THE BIOLOGICAL ROLE OF HUMAN TIMP IN MATRIX TURNOVER

细胞外基质的周转对维持 结缔组织的结构完整性，以及其中的像差 这一过程可能导致多种病理状态。一个人的活动 一组中性金属蛋白酶，包括间质胶原酶， 基质分解酶、72kD IV型胶原酶和92kD明胶酶/V型 胶原酶被认为控制细胞外基质的周转 组件。金属蛋白酶组织抑制因子(TIMP)的功能 作为这整个家族的主要，也可能是唯一的抑制者 结缔组织间质中的中性金属蛋白酶 纸巾。这项拨款建议的主要目的是研究 生化作用机制、分子调控和原位 在正常基质周转和疾病状态下产生TIMP。 对TIMP作用的生化机制的研究将包括 几个方面，包括其复合酶原与活性的能力 不同金属蛋白酶的形态，如评估的高效液相凝胶过滤 技巧。此外，TIMP与不同的 酶的测定将使用交联剂或动力学方法 对高度敏感的底物进行分析。的本地化 TIMP和金属蛋白酶中需要的结构位点 酶抑制物结合是一个主要的优先事项，并将得到解决 使用可加碘的共价交联剂， 将标记转移到第二蛋白质，并随后 碘化片段的分离/测序(S)。 对调控机制的研究将调查细胞- 基质相互作用在TIMP和胶原酶生物合成中的作用 对基质降解的后果。细胞因子和生物因素，如 将研究γ-干扰素和脂多糖的调节能力 TIMP和胶原酶在蛋白和mRNA水平均有表达。维甲酸 将进一步研究潜在的转录 它们的作用机制。 将对分离的细胞和整个细胞进行原位杂交 组织标本积极了解细胞类型 正常结缔组织中TIMP和胶原酶的合成 周转和疾病过程中。这些基因的细胞表达 胎儿发育、光化损伤和结缔组织中的蛋白质 组织疾病和大疱性表皮松解症将被研究。

The turnover of extracellular matrix is crucial for maintaining structural integrity of connective tissues, and aberrations in this process can lead to a variety of pathologic states. The activity of a group of neutral metalloproteinases, including interstitial collagenase, stromelysin, 72 kD type IV collagenase, and 92 kD gelatinase/type V collagenase is believed to control the turnover of extracellular matrix components. Tissue inhibitor of metalloproteinases, or TIMP, functions as the major, and probably, only, inhibitor of this entire family of neutral metalloproteinases within the interstitial spaces of connective tissues. The broad objective of this grant proposal is to study the biochemical mechanisms of action, molecular regulation, and in situ production of TIMP during normal matrix turnover and in disease states. Studies of the biochemical mechanisms of TIMP action will encompass several areas, including its capacity to complex zymogen versus active forms of the various metalloproteases, as assessed HPLC gel-filtration techniques. Furthermore, affinities between TIMP and the different enzymes will be determined using cross-linking reagents or by kinetic analyses against highly susceptible substrates. Localization of the structural sites in TIMP and metalloproteinases that are required for enzyme-inhibitor binding is a major priority and will be approached using covalent cross-linking reagents that can be iodinated, with transfer of the label to a second protein, and subsequent isolation/sequencing of the iodinated fragment(s). Studies of regulatory mechanisms will investigate the role of cell- matrix interactions in TIMP and collagenase biosynthesis and the consequences to matrix degradation. Cytokines and biologic factors such as gamma-interferon and LPS will be studied for capacity to regulate TIMP and collagenase expression at protein and mRNA levels. Retinoids will be further investigated with regards to potential transcriptional mechanisms of their effects. In situ hybridization will be performed on isolated cells and whole tissues specimens to gain insights in the cell types actively synthesizing TIMP and collagenase during normal connective tissue turnover and in disease processes. The cellular expression of these proteins during fetal development, actinic injury and in connective tissue diseases, and epidermolysis bullosa will be studied.