TISSUE INHIBITORS OF METALLOPROTEINASES

金属蛋白酶组织抑制剂

基本信息

批准号：
2668244
负责人：
JEFFREY ALLEN ENGLER
金额：
$ 18.77万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-03-01 至 2000-02-29
项目状态：
已结题

项目摘要

Tissue Inhibitors of Metalloproteinases (TIMP-l and TIMP-2) regulate the activity of matrix metalloproteinases in the extracellular milieu. Matrix metalloproteinases collectively degrade most if not all matrix macromolecules and appear to be responsible for stromal remodeling in many normal and pathological events. The inhibitors function at two important levels: by blocking activation of the latent matrix metalloproteinase precursors and by forming inhibited complexes with the activated forms of the enzymes. We wish to understand the molecular basis for these functions and to examine how TIMPs regulate extracellular matrix degradation by live cells. In Aim 1, we propose to identify those domains and subdomains of TIMP-1 and TIMP-2 that are important to inhibitory function by peptide- and antibody- competition approaches and by site-directed mutagenesis of sequences targeted by initial competition experiments. We also propose to identify those domains of TIMP-1 and TIMP-2 that are important to forming complexes with the Mr 72K GL and Mr 92K GL zymogens, and to understand the basis for these highly specific interactions. In Aim 2, we propose to examine how TIMPs maintain latency of MMP precursors. We have formulated the novel hypothesis that TIMPs form zymogen complexes with all MMP precursors and that they regulate the rate of opening of the Cys-Zn++- switch which is a prequisite for MMP activation. We will specifically quantify the extent to which TIMPs modify the rate of switch-opening and we will seek to demonstrate formation of dissociable complexes between fibroblast-type collagenase and stromelysin-1 and TIMPs. In Aim 3, the role of TlMPs in regulating the degradation of collagen fibrils by live cells will be analyzed. Two cell based systems will be used, one in which collagen breakdown is naturally blocked because of coexpression of TIMPs and one in which rapid collagen breakdown is induced by addition of plasminogen. The function of specific mutant and wildtype TIMPs in controlling collagen degradation in the plasminogen-supplemented system will be examined by addition or overexpression of these TIMPs by means of a vaccinia virus system. In addition, we will seek to establish that collagen breakdown is blocked in the absence of plasminogen because of endogenous AMP expression. Our approach will be to downregulate either the expression (antisense oligonucleotides or RNA) or the activity (neutralizing peptides antibodies) of one or both TIMPs and to monitor the effect on collagen degradation. The proposed studies will yield a better understanding of the natural control and regulation mechanisms that serve to limit tissue destruction during normal and pathological developments.

金属蛋白酶（TIMP-L和TIMP-2）的组织抑制剂调节基质金属蛋白酶在细胞外环境中的活性。矩阵金属蛋白酶共同降解大多数矩阵（如果不是全部）大分子，似乎负责许多人的基质重塑正常事件和病理事件。抑制剂在两个重要的功能级别：通过阻断潜在基质金属蛋白酶的激活前体并通过形成激活形式的抑制复合物酶。我们希望了解这些功能的分子基础并检查TIMP如何通过现场调节细胞外基质降解细胞。在AIM 1中，我们建议确定 TIMP-1和TIMP-2对于肽 - 抑制功能很重要和抗体竞争方法以及通过位于位置的诱变最初竞争实验针对的序列。我们还建议识别TIMP-1和TIMP-2的域，这些领域对于形成很重要 MR 72K GL和MR 92K GL Zymogens的复合物，并了解这些高度特定的相互作用的基础。在AIM 2中，我们建议检查TIMP如何保持MMP前体的延迟。我们已经制定了 Timps与所有MMP形成Zymogen复合物的新假设前体并调节Cys-Zn ++的打开率 - 开关是MMP激活的先决条件。我们将具体量化Timps修改开关率的程度和我们将寻求证明在成纤维细胞型胶原酶和Stromelysin-1和Timps。在AIM 3中 TLMP在调节现场调节胶原原纤维降解中的作用细胞将进行分析。将使用两个基于单元的系统，其中一个由于TIMP的共表达，胶原蛋白分解自然被阻塞以及通过添加诱导快速胶原蛋白分解的一种纤溶酶原。特定突变体和野生型TIMP的功能控制纤溶酶原系统中的胶原蛋白降解通过添加或过表达这些TIMP的检查一种离子病毒系统。此外，我们将寻求确定由于没有纤溶酶原，胶原蛋白分解被阻塞内源性AMP表达。我们的方法是下调表达（反义寡核苷酸或RNA）或活性（中和肽抗体）一个或两个Timps并监测对胶原蛋白降解的影响。拟议的研究将产生更好的了解服务的自然控制和调节机制在正常和病理发展期间限制组织破坏。