STRUCTURE AND FUNCTION OF COLLAGENASE INHIBITORS

胶原酶抑制剂的结构和功能

• 批准号: 2732844
• 负责人: HIDEAKI NAGASE
• 金额: $ 29.13万
• 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/2732844
• 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：（改编自申请人的摘要）- MMPs发挥核心作用， 在疾病过程中细胞外基质分解中的作用， 关节炎、肾小球肾炎、组织溃疡和肿瘤细胞转移。 MMP活性受到蛋白质抑制剂家族的特异性调节 命名为TIMPs，已知其三种形式（TIMPs-1、-2和-3）。 最近的工作已经确定TIMP-3的突变是导致显性免疫缺陷的原因。 早发性黄斑变性，Sorsby眼底营养不良（SFD）， 人类 首席研究员的长期目标是阐明 TIMPs调节MMP活性的结构基础和机制， 这是如何被操纵以增强其特异性或被干扰， 病理状态 该提案的具体目标如下： (1)TIMP抑制机制的研究。 这将是 通过定点诱变进行，使用N-末端结构域的 TIMP-1（N-TIMP-1）作为原型。 突变体的设计将基于 从生物化学研究中获得的信息和即将到来的晶体 TIMP-1-MMP-3复合物的结构，这是由一个 合作者，德国马克斯-普朗克研究所的Wolfram Bode博士。 （二） 特异性MMP的选择性抑制剂的分离。 功能性网站 将鉴定的N-TIMP-1进行饱和诱变， 在M13噬菌体和抑制剂具有增强的特异性将被分离， 用固定化MMPS选择。 (3)函数的确定 TIMP-3及其结构域的性质。 TIMP-3及其两个结构域将被 表达并表征了对MMP的抑制作用， 与细胞外基质成分结合。 SFD突变的影响 对这些性质，包括可能的新共价相互作用，将是 评估。 (4)TIMP-2与细胞表面结合的表征，a 这种现象与明胶酶A（proMMP-2）的活化有关。 这将通过表征一种新的TIMP-2结合蛋白来研究 在细胞表面表达。 这种蛋白质将被分离、克隆， 测序 (5)蛋白质、突变体和酶抑制剂的产生 为合作者提供TIMP-MMP结构研究的复合物 通过X射线晶体学和多核NMR光谱学研究相互作用。 这些研究将产生有关的机制和结构信息， TIMP-2与MMPs的相互作用以及TIMP-2在 调节细胞外基质中的细胞周围蛋白水解。