EXTRACELLULAR REGULATION OF MATRIX METALLOPROTEINASES

基质金属蛋白酶的细胞外调节

• 批准号: 3305522
• 负责人: HAROLD E VAN WART
• 金额: $ 14.71万
• 依托单位: SYNTEX (USA), INC.-RESEARCH DIVISION
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305522
• 关键词: active sites; cadmium; cobalt; collagenase; conformation; cysteine; endopeptidases; enzyme induction /repression; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; extracellular matrix; extracellular matrix proteins; human tissue; metalloenzyme; nuclear magnetic resonance spectroscopy; stromelysin; zinc; zymogens

The long range goal of this research is to elucidate the mechanisms by which the matrix metalloproteinases (NM) are regulated extracellularly. The NM are a homologous group of zinc proteinases that play a major role in the normal and pathological breakdown of the extracellular matrix. The MMP are produced by resident and inflammatory-cells as zymogens. The basis for the latency of these zymogens and the means by which they are activated extracellularly are incompletely understood. The proposed experiments will examine various aspects of the "cysteine-switch" model for the latency and activation of pro-MMP recently proposed by us. The model attributes the inactivity of the pro-MMP to a novel complex between a cysteine residue in their propeptide domains and the active-site zinc atom in their catalytic domains. The pro-MMP exhibit multiple modes of activation that share the property that they disrupt this complex. To investigate this new hypothesis, the metal contents of five major human MMP will be measured and the functional role of each metal ion examined. In particular, the presence of zinc at the active site will be investigated. Active-site metal-substituted pro-MMP will be prepared and the presence of a cysteine-zinc bond in the pro-MMP investigated by EXAFS studies on the native species, 113Cd NMR studies on the Cd-substituted enzymes and optical studies of the Co(II)-substituted species. The, efficiency of a variety of known activation routes (proteases, oxidants, disulfide exchange, heavy metals, etc.) will be quantitated and correlated with the dissociation of the zinc-cysteine bond. These events will also be correlated with the autolytic loss of the propeptide domain in these MMP. The source of the small amount of "residual" activity in the pro-MMP will be carefully studied to assess whether it is due to a conformational equilibrium in these zymogens that could lead to their activation by substrates. To provide a structural model for the zinc site in pro-MMP, model peptides will be synthesized that mimic both the cysteine propeptide and zinc binding regions. Their structures and that of a possible complex between them will be elucidated by two dimensional (1)H-NMR experiments.

这项研究的长期目标是通过以下方式阐明其机制： 其中基质金属蛋白酶（NM）在细胞外受到调节。 NM 是锌蛋白酶的同源组，在 细胞外基质的正常和病理性分解。 这 MMP 由常驻细胞和炎症细胞作为酶原产生。 基础 了解这些酶原的潜伏期及其激活方式 细胞外的机制尚不完全了解。 拟议的实验将 检查“半胱氨酸开关”模型的各个方面的潜伏期和 我们最近提出了pro-MMP的激活。 该模型归因于 前 MMP 对半胱氨酸残基之间的新型复合物的失活 它们的前肽结构域和催化活性位点锌原子 域。 pro-MMP 表现出多种激活模式，这些模式共享 他们扰乱了这个建筑群的财产。 为了调查这个新 假设，将测量五种主要人类 MMP 的金属含量，并 所检查的每种金属离子的功能作用。 特别是， 将研究活性位点锌的存在。 活性位点 将制备金属取代的 pro-MMP，并且存在 EXAFS 研究调查了 pro-MMP 中的半胱氨酸-锌键 本地物种，Cd 取代酶的 113Cd NMR 研究和光学 Co(II) 取代物质的研究。 各种效率 已知的激活途径（蛋白酶、氧化剂、二硫键交换、重 金属等）将被定量并与解离相关 锌-半胱氨酸键。 这些事件也将与 这些 MMP 中前肽结构域的自溶性丢失。 的来源 pro-MMP 中的少量“残留”活性将被仔细处理 研究评估这是否是由于构象平衡 这些酶原可能会被底物激活。 到 提供 pro-MMP 中锌位点的结构模型，模型肽 将合成模拟半胱氨酸前肽和锌 结合区域。 它们的结构以及它们之间可能存在的复合体的结构 它们将通过二维 (1)H-NMR 实验来阐明。