THE BIOLOGICAL ROLE OF HUMAN TIMP IN MATRIX TURNOVER

细胞外基质的周转对于维持 结缔组织的结构完整性，以及这种结构的畸变， 这一过程可导致多种病理状态。 的活性 中性金属蛋白酶组，包括间质胶原酶， 溶基质素、72 kD IV型胶原酶和92 kD明胶酶/V型 胶原酶被认为控制细胞外基质的周转 件. 金属蛋白酶组织抑制剂（TIMP）的功能 作为主要的，也可能是唯一的， 结缔组织间隙内的中性金属蛋白酶 组织中 这项拨款建议的主要目标是研究 作用的生化机制、分子调控和原位 在正常基质周转期间和疾病状态下产生TIMP。 TIMP作用的生化机制的研究将包括 几个领域，包括其能力，以复杂的酶原与活性 各种金属蛋白酶的形式，如HPLC凝胶过滤评估 技术. 此外，TIMP和不同的细胞因子之间的亲和力也不同。 酶将使用交联试剂或通过动力学测定 对高度敏感的底物进行分析。 定位 TIMP和金属蛋白酶中的结构位点， 酶抑制剂的结合是一个主要的优先事项， 使用可碘化的共价交联试剂， 将所述标记转移至第二蛋白，以及随后 碘化片段的分离/测序。 调节机制的研究将调查细胞的作用- 基质在TIMP和胶原酶生物合成中的相互作用， 基质降解的后果。 细胞因子和生物因子， 因为将研究γ-干扰素和LPS调节 TIMP和胶原酶在蛋白和mRNA水平的表达。 类视黄醇 将进一步研究关于潜在的转录 其影响的机制。 将对分离的细胞和整个细胞进行原位杂交。 组织标本，以获得在细胞类型的见解积极 正常结缔组织合成TIMP和胶原酶 周转和疾病过程。 这些基因的细胞表达 蛋白质在胎儿发育，光化损伤和结缔组织 组织疾病和大疱性表皮病。

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.