REGULATION OF MATRIX METALLOPROTEINASE ACTIVATION

肿瘤细胞的侵袭依赖于蛋白水解酶的级联。 能够降解细胞外基质成分。72 kDa(基质金属蛋白酶-2) 和92 kDa(MMP9)酶是基质中的两个成员 与肿瘤侵袭相关的金属蛋白酶家族 和转移。这项提议的长期目标是揭开 控制细胞活化的生化和生物学机制 基质金属蛋白酶-2和基质金属蛋白酶-9的抑制作用及其与细胞降解的关系 人类疾病中的细胞外基质。72 kDa和92 kDa酶的活性为 受活性物种的产生及其与 金属蛋白酶组织抑制因子(TIMPs)。我们和其他人 结果表明，这些酶的C末端是TIMP结合 酶原形式的结构域，是质膜所必需的- 依赖激活的基质金属蛋白酶-2。初步研究表明， 激活过程产生缺少C-末端的活性形式。 然而，这些物种的生化和生物学特性是 人们对此知之甚少。我们假设72和72的C末端 92 kDa酶是激活和酶活性的关键调节因子 它的裂解产生活性酶，降低了对 TIMP抑制作用。 为了定义活性物种的功能性质和 C-末端结构域在这些酶调控中的意义 我们建议：(1)研究活性物种的分子性质 裂解C-末端结构域后形成的基质金属蛋白酶-2和基质金属蛋白酶-9 分析和生化方法；(2)表达分泌的基质金属蛋白酶-2C-C- 末端在痘苗病毒表达系统和肿瘤细胞中的研究 该结构域在激活、抑制TIMP-2和体外实验中的作用 细胞侵袭；(3)构建和表达嵌合的基质金属蛋白酶-2和基质金属蛋白酶-9 带有异源C-末端的酶在痘苗病毒中的表达 系统和在肿瘤细胞中确定C-末端的重要性 膜激活中的结构域及其与TIMPs的相互作用。建议数 研究将提供关于基质金属蛋白酶调控的基本信息 活性和抑制，并可能有助于特定的发展 控制结缔组织中这些酶活性的抑制剂 在疾病和恶性过程中。

The invasion of tumor cells depends on a cascade of proteolytic enzymes capable of degrading extracellular matrix components. The 72 kDa (MMP-2) and 92 kDa (MMP-9) enzymes are two members of the matrix metalloproteinase family which have been associated with tumor invasion and metastasis. The long term objective of this proposal is to unveil the biochemical and biological mechanisms controlling the activation and inhibition of MMP-2 and MMP-9 and their relevance to the degradation of ECM in human diseases. The activity of the 72 and 92 kDa enzymes is regulated by the generation of active species and their interactions with the tissue inhibitors of metalloproteinases (TIMPs). We and other have shown that the C-terminal end of these proteinases is the TIMP binding domain in the proenzyme form and is necessary for the plasma membrane- dependent activation of MMP-2. Preliminary studies suggest that the activation process generates active forms lacking the C-terminal end. However, the biochemical and biological properties of these species is poorly understood. We hypothesize that the C-terminal end of the 72 and 92 kDa enzymes is a key regulator of activation and enzymatic activity and its cleavage generates active enzymes with a reduced sensitivity to TIMP inhibition. To define the functional properties of the active species and the significance of the C-terminal domain in the regulation of these enzymes we propose (1) to study the molecular properties of the active species of MMP-2 and MMP-9 formed after cleavage of the C-terminal domain using analytical and biochemical methods; (2) to express a secreted MMP-2 C- terminal end in a vaccinia expression system and in tumor cells to study the role of this domain on activation, TIMP-2 inhibition and in vitro cell invasion and, (3) to construct and express chimeric MMP-2 and MMP-9 enzymes with a heterologous C-terminal end in a vaccinia expression system and in tumor cells to determine the importance of the C-terminal domain in membrane activation and interactions with TIMPs. The proposed studies will provide fundamental information on the regulation of MMP activity and inhibition and may contribute to the development of specific inhibitors to control the activity of these enzymes in connective tissue diseases and in malignant processes.