or met); hemolysin of Listeria monocytogenes allows dephosphorylation of histone H3S10 of epithelial cells; the protein effector OspF of Shigella flexneri can be directly injected into the nucleus of epithelial cells through type III secretion, enables chromatin remodeling of NF-κB promoter site by blocking the phosphorylation of histone H3. These unexpected results provide a new clue for the understanding of the biological characteristics of eukaryotic cells,so in a sense some bacterial protein effectors can be used as powerful tools for the exploration of biological functions of eukaryotic cells. ibeB of K1 strains of E.coli is a gene contributing to the invasion to human brain microvascular endothelial cells. Splicing of its mature protein is similar to Gram-negative bacteria typeⅣsecretion protein. Our lab have transfected ibeB gene to eukaryotic cells to explore its effects on biological activities of these eukaryotic cells.The results have showed that:(1) HeLa cells transfected ibeB gene appeared glial fibrillary acidic protein (GFAP) positive neural-like changes; (2) mouse embryonic stem cells AB2.1 transfected stablely ibeB gene differentiated to neural precursor-like cells.Our aim is to screen target proteins of exogenous IbeB in embryonic stem cells and to explore the mechanism how ibeB control embryonic stem cells differentiation into neural precursor cells. Our study may accumulate information for the research of the regulatory mechanism of neural differentiation.Methods1. Screen IbeB target proteins by Yeast two-hybrid.2. Confirm interaction of IbeB and Cathepsin A in yeast.3. Express and purify the GST-IbeB fusion protein in E.coli BL21.4. Confirm interaction of IbeB and Cathepsin A in vitro by GST-pull down.5. Detect the expression of Cathepsin A in brain during mouse development by Western blot and Immunohistochemistry.Results1. The result of Yeast two-hybrid showed that Cathepsin A may be the target protein of exogenous IbeB.2. IbeB can interact with Cathepsin A in yeast.3. We have confirmed the interaction of IbeB and Cathepsin A in vitro by GST-pull down.4. The level of Cathepsin A differed during different phases of brain development in mice, of which showed the highest level on the 14.5th day of embryonic period. A small amount of Cathepsin A was detectable in the nucleus.ConclusionCathepsin A was the new interacting protein of exogenous IbeB.