ECM PROTEASES IN INFLAMMATION, FIBROSIS & TUMORIGENESIS

描述：分泌型金属蛋白水解酶在过程中起主导作用。 通过启动细胞外降解来实现组织重塑 矩阵(ECM)。这项提案概述了调查 这些酶的生物学功能。中描述的实验 此应用程序基于存在交互作用的假设 在细胞表面和/或纤维上具有固定的分子结构 ECM。这一假设隐含着这样一种理解，即 酶在这些部位的隔离是它们生理上所必需的 激活。因此，胞外酶的净活性 取决于此类站点的可用性和激活条件。这个 对这种相互作用的机理研究一直是 引起了调查人员的注意，最近才产生了隔离 与细胞有关的新的膜结合金属蛋白酶MT-MMPs 72 kDa IV型胶原酶(72T4Cl)的表面活化。澄清 72T4Cl的活化机制证实了一个新兴的概念 这种转移性侵袭需要上皮和间质的合作。 基质金属蛋白水解酶在体内由基质细胞表达。 上皮性肿瘤细胞表达细胞表面的蛋白水解酶受体S。 它们之间的相互作用会导致高度特异的激活 本地化，从而为药物开发提供了一个新的目标。研究 人类72T4CL的结构-功能关系，特别是 羧基末端类血凝蛋白结构域的功能将是一个重点 了解酶的激活机制及其与细胞的相互作用 特异性抑制剂TIMP2。定点诱变与基因工程的结合 确定这个领域的三级结构将是主要的 在这些研究中采用的方法。调查员在提纯过程中的成功 并将重组羧基末端结构域结晶 对这些研究的成功起到了重要作用。几个有希望的途径 已经开展了一系列旨在阐明细胞表面的研究 间质胶原酶(CL1)的激活机制一种新设计的 体外实验免疫荧光法检测血管内皮细胞的分布 在组织培养中添加外源纯化酶将是有益的 在间质胶原酶与细胞表面和 在开发特定的竞争性配体方面。数据获取自 这些研究将提供对分子机制的理解。 参与调控的胞外蛋白分解及细胞外基质的作用 金属蛋白酶在形态发生、伤口愈合和多种病理中的作用 条件。

DESCRIPTION: Secreted metalloproteases play a leading role in processes of tissue remodeling by initiating the degradation of extracellular matrix (ECM). This proposal outlines the plan for investigating the biological function of these proteases. The experiments described in this application are based on the hypothesis that there is interaction with fixed molecular structures on the cell surfaces and/or fibrils of the ECM. Implicit in this hypothesis is the understanding that sequestering of enzymes at such sites is required for their physiological activation. Consequently, the net extracellular enzymatic activity depends on the availability of such sites and activation conditions. The mechanistic study of such interactions has been a focus of the investigator's attention, and just recently resulted is isolation of the novel membrane bound metalloprotease MT-MMP that is responsible for cell surface activation of 72 kDa type IV collagenase (72T4Cl). Elucidation of the activation mechanism of 72T4Cl substantiates an emerging concept that metastatic invasion requires epithelial-mesenchymal cooperation. Matrix metalloproteases are expressed in vivo by stromal cells. Epithelial tumor cells express protease receptor(s) on the cell surface. Interaction between these leads to activation that is highly specific and localized, thus presenting a new target for drug development. Studies of structure-function relationships in the human 72T4Cl, particularly the function of the carboxyl-end hemopexin-like domain, will be a focus for understanding of mechanisms of enzyme activation and interaction with the specific inhibitor TIMP2. Combination of site directed mutagenesis with determination of the tertiary structure of this domain will be main approach in these studies. The investigator's success in the purification and crystallization of the recombinant carboxyl end domain will be instrumental for the success of these studies. Several promising avenues of investigation has been developed aimed at elucidation of cell surface activation mechanism of interstitial collagenase (Cl1). A newly designed experimental immunofluorescence in vitro to detect the distribution of exogenously added purified enzymes in tissue culture will be instrumental in studies of interstitial collagenase interaction with cell surface and in the development of specific competitive ligands. Data obtained from these studies will provide an understanding of the molecular mechanisms involved in regulated extracellular proteolysis and the role of ECM metalloproteases in morphogenesis wound healing and numerous pathologic conditions.