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.

摘要 血管外凝血酶活性是导致动脉粥样硬化和支架内许多过程的基础。 血管再狭窄。糖胺聚糖(GAGS)、皮肤素和硫酸肝素加速局部失活 凝血酶由肝素辅因子II(HCII)引起，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。与临床和体内的情况一致 研究表明，HCII对动脉粥样硬化和再狭窄有保护作用。AT机制已经被 详细分析，但公认的HCII机制不能解释其结合和动力学行为。不像 在AT，HCII有一个分子内隔离的N末端尾巴，被认为是通过GAG结合释放的，所以它可以 使凝血酶(T)进入米氏复合体。HCIIGAG结合被认为可触发凝血酶失活 通过HCII，而不是凝血酶和丝氨酸之间的缝隙桥接，这是AT的基础 机制。小插管也可加速HCII的凝血酶失活，但AT不能，后者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，大小恶作剧的绑定到 HCII对凝血酶或AT的作用比凝血酶或AT弱得多，这意味着在Gag上存在一个稀疏的HCII·Gag复合体 造成最大抑制的浓度。失活速率平行于T·Gag复合体的形成，且长 GAG表现出模板动力学，与公认的机制不一致。我们的目标是定义是否/如何疲软 HCII·Gag结合可驱动催化作用。在游离的HCII和T·HCII米氏复合体结构中70%的尾巴 是悬而未决的，而且HCIIGag结构在实验上是无法获得的。我们将识别尾部身体接触者 保持循环中的HCII处于低反应状态，并定义GAG与氢结合时的结构变化- 氢交换(HDX)MS和圆二色谱，它们允许在 结晶学(目标1)。我们将首次确定THCII Michaelis上的大插曲绑定位置 复杂的，并确定复杂界面上的小插嘴的潜在捆绑口袋(目标2)。我们会 定量测定Gag与HCII和凝血酶结合的速率步长，阐明Michaelis的动力学途径 以及通过停流动力学、平衡结合、凝血酶失活、HCII形成共价络合物 诱变和FRET(目标3)。我们将检验以下假设：a)HCII分子内尾体和C片断- 铰链相互作用维持循环中的HCII处于低活性构象，可激活到抑制状态；b) T·HCII界面与GaG的接触使Michaelis络合物处于开闭平衡状态 反映了Exosite I与HCII尾部的结合；以及c)凝血酶在共价键上的移位程度 复合体可能是HCII-凝血酶对所特有的。预期的结果将阐明GAG的作用机制 催化作用，并表征没有结构的共价络合物构象。长的- 学期目标是将机械信息应用于基于HCII和T·HCII特异性GAG的治疗设计。 这一发现将对开发动脉粥样硬化和再狭窄的新治疗方法具有重要意义。