MMP-2 and Fibronectin Fragmentation in Periodontal Diseases and Oral Cancer

描述（由申请人提供）：基质金属蛋白酶（MMP）家族中的酶通过其降解主要组织分子和激活生长因子的集体能力对定期组织维护很重要。MMP由此有益地促进正常发育和组织适应。然而，某些MMP，包括MMP-2，已被发现在升高的水平，并可能异常降解组织在几种疾病，如牙周病，关节炎和癌症。近年来，我们和其他研究者的研究表明，在这些疾病中，主要的细胞粘附分子纤连蛋白被降解，MMP-2可以有效地切割纤连蛋白。这种降解需要纤连蛋白和MMP-2的独特亚结构域之间的特异性结合。由于细胞的行为在暴露于纤连蛋白切割片段时发生变化，因此此类片段可改变疾病进展。因此，了解这两种蛋白质之间结合的确切机制可能为开发能够阻断MMP-2降解纤连蛋白的抑制剂提供基础。在涉及来自不同学科和机构的研究人员的合作努力中，我们将应用分子生物学，生物化学，质谱和蛋白质工程方法来定义纤连蛋白和MMP-2的精确结合位点。基于噬菌体展示随机肽库筛选的结果，我们将设计模拟结合位点的合成肽，并测试其阻断MMP-2结合和纤连蛋白降解的能力。在结合结构域中引入位点特异性突变将确认结合位点。在精确绘制纤连蛋白切割位点和片段后，我们将通过多个参数、MMP表达和细胞凋亡来表征与一系列重组纤连蛋白切割片段接触后牙周和癌细胞行为的改变。然后，我们将测试抑制肽拯救细胞功能的能力。这种实验策略应该定义MMP-2和纤连蛋白之间相互作用的特异性结合位点，并寻求一种新的策略来抑制单个MMP降解特定分子，例如MMP-2切割纤连蛋白。成功地抑制纤维连接蛋白断裂可能对牙周病和口腔癌的治疗都有显著的益处。

DESCRIPTION (provided by applicant): The enzymes in the family of matrix metalloproteinases (MMPs) are important to regular tissue maintenance by their collective capacity to degrade major tissue molecules and activating growth factors. The MMPs contribute hereby beneficially to normal development and tissue adaptation. Nevertheless, certain MMPs, including MMP-2, have been found at elevated levels and may abnormally degrade tissues in several diseases, such as periodontal disease, arthritis, and cancer. Recent studies by ourselves and other investigators have shown that the major cell adhesion molecule fibronectin is degraded in those diseases and that MMP-2 can efficiently cleave fibronectin. This degradation requires specific binding between fibronectin and a unique subdomain of MMP-2. Since the behavior of cells changes upon exposure to the fibronectin cleavage fragments, such fragments may alter the disease progression. Therefore, understanding the precise mechanism of binding between the two proteins may provide the basis for developing inhibitors that can block MMP-2 degradation of fibronectin. In a collaborative effort that involves investigators from different disciplines and institutions, we will apply molecular biology, biochemical, mass spectrometry, and protein engineering methods to define the precise binding sites for fibronectin and MMP-2. Based on results gained from screening of phage-displayed random peptide libraries, we will design synthetic peptides, which mimic the binding sites, and test their capacity to block both MMP-2 binding and degradation of fibronectin. Introduction of site-specific mutations in binding domains will confirm the binding sites. After precisely mapping the fibronectin cleavage sites and fragments, we will characterize altered periodontal and cancer cell behavior by multiple parameters, MMP expression, and apoptosis in response to contact with a series of recombinant fibronectin cleavage fragments. We will then test the capacity of the inhibitory peptides to rescue cell functions. This experimental strategy should define the specific binding sites for the interactions between MMP-2 and fibronectin and pursues a novel strategy to inhibit degradation of specific molecules by individual MMPs, such as cleavage of fibronectin by MMP-2. Successful inhibition of fibronectin fragmentation could be of significant benefit for the management of both periodontal disease and oral cancer.