Analysis of the proteolytic events on cell surface through proteomic approach

点击翻译按钮获取中文摘要

We established a transfected human squemous carcinoma A431 cell line m which expression of FLAG-tagged MT1-MMP or FIAG-tagged integrins beta-1 is inducible. We set up conditions to isolate the FLAG-tagged MT1-MMP including various associating molecules using different types of detergents. We performed systemic analysis of the proteins in the complex. Five secreted proteins, 64 membrane proteins, 87 cytoplasmic proteins, and 7 unknown proteins were identified Then, we examined 18 membrane proteins whether they can be substrates of MT1-MMP by expressing them in COS-1 cells. Nine of them were found to be cleaved by the expression of MT1-MMP generating processed fragments. One of the processed proteins has been reported to be overexpressed many tumors and be shed by metalloproteinases. We confirmed that this protein also cleaved by endogenous MT1-MMP in human tumor cell lines that express MT1-MMP. Thus, we believe this approach is useful to identify MT1-MMP substrates and regulators by associating with MT1-MMP We are trying to know the functions of the substrates and associating proteins by the knock-down experiments using siRNA. The association of the identified proteins with MT1-MMP is further tested using cross-linkers. We are continuing analysis of integrin-assoaating proteins as well.