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.

概括血管外凝血酶活性是导致动脉粥样硬化和支架内发生的许多过程的基础糖胺聚糖（GAG）皮肤素和硫酸乙酰肝素加速局部失活。肝素辅因子 II (HCII) 可与凝血酶结合，但血管组织中只有不到 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。详细分析，但公认的 HCII 机制并没有解释其结合和动力学行为。 AT、HCII 具有分子内隔离的 N 末端尾部，被认为是通过 GAG 结合释放的，因此它可以使凝血酶 (T) 参与米氏复合体 (HCII) GAG 结合被认为会触发凝血酶失活。通过 HCII，而不是凝血酶和丝氨酸蛋白酶抑制剂之间的 GAG 桥接，后者是 AT 的基础小 GAG 还可以加速 HCII 而非 AT 的凝血酶失活，从而加强了凝血酶的失活。假设 GAG 模板在 HCII 反应中不起任何作用，但是，大和小 GAG 的结合。 HCII 比凝血酶或 AT 弱得多，这表明 GAG 上存在稀疏的 HCII·GAG 复合物引起最大抑制的浓度与 T·GAG 复合物形成平行，且时间较长。 GAG 显示模板动力学，与公认的机制不同，我们的目标是定义是否/有多弱。 HCII·GAG结合可以驱动游离HCII和T·HCII米氏复合物结构中70%的尾部的催化作用。尚未解决，并且 HCII GAG 结构在实验上无法实现，我们将确定尾体接触。保持循环 HCII 处于低反应状态，并定义 GAG 与氢结合后的结构变化氘交换 (HDX) MS 和圆二色性，允许在我们将首次确定大 GAG 在 T HCII Michaelis 上的结合位置。复杂，并确定复杂界面上小 GAG 的潜在结合袋（目标 2）。定量 GAG 与 HCII 和凝血酶结合的速率步长，并阐明 Michaelis 的动力学途径和通过停流动力学、平衡结合、凝血酶失活、HCII 形成共价复合物诱变和 FRET（目标 3）我们将测试以下假设：a) HCII 分子内尾体和 C 片-。铰链相互作用将循环 HCII 维持在低反应性构象，可激活至抑制状态 b) T·HCII 界面与 GAG 的接触使 Michaelis 复合体保持开闭平衡反映 HCII 尾部的外位点 I 结合；以及 c) 共价凝血酶易位的程度复合物可能对 HCII-凝血酶对具有特异性，预期结果将阐明 GAG 的机制。催化，并表征没有可用结构的共价复合构象。术语目标是应用机制信息来设计基于 HCII 和 T·HCII 特异性 GAG 的疗法。这些发现对于开发动脉粥样硬化和再狭窄的新治疗方法具有重要意义。