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

• 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
• 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

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 血管外凝血酶活性是导致动脉粥样硬化和支架内血栓形成的许多过程的基础。 再狭窄糖胺聚糖（GAG）、皮肤素和硫酸乙酰肝素可加速局部 凝血酶通过肝素辅因子II（HCII），但血管组织中不到0.1%的GAG是高亲和力肝素 通过与抗凝血酶（AT）紧密结合来催化凝血酶抑制。与临床和体内一致 研究表明，HCII可以预防动脉粥样硬化和再狭窄。AT机制已被 详细分析，但公认的HCII机制并不能解释其结合和动力学行为。不像 AT，HCII有一个分子内隔离的N-末端尾，被认为是通过GAG结合释放的，因此它可以 使凝血酶（T）参与米氏复合物。HCII β-GAG结合被认为触发凝血酶失活 通过HCII，而不是在凝血酶和丝氨酸蛋白酶抑制剂之间桥接GAG，这是AT的基础 机制小GAG也加速了HCII对凝血酶的失活，但AT对凝血酶的失活没有影响，这增强了凝血酶的活性。 假设GAG模板在HCII反应中不起作用。然而，将大小GAG结合到 HCII比凝血酶或AT弱得多，暗示在GAG处存在稀疏分布的HCII·GAG复合物 导致最大抑制的浓度。灭活率与T·GAG复合物形成平行， GAG显示模板动力学，与公认的机制不一致。我们的目标是定义是否/如何弱 HCII·GAG结合可驱动催化。在游离HCII和T·HCII的米氏复合体结构中有70%的尾部 是未解决的，和HCII β-GAG结构是实验上无法实现的。我们将识别尾体接触， 使循环HCII保持在低反应性状态，并在GAG与氢结合后确定结构变化， 氘交换（HDX）MS和圆二色性，这使得条件是禁止在 晶体学（目标1）。我们将首次确定大GAG在T细胞HCII米氏膜上的结合位置。 复合物，并确定潜在的结合口袋的小GAG在复杂的界面（目的2）。我们将 定量GAG与HCII和凝血酶结合的速率步骤，并阐明米氏动力学途径 和通过停流动力学、平衡结合、凝血酶灭活、HCII形成共价复合物 诱变和FRET（目的3）。我们将测试以下假设：a）HCII分子内尾体和C片层- 铰链相互作用将循环HCII维持在低反应性构象，可活化至抑制状态; B） T·HCII界面与GAGs的接触使米氏络合物稳定在开-闭平衡状态 反映了HCII尾的外切位点I结合;和c）共价结合中凝血酶易位的程度 复合物可以特异于HCII-凝血酶对。预期结果将阐明GAG的机制 催化，并表征没有结构可用的共价复合物构象。很长的- 长期目标是将机制信息应用于设计基于HCII和T·HCII特异性GAG的治疗。 这一发现对于开发新的动脉粥样硬化和再狭窄的治疗方法具有重要意义。