BIOLOGICAL ROLE OF THE 92KDA TYPE IV COLLAGENASE

金属蛋白酶在细胞外基质重塑中起重要作用 矩阵（ECM）。 该提案旨在解决生物学作用， 一种特殊的酶，人92 kDa IV型胶原酶。 92 kda 金属蛋白酶是角质形成细胞的主要分泌产物，单核 吞噬细胞和许多转化细胞系。 我们最近净化了 这种酶的同质性，并确定其完整的一级结构。 为了进一步研究这种酶的生物学作用，我们建议 探讨酶抑制剂复合物的分子机理 阵 我们还将研究92 kDa IV型胶原酶在 细胞外基质降解通过研究分泌的 酶与ECM和细胞表面。 最后，92 kDa IV型 将研究胶原酶在组织重塑中的作用，并与 通过用野生型转染cDNA克隆来检测其它分泌型金属蛋白酶 型酶与突变体酶转染亲本肿瘤和E1 A转染的细胞。 我们的实验方法将包括诱变， 蛋白质水平上的分子间相互作用，交联和 使用合成肽的竞争实验。 定点 野生型酶的诱变将用于产生点突变体 无法与TIMP相互作用。 92 kDa酶相互作用的研究 将利用“自杀”配体技术 最近在我们的实验室里开发的。 能够竞争的合成肽 与酶-ECM和/或酶-细胞表面相互作用将是 化学修饰以结合亲和性和放射性标记 可光活化的交联基团，以特异性标记 分子间缔合 用腺病毒E1 A基因转染肿瘤细胞， 转录抑制的内源性间质和IV型 胶原酶启动子。 通过允许每种酶的表达 在人肿瘤细胞的亲本HT 1080和E1 A转染子中，在 控制一个不受E1 A抑制的启动子， 单个酶表达对细胞表型的特定影响。 从这些研究中获得的信息将提供进一步的 了解参与分泌的分子机制 在正常和病理条件下的金属蛋白酶作用。

Metalloproteases play an important role in the remodeling of extracellular matrix (ECM). This proposal is designed to address the biologic role of one particular enzyme, human 92 kDa type IV collagenase. The 92 kda metalloprotease is a major secreted product of keratinocytes, mononuclear phagocytes, and many transformed cell lines. We have recently purified this enzyme to homogeneity and determined its complete primary structure. To further study the biologic role of this enzyme, we propose to investigate the molecular mechanism of proenzyme-inhibitor complex formation. We will also examine the role of 92 kDa type IV collagenase in extracellular matrix degradation by studying interaction of the secreted enzyme with ECM and cell surface. Finally, the role of the 92 kDa type IV collagenase in tissue remodeling will be studied and compared to that of other secreted metalloproteases by transfection of cDNA clones with wild type versus mutant enzymes into parental tumor and E1A transfected cells. Our experimental approaches will include mutagenesis, characterization of intermolecular interactions on the protein level, crosslinking and competition experiments using synthetic peptides. Site-directed mutagenesis of wild type enzyme will be used to generate point mutants unable to interact with TIMP. The studies of 92 kDa enzyme interactions with matrix and/or cell surfaces will utilize a "suicide" ligand technology recently developed in our laboratory. Synthetic peptides able to compete with the enzyme-ECM and/or enzyme-cell surface interactions will be chemically modified to incorporate affinity and radiolabeled photoactivatable crosslinking groups to specifically tag the sites of intermolecular associations. Transfection of tumor cells with the adenovirus E1A gene results in transcriptional repression of the endogenous interstitial and type IV collagenase promoters. By allowing the expression of each of the enzymes in parental HT1080 and E1A transfectants of human tumor cells, under control of a promoter not subject to E1A repression, we can gain insights into specific effects of individual enzyme expression on cell phenotype. The information obtained from these studies will provide a further understanding of the molecular mechanisms involved in secreted metalloprotease action in normal and pathologic conditions.