BIOLOGICAL ROLE OF THE 92KDA TYPE IV COLLAGENASE

92KDA IV 型胶原酶的生物学作用

• 批准号: 3161065
• 负责人: GREGORY I GOLDBERG
• 金额: $ 17.37万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161065
• 关键词: affinity labeling; collagenase; connective tissue metabolism; crosslink; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme substrate complex; extracellular matrix; intermolecular interaction; laboratory mouse; laboratory rabbit; molecular cloning; molecular site; neoplastic cell culture for noncancer research; site directed mutagenesis; synthetic peptide; transfection

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.

金属蛋白酶在细胞外基质的重塑中发挥重要作用 矩阵（ECM）。 该提案旨在解决 一种特殊的酶，人类 92 kDa IV 型胶原酶。 92kda 金属蛋白酶是角质形成细胞、单核细胞的主要分泌产物 吞噬细胞和许多转化细胞系。 我们最近净化了 该酶的同质性并确定了其完整的一级结构。 为了进一步研究这种酶的生物学作用，我们建议 研究酶原-抑制剂复合物的分子机制 形成。 我们还将研究 92 kDa IV 型胶原酶在 通过研究分泌物的相互作用来降解细胞外基质 酶与 ECM 和细胞表面。 最后，92 kDa IV 型的作用 将研究胶原酶在组织重塑中的作用并与 通过用野生型 cDNA 克隆转染其他分泌型金属蛋白酶 类型与突变型酶进入亲本肿瘤和E1A转染细胞。 我们的实验方法将包括诱变、表征 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。 现场定向 野生型酶的诱变将用于产生点突变体 无法与 TIMP 交互。 92 kDa酶相互作用的研究 与基质和/或细胞表面将利用“自杀”配体技术 最近在我们的实验室开发。 能够竞争的合成肽 与酶-ECM 和/或酶-细胞表面相互作用将是 经过化学修饰以结合亲和力和放射性标记 可光活化的交联基团专门标记的位点 分子间缔合。 用腺病毒 E1A 基因转染肿瘤细胞会产生 内源性间质和 IV 型的转录抑制 胶原酶促进剂。 通过允许每种酶的表达 在人类肿瘤细胞的亲本 HT1080 和 E1A 转染子中， 控制不受 E1A 抑制的启动子，我们可以获得见解 研究个体酶表达对细胞表型的具体影响。 从这些研究中获得的信息将提供进一步的信息 了解参与分泌的分子机制 金属蛋白酶在正常和病理条件下的作用。