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: 8141806
• 负责人: Paolo Mignatti
• 金额: $ 3.17万
• 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/8141806
• 关键词: NON; PROTEOLYTIC; INTERACTIONS; TIMP; MT1

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.

描述（由申请人提供）：促侵袭、致瘤膜 1 型基质金属蛋白酶 (MT1-MMP) 是一种跨膜蛋白酶，具有细胞外催化、血红素结合蛋白 (PEX) 和铰链结构域以及短胞质尾部，与其生理蛋白抑制剂、组织抑制剂形成化学计量复合物。 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侵袭和增殖所必需的，并且 TIMP-2 与 MT1-MMP 的催化结构域结合并阻断其蛋白水解活性。我们最近发表的广泛研究表明，除了细胞外蛋白水解之外，MT1-MMP 和 TIMP-2 通过非蛋白水解机制控制细胞增殖和迁移。 TIMP-2 与 MT1-MMP 结合，通过独立于 MT1-MMP 催化结构域和 TIMP-2 抑制结构域的机制，诱导细胞外信号调节激酶 1/2 (ERK1/2) 的激活。这种效应涉及 TIMP-2 与血红素结合蛋白和/或铰链结构域的结合，并由 MT1-MMP 的细胞质尾介导。 MT1-MMP 介导的 ERK1/2 激活在体外上调细胞迁移和增殖，独立于细胞外基质蛋白水解。与这一发现一致，蛋白水解失活的 MT1-MMP 促进体内肿瘤生长，其效果与野生型 MT1-MMP 相当。虽然这些观察结果并没有削弱涉及 TIMP-2 和 MT1-MMP 的蛋白水解相互作用的既定重要性，但我们新颖的、意想不到的发现强烈主张我们确定的独立于蛋白水解的信号机制具有非常重要的作用。由于 ERK1/2 信号通路控制多种肿瘤细胞功能，包括增殖和迁移，因此详细了解 TIMP-2 - MT1-MMP 相互作用激活该信号通路的分子机制可以为设计新型抑制剂来阻止肿瘤进展提供基础信息。因此，我们建议通过制定以下具体目标来详细探索这种新颖的信号传导机制：1）识别介导TIMP-2结合和ERK1/2激活的MT1-MMP PEX和/或铰链结构域的区域，2）识别与MT1-MMP的PEX和/或铰链结构域结合所需的TIMP-2区域； 3) 表征MT1-MMP至Ras-ERK1/2通路的信号转导机制； 4)确定TIMP-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的肿瘤异种移植模型。我们的研究结果将有助于了解 MT1-MMP 和 TIMP-2 在肿瘤生长中的非蛋白水解作用，并有助于开发新型抑制剂，旨在阻断 MT1-MMP - TIMP-2 相互作用以及促进肿瘤进展的细胞内信号传导的产生。公共健康相关性：我们发现了一种意想不到的范式转变机制，可以控制肿瘤细胞增殖和迁移，以及体内实验模型中的肿瘤生长。因此，我们建议详细研究这种新机制及其在肿瘤生长中的功能作用。从我们的研究中获得的知识对于设计阻止肿瘤进展的新型药物具有重要意义。