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(申请人提供)：侵袭促进，致癌膜型基质金属蛋白酶(MT1-MMP1)，一种具有细胞外催化、血凝蛋白(PEX)和铰链结构域的跨膜蛋白酶，以及一条短的细胞质尾巴，与其生理蛋白抑制物金属蛋白酶组织抑制物-2(TIMP-2)形成化学计量复合体。众所周知，MT1-MMPs的蛋白水解性是肿瘤细胞侵袭和增殖所必需的，而TIMP-2结合在MT1-MMPs的催化结构域上并阻断其蛋白水解性。我们最近发表的大量研究表明，除了细胞外的蛋白分解，MT1-MMPs和TIMP-2还通过非蛋白分解机制控制细胞的增殖和迁移。TIMP-2通过TIMP-2的催化结构域和抑制结构域外信号调节的蛋白激酶1/2(ERK1/2)的激活，参与TIMP-2与血粘蛋白和/或铰链域的结合，并通过MT1-MMP的胞浆尾部介导。MT1-MMP介导的ERK1/2的激活在体外上调细胞的迁移和增殖，而不依赖于细胞外基质蛋白的降解。与这一发现一致的是，非蛋白水解性MT1-MMP体内促进肿瘤生长的效果与野生型MT1-MMP相媲美。虽然这些观察结果并没有削弱涉及TIMP-2和MT1-MMP的蛋白水解性相互作用的重要性，但我们新的、意想不到的发现有力地支持了我们确定的蛋白质分解非依赖性信号机制的非常重要的作用。由于ERK1/2信号通路控制着肿瘤细胞的增殖和迁移等多种功能，深入了解TIMP-2-MT1-MMP相互作用激活这一信号通路的分子机制可以为设计新型的抑制肿瘤进展的药物提供基础信息。因此，我们建议通过以下具体目标来详细探讨这一新的信号机制：1)确定介导TIMP-2结合和ERK1/2激活的TIMP-2 PEX和/或铰链结构域的区域(S)；2)确定TIMP-2与MT1-MMPPEX和/或铰链结构域结合所需的区域(S)；3)研究MT1-MMPPEX到Ras-ERK1/2通路的信号转导机制；4)确定TIMP-2-MMP1-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理解MT1-MMPs和TIMP-2在肿瘤生长中的非蛋白水解性作用，并为开发旨在阻断MT1-MMPTIMP-2相互作用和促进肿瘤进展的细胞内信号产生的新型抑制剂提供依据。公共卫生相关性：我们在体内实验模型中发现了一种意想不到的、范式转换的机制，它控制着肿瘤细胞的增殖和迁移，以及肿瘤的生长。因此，我们建议详细研究这种新的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将对设计阻止肿瘤进展的新型药理药物具有重要意义。