REGULATION OF CELL MIGRATION IN VASCULAR REMODELING

血管重塑中细胞迁移的调节

• 批准号: 6390276
• 负责人: ERIC W HOWARD
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390276
• 关键词: active sites; binding sites; cell migration; chimeric proteins; collagenase; crosslink; enzyme activity; fibroblasts; integrins; muscle cells; protein binding; protein sequence; site directed mutagenesis; tissue /cell culture; tissue inhibitor of metalloproteinases; vascular smooth muscle

The pathogenic remodeling of blood vessels often involves smooth muscle proliferation and migration. As in other instances of cell movement, smooth muscle cell migration into the neointima involves a regulated series of adhesion and de-adhesion events regulated by cell-surface receptors and surface-associated proteases. The matrix metalloproteinase (MMP), gelatinase A, plays a significant role in this process by enabling cells to overcome matrix barriers, and also regulates cell-matrix adhesion during migration. It does this through its interaction with its specific activator, MT1-MMP, a membrane-associated proteinase that initiates cleavage of the gelatinase A pro-peptide in a complex process involving multiple interactions with cell-surface proteins. One such interaction involves MT1-MMP itself, which bindings gelatinase A through a bridge with the MMP inhibitor, TIMP-2, a critical mediator of gelatinase A activation. TIMP-2 binds the active site of MT1-MMP and the carboxyl domain of gelatinase A, forming a triplex which positions gelatinase A appropriate for subsequent activation by another MT1-MMP molecule. The carboxyl domain of gelatinase A also binds the integrin, alphavbeta3, which also plays a role in the activation process. Finally, cell bind TIMP-2 to other, as yet unknown sites which may also be key elements of gelatinase A activation. Interestingly, fibroblasts and smooth muscle cells rapidly respond to changes in cell shape by activating and binding gelatinase A in a process independent of new protein synthesis, suggesting that the components of the activation mechanism are present on cells but preventing from interacting appropriately. In this study, the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for TIMP-2's specificity for MT1-MMP will be deduced using chimeric TIMP molecules in which TIMP-2 sequence will be exchanged for sites within TIMP-2, a highly homologous inhibitor with virtually no specificity for MT1-MMP. Net, the non-MMP cell-surface binding sites for TIMP-2 will be characterized, and the critical amino acid sequences within TIMP-2 that confer this binding will be determined. Finally, the contribution of TIMP-2 to gelatinase A binding and activation will be modeled. Understanding this mechanism will be important in defining the biochemical events that occur during vascular remodeling in both normal and disease processes.

血管的致病重塑通常涉及平滑肌增殖和迁移。与其他细胞运动的情况一样，平滑肌细胞迁移到新内膜中涉及受细胞表面受体和表面相关蛋白酶调节的一系列粘附和去粘附事件。基质金属蛋白酶（MMP），明胶酶A通过使细胞能够克服基质屏障，并调节迁移过程中细胞 - 基质粘附，从而在此过程中起重要作用。它通过与特定活化剂MT1-MMP（一种与膜相关的蛋白酶）相互作用来做到这一点，该蛋白酶在复杂的过程中启动明胶酶A促肽的裂解，涉及与细胞表面蛋白的多种相互作用。一种这样的相互作用涉及MT1-MMP本身，它通过桥梁与MMP抑制剂TIMP-2（明胶酶A激活的关键介体）结合明胶酶A。 TIMP-2结合MT1-MMP的活性位点和明胶酶A的羧基结构域，形成了三折射，该三体将明胶酶A定位，适合于另一种MT1-MMP分子激活。明胶酶A的羧基结构域还结合了整合素Alphavbeta3，这也在激活过程中起作用。最后，细胞将TIMP-2与其他结合，而未知位点也可能是明胶酶A激活的关键元素。有趣的是，成纤维细胞和平滑肌细胞通过在与新蛋白质合成的过程中激活和结合明胶酶A的激活和结合明胶酶A迅速响应细胞形状的变化，这表明激活机制的成分存在于细胞上，但可以防止适当相互作用。在这项研究中，将探索TIMP-2在明胶酶A的细胞结合和激活中的作用。首先，将探索TIMP-2在明胶A的细胞结合和激活中的作用的生化基础。首先，将使用嵌合TIMP分子来推导TIMP-2对MT1-MMP的特异性的生化基础，其中TIMP-2序列将用于TIMP-2中的位点，TIMP-2（一种高度同源抑制剂，实际上没有MT1-MMP的特异性）。将表征NET，TIMP-2的非MMP细胞表面结合位点，并且将确定TIMP-2内的临界氨基酸序列，以确定这种结合。最后，将建模TIMP-2对明胶酶A结合和激活的贡献。了解这种机制对于定义正常和疾病过程中血管重塑期间发生的生化事件至关重要。