REGULATION OF CELL MIGRATION IN VASCULAR REMODELING

血管重塑中细胞迁移的调节

• 批准号: 2826076
• 负责人: ERIC W HOWARD
• 金额: $ 22.78万
• 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/2826076
• 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.

血管的致病性重塑通常涉及平滑肌增殖和迁移。与细胞运动的其他情况一样，平滑肌细胞迁移到新内膜涉及一系列受细胞表面受体和表面相关蛋白酶调节的粘附和脱粘附事件。基质金属蛋白酶 (MMP)、明胶酶 A 在此过程中发挥着重要作用，使细胞能够克服基质屏障，并在迁移过程中调节细胞与基质的粘附。它通过与其特定激活剂 MT1-MMP 相互作用来实现这一点，MT1-MMP 是一种膜相关蛋白酶，在涉及与细胞表面蛋白多重相互作用的复杂过程中启动明胶酶 A 前肽的裂解。其中一种相互作用涉及 MT1-MMP 本身，它通过与 MMP 抑制剂 TIMP-2（明胶酶 A 激活的关键介质）的桥结合明胶酶 A。 TIMP-2 结合 MT1-MMP 的活性位点和明胶酶 A 的羧基结构域，形成三链体，将明胶酶 A 定位为适合随后被另一个 MT1-MMP 分子激活的位置。明胶酶 A 的羧基结构域还结合整合素 alphavbeta3，它也在激活过程中发挥作用。最后，细胞将 TIMP-2 与其他未知位点结合，这些位点也可能是明胶酶 A 激活的关键要素。有趣的是，成纤维细胞和平滑肌细胞通过在独立于新蛋白质合成的过程中激活和结合明胶酶 A 来快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，但阻止了适当的相互作用。在本研究中，将探讨 TIMP-2 在明胶酶 A 的细胞结合和激活中的作用。首先，将探讨 TIMP-2 在明胶酶 A 的细胞结合和激活中的作用的生化基础。首先，TIMP-2 对 MT1-MMP 特异性的生化基础将使用嵌合 TIMP 分子来推断，其中 TIMP-2 序列将替换 TIMP-2 内的位点，TIMP-2 是一种高度同源的抑制剂，对 MT1-MMP 实际上没有特异性。 Net，将表征 TIMP-2 的非 MMP 细胞表面结合位点，并将确定 TIMP-2 内赋予这种结合的关键氨基酸序列。最后，将对 TIMP-2 对明胶酶 A 结合和激活的贡献进行建模。了解这一机制对于定义正常和疾病过程中血管重塑过程中发生的生化事件非常重要。