REGULATION OF MATRIX METALLOPROTEINASE ACTIVATION

肿瘤细胞的侵袭依赖于一系列蛋白水解酶 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。该提案的长期目标是揭示 控制激活和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 人类疾病中的 ECM。 72 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剂 (TIMP)。我们和其他人有 显示这些蛋白酶的 C 末端是 TIMP 结合 结构域以酶原形式存在，并且是质膜所必需的- MMP-2 的依赖性激活。初步研究表明 激活过程产生缺乏 C 末端的活性形式。 然而，这些物种的生化和生物学特性是 不太了解。我们假设 72 的 C 末端和 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，其敏感性降低 TIMP 抑制。 定义活性物质的功能特性和 C 末端结构域在这些酶的调节中的重要性 我们建议（1）研究活性物质的分子特性 使用切割 C 末端结构域后形成的 MMP-2 和 MMP-9 分析和生化方法； (2) 表达分泌型 MMP-2 C- 牛痘表达系统和待研究肿瘤细胞中的末端 该结构域对激活、TIMP-2 抑制和体外的作用 细胞侵袭，(3)构建并表达嵌合MMP-2和MMP-9 牛痘表达中具有异源 C 末端的酶 系统和肿瘤细胞中确定 C 末端的重要性 膜激活和与 TIMP 相互作用的结构域。拟议的 研究将提供有关 MMP 监管的基本信息 活性和抑制，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 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.