STRUCTURE-FUNCTION OF HUMAN SERPIN REGULATORY DOMAINS

人丝氨酸蛋白酶抑制剂调节域的结构-功能

• 批准号: 2177252
• 负责人: DANIEL J. KNAUER
• 金额: $ 21.67万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177252
• 关键词: alpha 1 antitrypsin; amyloid proteins; antithrombin III; chemical binding; enzyme activity; enzyme complex; extracellular matrix; heparin; protease inhibitor; protein sequence; protein structure function; receptor binding; site directed mutagenesis; synthetic peptide; tissue /cell culture; transfection

Human antithrombin III and human protease nexin I are members of the large serine protease inhibitor (SERPIN) family. The present studies are focused on the elucidation and genetic manipulation of two important regulatory domains in these SERPINS. The heparin binding domain mediates the activation of the anti-protease activities of both by heparin, and proposedly is responsible for extracellular matrix localization. The loss of heparin activation has been genetically linked to several thrombotic disorders involving ATIII. This domain has been biochemically and immunologically localized to the D helix and adjacent regions in both ATIII and PN1. A series of site-directed mutants, targeting lysine and arginine residues, will be constructed and expressed in baculovirus to determine the essential residues for heparin binding and activation, and to evaluate the role of heparin binding in extracellular matrix localization. The other region of interest is the clearance receptor binding domain. This domain has been biochemically localized to the carboxy terminal region of a closely related SERPIN, alpha-1-antitrypsin. Two approaches will be used to determine if the receptor binding domains of ATIII and PN1 are similarly located. The relationship between the ATIII and PN1 clearance receptors, and the clearance receptor for alpha- 1 -antitrypsin identified in liver cells, will also be investigated. In the first approach, site-directed deletion mutants in the carboxy terminal regions of ATIII and PN1 will be constructed and expressed in baculovirus to evaluate the potential role of these regions of ATIII and PNl clearance receptor binding. As a complementary approach, synthetic peptide libraries representing the entire amino acid sequences of ATIII and PN1, will be screened immunologically and by receptor binding assays to identify this site independently. The final goal of these studies is to begin a characterization and purification of the clearance receptor(s) that mediate the uptake and degradation of ATIII:Protease and PN1:Protease complexes.

人抗凝血酶III和人蛋白酶连接蛋白I是人抗凝血酶基因组的成员。 大丝氨酸蛋白酶抑制剂（SERPIN）家族。 目前的研究是 集中在阐明和遗传操作的两个重要的 在这些SERPINS中的调节域。 肝素结合结构域介导 肝素对两者的抗蛋白酶活性的活化，以及 脯氨酸负责细胞外基质定位。 的 肝素活化的丧失与几种基因相关， 涉及ATIII的血栓性疾病。 这个领域已经被生物化学 免疫学定位于D螺旋和邻近区域， ATIII和PN 1。 一系列定点突变体，靶向赖氨酸和 精氨酸残基，将在杆状病毒中构建和表达， 确定肝素结合和激活的必需残基，以及 评估肝素结合在细胞外基质中的作用， 本地化 另一个感兴趣的区域是清除受体 结合域 这个结构域在生物化学上定位于 与SERPIN密切相关的α-1-抗胰蛋白酶的羧基末端区域。 将使用两种方法来确定受体结合结构域是否 ATIII和PN 1的位置相似。 的关系 ATIII和PN 1清除受体，以及α- 还将研究在肝细胞中鉴定的1 -抗胰蛋白酶。 在 第一种方法，羧基中的定点缺失突变体， 将构建ATIII和PN 1的末端区域，并在 杆状病毒，以评估ATIII的这些区域的潜在作用， PN 1清除受体结合。 作为一种补充方法， 代表ATIII的整个氨基酸序列的肽文库 和PN 1，将通过免疫学和受体结合试验进行筛选 独立地识别这个网站。 这些研究的最终目标是 开始清除受体的表征和纯化 介导ATIII的摄取和降解：蛋白酶， PN 1：蛋白酶复合物。