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鳞癌 A431 细胞系，其中可诱导 FLAG 标记的 MT1-MMP 或 FIAG 标记的整联蛋白 beta-1 的表达。我们设置了条件来使用不同类型的去污剂分离带有 FLAG 标记的 MT1-MMP，包括各种缔合分子。我们对复合物中的蛋白质进行了系统分析。鉴定出5种分泌蛋白、64种膜蛋白、87种细胞质蛋白和7种未知蛋白。然后，我们通过在COS-1细胞中表达18种膜蛋白来检查它们是否可以作为MT1-MMP的底物。发现其中九个被 MT1-MMP 的表达切割，产生加工片段。据报道，其中一种经过加工的蛋白质在许多肿瘤中过度表达，并被金属蛋白酶排出。我们证实，在表达 MT1-MMP 的人类肿瘤细胞系中，该蛋白也被内源性 MT1-MMP 裂解。因此，我们相信这种方法可用于通过与 MT1-MMP 关联来识别 MT1-MMP 底物和调节剂。我们正试图通过使用 siRNA 的敲除实验来了解底物和关联蛋白的功能。使用交联剂进一步测试已鉴定的蛋白质与 MT1-MMP 的关联。我们也在继续分析整合素相关蛋白。

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

Impaired alveolization in mice deficient in membrane-type matrix metalloproteinase 1 (MT1-MMP)

• DOI: 10.1007/s00795-004-0277-9
• 发表时间: 2005-03-05
• 期刊: Medical Molecular Morphology
• 影响因子: 1.8
• 作者: Irie, Kazuharu;Komori, Kiyoshi;Yajima, Toshihiko
• 通讯作者: Yajima, Toshihiko

Matsuki H., et al.: "Monoclonal antibodies with defined recognition sequences in the stem region of CD44 : detection of differential glycosylation of CD44 between tumor and stromal cells in tissue."Cancer Res.. 63. 8278-8283 (2003)

Matsuki H. 等人：“CD44 干区中具有确定识别序列的单克隆抗体：检测组织中肿瘤和基质细胞之间 CD44 的差异糖基化。”Cancer Res.. 63. 8278-8283 (2003)

Matrix metalloproteinase expression and function during fin regeneration in zebrafish: Analysis of MT1-MMP, MMP2 and TIMP2

• DOI: 10.1016/j.matbio.2005.03.007
• 发表时间: 2005-06-01
• 期刊: MATRIX BIOLOGY
• 影响因子: 6.9
• 作者: Bai, S;Thummel, R;Sarras, MP
• 通讯作者: Sarras, MP