BIOLOGICAL ROLE OF THE 92KDA TYPE IV COLLAGENASE

92KDA IV 型胶原酶的生物学作用

基本信息

批准号：
2080167
负责人：
GREGORY I GOLDBERG
金额：
$ 18.89万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/2080167
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

项目摘要

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。