BIOLOGICAL ROLE OF THE 92KDA TYPE IV COLLAGENASE

92KDA IV 型胶原酶的生物学作用

• 批准号: 2080166
• 负责人: GREGORY I GOLDBERG
• 金额: $ 18.16万
• 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/2080166
• 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重构中的重要作用 矩阵(ECM)。这项提议旨在解决生物角色的问题 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，为单核细胞。 吞噬细胞，以及许多转化的细胞系。我们最近提纯了 该酶均一，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 酶原-抑制物复合体的分子机制研究 队形。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降解细胞外基质 含有细胞外基质和细胞表面的酶。最后，92 kDa IV型的作用 胶原酶在组织重塑中的作用将被研究，并与 用野生型基因转化其他分泌型金属蛋白酶 类型与突变的酶进入亲本肿瘤细胞和E1a基因的细胞。 我们的实验方法将包括突变、表征 蛋白质水平上的分子间相互作用、交联和 使用合成多肽的竞争实验。站点定向 野生型酶的诱变将用于产生点突变 无法与TIMP交互。92 kDa酶相互作用的研究 具有基质和/或细胞表面的材料将使用“自杀式”配基技术 是我们实验室最近开发出来的。具有竞争能力的合成肽 随着酶-ECM和/或酶-细胞表面的相互作用 经过化学修饰以结合亲和力和放射性标记 可光激活的交联基，用于特定标记 分子间关联。 腺病毒E1a基因转染腺病毒感染肿瘤细胞 内源性间质和IV型转录抑制 胶原酶促进剂。通过允许每种酶的表达 在人肿瘤细胞的亲本HT1080和E1A转染体中， 控制不受E1a抑制的启动子，我们可以获得洞察力 单个酶的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 对分泌型糖尿病的分子机制的认识 金属蛋白酶在正常和病理条件下的作用。