Analysis of the proteolytic events on cell surface through proteomic approach

通过蛋白质组学方法分析细胞表面的蛋白水解事件

• 批准号: 15209011
• 负责人: SEIKI Motoharu
• 金额: $ 31.95万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15209011/
• 关键词: Membrane-type MMP; Integrins; Menbrane proteins; Proteomic analysis; MT1-MMP; 浸潤装置; がん細胞; プロテアーゼ; 細胞接着分子; Matrix metalloproteinase; Proteolysis; Membrane proteins; proteomics; nano-LC-MS; MS

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.

我们建立了转染的人形癌A431细胞系M，该细胞系M可诱导标记为标记的MT1-MMP或FIAG标记的整联蛋白β-1的表达。我们设置了条件，以隔离使用不同类型的洗涤剂的各种关联分子，隔离标记标记的MT1-MMP。我们对复合物中的蛋白质进行了全身分析。五种分泌的蛋白质，64种膜蛋白，87种细胞质蛋白和7种未知蛋白被鉴定出来，我们检查了18种膜蛋白是否可以通过在COS-1细胞中表达MT1-MMP的底物。发现其中有9个被MT1-MMP生成已加工的片段的表达裂解。据报道，一种加工的蛋白质过表达了许多肿瘤，并被金属蛋白酶脱落。我们证实，这种蛋白也被表达MT1-MMP的人肿瘤细胞系中的内源性MT1-MMP裂解。因此，我们认为这种方法对于通过与MT1-MMP关联来识别MT1-MMP底物和调节剂很有用，我们试图通过使用siRNA的敲低实验来了解底物的功能和关联蛋白质的功能。使用交联的蛋白质与MT1-MMP的关联进行了进一步测试。我们也在继续分析整联蛋白宣传蛋白。

项目成果

Palmitoylation at Cys574 is essential for MT1-MMP to promote cell migration

• DOI: 10.1096/fj.04-3651fje
• 发表时间: 2005-06-01
• 期刊: FASEB JOURNAL
• 影响因子: 4.8
• 作者: Anilkumar, N;Uekita, T;Itoh, Y
• 通讯作者: Itoh, Y

CD44 binding through the hemopexin-like domain is critical for its shedding by membrane-type 1 matrix metalloroteinase

CD44 通过血红素结合蛋白样结构域的结合对于膜 1 型基质金属蛋白酶的脱落至关重要

• 发表时间: 2005
• 期刊: Oncogene 24
• 作者: Suenaga;N.他3人
• 通讯作者: N.他3人

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