NON-PROTEOLYTIC INTERACTIONS OF TIMP-2 AND MT1-MMP

TIMP-2 和 MT1-MMP 的非蛋白水解相互作用

• 批准号: 8034818
• 负责人: Paolo Mignatti
• 金额: $ 34.12万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8034818
• 关键词: Address; Advocate; Binding; C-terminal; CD44 gene; Catalytic Domain; Cell Proliferation; Cell physiology; Cells; Chimera organism; Complex; Cytoplasmic Tail; Development; Extracellular Matrix; Favorable Clinical Outcome; Gelatinase A; Generations; Growth Factor Receptors; Health; Hemopexin; Immunodeficient Mouse; Immunologic Techniques; In Vitro; Integral Membrane Protein; Integrins; Knowledge; Lead; Matrix Metalloproteinases; Mediating; Mitogen-Activated Protein Kinases; Molecular; Mutation; N-terminal; Pathway interactions; Peptide Hydrolases; Phosphorylation; Physiological; Play; Process; Proteins; Proteolysis; Publishing; Role; Signal Pathway; Signal Transduction; Testing; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tumor Cell Invasion; Tumorigenicity; Up-Regulation; Xenograft Model; cell motility; design; extracellular; human MMP14 protein; in vivo; in vivo Model; inhibitor/antagonist; knockout gene; malignant breast neoplasm; migration; mutant; neoplastic cell; novel; outcome forecast; proMMP-2; tumor; tumor growth; tumor progression; tumor xenograft; tumorigenic

DESCRIPTION (provided by applicant): The invasion-promoting, tumorigenic membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin (PEX) and hinge domains, and a short cytoplasmic tail, forms a stoichiometric complex with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). It is well established that the proteolytic activity of MT1-MMP is required for tumor cell invasion and proliferation, and that TIMP-2 binds to the catalytic domain of MT1-MMP and blocks its proteolytic activity. Our extensive, recently published studies have shown that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of Extracellular signal-Regulated Kinase 1/2 (ERK1/2) by a mechanism independent of the MT1-MMP catalytic domain and the inhibitory domain of TIMP- 2. This effect involves TIMP-2 binding to the hemopexin and/or hinge domain and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 upregulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Consistent with this finding, proteolytically inactive MT1-MMP promotes tumor growth in vivo with an effect comparable to that of wild- type MT1-MMP. While these observations do not diminish the well-established importance of the proteolytic interactions involving TIMP-2 and MT1-MMP, our novel, unexpected findings strongly advocate a very important role for the proteolysis-independent signaling mechanism we identified. Because the ERK1/2 signaling pathway controls a variety of tumor cell functions including proliferation and migration, a detailed understanding of the molecular mechanism by which TIMP-2 - MT1-MMP interaction activates this signaling pathway can provide fundamental information for designing novel inhibitors to block tumor progression. Therefore, we propose to explore this novel signaling mechanism in detail by developing the following Specific Aims: 1) To identify the region(s) of the MT1-MMP PEX and/or hinge domains that mediate TIMP-2 binding and ERK1/2 activation, 2) To identify the region(s) of TIMP-2 required for binding to the PEX and/or hinge domain of MT1-MMP; 3) To characterize the mechanism of signal transduction from MT1-MMP to the Ras-ERK1/2 pathway; 4) To determine the functional role of TIMP-2 - MT1-MMP interaction in tumor growth in vivo. For this purpose we will use state-of-the-art molecular, cellular and immunological techniques and a tumor xenograft model in immunodeficient mice. The results of our study will lead to the understanding of the non-proteolytic role of MT1-MMP and TIMP-2 in tumor growth, and afford the development of novel inhibitors aimed to block MT1-MMP - TIMP-2 interaction and the generation of intracellular signaling that promotes tumor progression. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls tumor cell proliferation and migration, as well as tumor growth in an experimental in vivo model. Therefore, we propose to study in detail this novel mechanism and its functional role in tumor growth. The knowledge derived from our study will be of fundamental importance for designing novel pharmacological agents to block tumor progression.

DESCRIPTION (provided by applicant): The invasion-promoting, tumorigenic membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin (PEX) and hinge domains, and a short cytoplasmic tail, forms a stoichiometric complex with its physiological protein inhibitor, tissue inhibitor of金属蛋白酶-2（TIMP-2）。众所周知，MT1-MMP的蛋白水解活性是肿瘤细胞浸润和增殖所必需的，并且TIMP-2与MT1-MMP的催化结构域结合并阻止其蛋白水解活性。我们广泛发表的研究表明，除了细胞外蛋白水解外，MT1-MMP和TIMP-2控制细胞的增殖以及通过非蛋白水解机制的迁移。 TIMP-2与MT1-MMP的结合通过与MT1-MMP催化域和TIMP-2的抑制结构域无关的机制诱导细胞外信号调节的激酶1/2（ERK1/2）的激活。这种作用涉及TIMP-2与hemopexin和/heringe-dimain的结合，并通过呼吸症和呼吸剂迁移。 MT1-MMP介导的ERK1/2的激活上调了细胞迁移和体外的增殖，独立于细胞外基质蛋白水解。与这一发现一致，蛋白水解的MT1-MMP在体内促进肿瘤的生长，其作用与野生型MT1-MMP相当。尽管这些观察结果并没有减少涉及TIMP-2和MT1-MMP的蛋白水解相互作用的良好重要性，但我们的新颖的，意外的发现强烈建议我们确定的无蛋白水解信号传导机制非常重要。由于ERK1/2信号传导途径控制着多种肿瘤细胞功能，包括增殖和迁移，因此对TIMP-2-2-MT1-MMP相互作用激活该信号传导途径的分子机制的详细理解可以为设计新型抑制剂提供基本信息，以阻止肿瘤进展。因此，我们建议通过开发以下特定目的来详细探讨这种新颖的信号传导机制：1）确定MT1-MMP PEX和/或铰链域的区域，这些区域介导TIMP-2结合和ERK1/2激活，2），以识别与PEX和//或Hering domain domain domain domain domain domain domain domain dimp-2所需的timp-2区域所需的区域。 3）表征从MT1-MMP到RAS-ERK1/2途径的信号转导的机理； 4）确定TIMP-2-2-MT1-MMP相互作用在体内肿瘤生长中的功能作用。为此，我们将使用最先进的分子，细胞和免疫学技术以及免疫缺陷小鼠中的肿瘤异种移植模型。我们的研究结果将导致对MT1-MMP和TIMP-2在肿瘤生长中的非蛋白水解作用的理解，并提供旨在阻断MT1-MMP-TIMP-2相互作用的新型抑制剂的发展，以及促进肿瘤进展的细胞内信号传导的产生。公共卫生相关性：我们发现了一种意外的，范式转移的机制，可以控制肿瘤细胞增殖和迁移，以及实验性体内模型中的肿瘤生长。因此，我们建议详细研究这种新型机制及其在肿瘤生长中的功能。从我们的研究中得出的知识对于设计新型药理剂以阻断肿瘤进展至关重要。