REGULATION OF MATRIX METALLOPROTEINASE ACTIVATION

肿瘤细胞的侵袭依赖于蛋白水解酶的级联反应 能够降解细胞外基质成分。72 kDa（MMP-2） 和92 kDa（MMP-9）酶是基质的两个成员 与肿瘤侵袭相关的金属蛋白酶家族 和转移。这项提案的长期目标是揭示 控制激活的生化和生物学机制， MMP-2和MMP-9的抑制及其与降解的相关性 ECM与人类疾病72和92 kDa酶的活性是 受活性物质的产生及其与 金属蛋白酶组织抑制剂（TIMPs）。我们和其他人有 表明这些蛋白酶的C-末端是TIMP结合 结构域的酶原形式，是必要的质膜- MMP-2的依赖性活化。初步研究表明， 活化过程产生缺乏C-末端的活性形式。 然而，这些物种的生物化学和生物学特性是 不太了解。我们假设，72和74个氨基酸的C-末端， 92 kDa酶是活化和酶活性的关键调节剂 其裂解产生活性酶， TIMP抑制。 为了定义活性物质的功能特性， C-末端结构域在这些酶的调节中的重要性 我们建议（1）研究活性物种的分子性质 MMP-2和MMP-9的C-末端结构域切割后形成的， 分析和生物化学方法;（2）表达分泌的MMP-2 C- 在牛痘表达系统和肿瘤细胞中研究 该结构域在激活、TIMP-2抑制和体外 （3）构建并表达MMP-2和MMP-9嵌合体 在牛痘表达中具有异源C末端的酶 系统和肿瘤细胞中，以确定C-末端的重要性 结构域在膜激活和与TIMP的相互作用。拟议 研究将提供有关MMP调节的基本信息 活性和抑制作用，并可能有助于发展特定的 抑制剂来控制这些酶在结缔组织中的活性 疾病和恶性过程。

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.