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: 2177251
• 负责人: DANIEL J. KNAUER
• 金额: $ 20.46万
• 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/2177251
• 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