EXTRACELLULAR REGULATION OF MATRIX METALLOPROTEINASES

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)受细胞外调控。 NM是锌蛋白水解酶的同源基团，起主要作用 在细胞外基质的正常和病理分解中。这个 基质金属蛋白酶是由常驻细胞和炎性细胞作为发酵源产生的。其基础是 这些酵素的潜伏期以及它们被激活的方式 从细胞外的角度来看，人们对此并不完全了解。拟议中的实验将 检查“半胱氨酸开关”模型的各个方面的延迟和 我们最近提出的激活基质金属蛋白酶原的建议。该模型将 原-基质金属蛋白酶对半胱氨酸残基之间的新型络合物的失活 它们的前肽结构域及其催化活性中心锌原子 域名。前基质金属蛋白酶表现出多种激活模式，这些模式共享 他们破坏了这个建筑群的财产。为了调查这一新的 假设，将测量五种主要人体金属基质的金属含量并 考察了每种金属离子的功能作用。尤其是， 将对活性部位是否存在锌进行调查。活动站点 将制备金属取代的金属基质蛋白原，并在其存在下 半胱氨酸-锌键在基质金属蛋白酶原中的EXAFS研究 天然物种、Cd取代酶的~(113)Cd核磁共振研究及光学性质 Co(II)取代物种的研究。一种品种的效率 已知的激活途径(蛋白酶、氧化剂、二硫键交换、重 金属等)将被量化，并与解离 锌-半胱氨酸键。这些事件还将与 这些基质金属蛋白酶的前肽结构域的自溶丢失。该产品的来源 少量的“残留”活性的亲基质金属蛋白酶将被小心 研究以评估它是否是由于 这些酶原可能导致它们被底物激活。至 提供前基质金属蛋白酶中锌位点的结构模型，模型多肽 将合成同时模拟半胱氨酸前肽和锌的化合物 结合区。它们的结构和可能的复合体之间的结构 它们将通过二维(1)H-核磁共振实验加以阐明。

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.