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Mechanisms of Glycosaminoglycan-Catalyzed Protease Inactivation by Serpins

丝氨酸蛋白酶抑制剂 (Serpin) 糖胺聚糖催化的蛋白酶灭活机制

基本信息

批准号：
9335436
负责人：
INGRID M VERHAMME
金额：
$ 39.38万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9335436
关键词：
Active Sites Acylation Affinity Agreement Antithrombins Atherosclerosis Behavior Binding Binding Sites Blood Vessels Catalysis Chemicals Circular Dichroism Clinical Clinical Research Complex Coupled Crystallization Crystallography Data Dermatan Sulfate Deuterium Development Disease Docking Endothelium Enzymes Equilibrium Fluorescence Resonance Energy Transfer Future Generations Glycosaminoglycans Goals Hemorrhage Heparin Heparin Binding Heparin Cofactor II Heparitin Sulfate Huntington Disease Hydrogen Institutes Kinetics Ligands Link Maryland Measures Medical Research Modeling Molecular Molecular Conformation Mutagenesis N-terminal Outcome Pathology Pathway interactions Patients Peptide Hydrolases Pharmaceutical Preparations Plant Roots Plasma Play Positioning Attribute Process Property Proteins Reaction Research Personnel Risk Role Serpins Site Stents Structure System Tail Testing Therapeutic Thrombin Time Tissues base design experience implantation in vivo inhibitor/antagonist mutant novel novel strategies novel therapeutics restenosis therapy design

项目摘要

SUMMARY Extravascular thrombin activity is at the basis of many processes that cause atherosclerosis and in-stent restenosis. The glycosaminoglycans (GAGs) dermatan and heparan sulfate accelerate inactivation of localized thrombin by heparin cofactor II (HCII), but less than 0.1% of the GAGs in vascular tissue is high-affinity heparin that catalyzes thrombin inhibition by tight binding to antithrombin (AT). In agreement with clinical and in-vivo studies, we propose that HCII protects against atherosclerosis and restenosis. The AT mechanism has been analyzed in detail, but the accepted HCII mechanism does not explain its binding and kinetic behavior. Unlike AT, HCII has an intramolecularly sequestered N-terminal tail, thought to be released by GAG binding so it can engage thrombin (T) in the Michaelis complex. HCIIGAG binding is considered to trigger thrombin inactivation by HCII, rather than GAG bridging between thrombin and the serpin, which is at the basis of the AT mechanism. Small GAGs also accelerate thrombin inactivation by HCII but not by AT, which strengthened the assumption that GAG templates play no role in HCII reactions. However, binding of large and small GAGs to HCII is much weaker than to thrombin or AT, implicating a sparsely populated HCII·GAG complex at GAG concentrations that cause maximal inhibition. Inactivation rates parallel T·GAG complex formation, and long GAGs show template kinetics, in disagreement with the accepted mechanism. We aim to define if/how weak HCII·GAG binding can drive catalysis. In the free HCII and T·HCII Michaelis complex structures 70% of the tail is unresolved, and HCIIGAG structures are experimentally unattainable. We will identify tail-body contacts that keep circulating HCII in a low-reactive state, and define structural changes upon GAG binding by hydrogen- deuterium exchange (HDX) MS and circular dichroism, which allow conditions that are prohibitive in crystallography (Aim 1). We will identify for the first time where large GAGs bind across the THCII Michaelis complex, and identify potential binding pockets for small GAGs at the complex interface (Aim 2). We will quantitate the rate steps of GAG binding to HCII and thrombin, and elucidate the kinetic pathways of Michaelis and covalent complex formation by stopped-flow kinetics, equilibrium binding, thrombin inactivation, HCII mutagenesis and FRET (Aim 3). We will test the hypotheses that a) HCII intramolecular tail-body and C sheet- hinge interactions maintain circulating HCII in a low-reactive conformation, activatable to the inhibitory state; b) that T·HCII interface contacts with GAGs stabilize the Michaelis complex with an open-closed equilibrium reflecting exosite I binding of the HCII tail; and c) that the extent of thrombin translocation in the covalent complex may be specific for the HCII-thrombin pair. The expected outcomes will clarify the mechanism of GAG catalysis, and characterize the covalent complex conformation for which no structure is available. The long- term goal is to apply mechanistic information to designing therapies based on HCII and T·HCII-specific GAGs. The findings will be significant for developing novel management of atherosclerosis and restenosis.

总结