STRUCTURE AND FUNCTION OF COLLAGENASE INHIBITORS

胶原酶抑制剂的结构和功能

• 批准号: 2442814
• 负责人: HIDEAKI NAGASE
• 金额: $ 28.28万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2442814
• 关键词: X ray crystallography; active sites; chemical binding; collagenase; enzyme activity; enzyme mechanism; enzyme substrate complex; extracellular matrix proteins; gene expression; gene mutation; intermolecular interaction; macular degeneration; metalloproteins; molecular cloning; molecular site; nuclear magnetic resonance spectroscopy; protease inhibitor; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis; tissue /cell culture; tissue inhibitor of metalloproteinases; zymogens

DESCRIPTION: (Adapted from the applicant's abstract) - MMPs play a central role in extracellular matrix breakdown in disease processes such as arthritis, glomerulonephritis, tissue ulceration, and tumor cell metastasis. MMP activities are specifically regulated by a family of protein inhibitors designated TIMPs of which three forms (TIMPs-1, -2 and -3) are known. Recent work has identified mutations in TIMP-3 as the cause of a dominant early onset macular degeneration, Sorsby's fundus dystrophy (SFD), in humans. The principal investigator's long-term goals are to elucidate the structural basis and mechanisms by which TIMPs regulate MMP activities and how this can be manipulated to enhance its specificity or is disturbed in pathological condition. The specific aims of the proposal are as follows: (1) Investigation of the inhibition mechanisms of TIMPs. This will be carried out by site-directed mutagenesis using the N-terminal domain of TIMP-1 (N-TIMP-1) as a prototype. The design of mutants will be based on the information obtained from biochemical studies and a forthcoming crystal structure of the TIMP-1-MMP-3 complex that is being determined by a collaborator, Dr. Wolfram Bode at Max-Planck-Institut in Germany. (2) Isolation of selective inhibitors for specific MMPs. The functional sites identified will be subjected to saturation mutagenesis in N-TIMP-1 displayed on M13 phage and inhibitors with enhanced specificity will be isolated by selection with immobilized MMPS. (3) Determination of the functional properties of TIMP-3 and its domains. TIMP-3 and its two domains will be expressed and characterized with respect to inhibitory action on MMPs and binding to extracellular matrix components. The effect of the SFD mutation on these properties, including possible new covalent interactions, will be assessed. (4) Characterization of TIMP-2 binding to the cell surface, a phenomenon that is related to the activation of progelatinase A (proMMP-2). This will be investigated by characterizing a novel TIMP-2 binding protein expressed on the cell surface. This protein will be isolated, cloned and sequenced. (5) Generation of proteins, mutants and enzyme-inhibitor complexes to provide to collaborators for structural studies of TIMP-MMP interactions by X-ray crystallography and multinuclear NMR spectroscopy. These studies will yield mechanistic and structural information about the interaction of TIMPs and MMPs and new insights into the role of TIMP-2 in regulation of pericellular proteolysis in the extracellular matrix.

描述：(改编自申请者的摘要)-MMP扮演核心角色 在疾病过程中细胞外基质分解的作用 关节炎、肾小球肾炎、组织溃疡和肿瘤细胞转移。 基质金属蛋白酶活性受一系列蛋白抑制物的特异性调节 指定的TIMP，其中三种形式(TIMP-1、-2和-3)是已知的。 最近的研究发现，TIMP-3的突变是导致显性 早发性黄斑变性，Sorsby眼底营养不良(SFD)， 人类。首席调查员的长期目标是阐明 TIMP调节基质金属蛋白酶活性的结构基础和机制 如何操纵这一点以增强其特异性或在 病理状态。该建议的具体目标如下： (1)TIMPs抑制作用机制的研究。这将是 通过使用N端结构域的定点突变来实现 TIMP-1(N-TIMP-1)作为原型。突变体的设计将基于 从生化研究和即将到来的晶体中获得的信息 TIMP-1-基质金属蛋白酶-3复合体的结构 合作者，德国Max-Planck-Institut的Wolfram Bode博士。(2) 特异性MMPs选择性抑制剂的分离。功能部位 经鉴定将在N-TIMP-1展示中进行饱和诱变 在M13噬菌体上，通过以下方法分离具有增强特异性的噬菌体和抑制物 用固定化MMPs进行选择。(3)功能的确定 TIMP-3及其结构域的性质。TIMP-3及其两个结构域将是 表达和表征对MMPs和MMPs的抑制作用 与细胞外基质成分结合。SFD突变的影响 关于这些性质，包括可能的新的共价相互作用，将是 评估过了。(4)TIMP-2与细胞表面结合的特征，a 与孕激素酶A(ProMMP2)活化有关的现象。 这将通过鉴定一种新的TIMP-2结合蛋白来进行研究 在细胞表面表达。该蛋白将被分离、克隆和 已排序。(5)蛋白质、突变体和酶抑制剂的产生 提供给合作者进行TIMP-MMPs结构研究的络合物 用X射线结晶学和多核核磁共振波谱研究相互作用。 这些研究将产生关于 TIMP与MMPs的相互作用及其对TIMP-2作用的新认识 细胞外基质中细胞周围蛋白分解的调节。