STRUCTURAL STUDIES ON SERINE PROTEASES

丝氨酸蛋白酶的结构研究

• 批准号: 8172016
• 负责人: Enrico Di Cera
• 金额: $ 2.19万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172016
• 关键词: Address; Anticoagulants; Architecture; Binding; Blood; Blood Platelets; Blood coagulation; Complex; Computer Retrieval of Information on Scientific Projects Database; Enzyme Precursors; Enzymes; F2R gene; Factor Va; Funding; Grant; Inflammation; Institution; Molecular; PAWR gene; Pathway interactions; Peptide Hydrolases; Phase I Clinical Trials; Protein C; Proteinase-Activated Receptors; Prothrombin; Research; Research Personnel; Resources; Serine Protease; Source; Structure; Thrombin; Thrombomodulin; Thrombosis; United States National Institutes of Health; activated Protein C; base; clinically relevant; inhibitor/antagonist; meizothrombin; mutant; protein structure; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal focuses on biologically relevant serine proteases in complex with inhibitors, substrates and effectors for which we currently lack structural information. The focus is mainly on thrombin and activated protein C, two key proteases involved in the progression (thrombin) and inhibition (activated protein C) of blood coagulation. We plan to address a number of unsolved issues that will benefit tremendously from the availability of crystal structures. In the case of thrombin, we plan to crystallize the enzyme in complex with thrombomodulin and protein C to establish the molecular basis of its anticoagulant activity. We plan to crystallize the thrombin mutant W215A/E217A, that is entering Phase I clinical trials, in complex with the platelet receptor GPIb to identify the mode of binding underscoring this newly discovered interaction. We plan to crystallize prothrombin in order to obtain information on the architecture of the zymogen form of thrombin. We also plan to crystallize meizothrombin, the most relevant intermediate along the prothrombin pathway, in complex with GPIb and fragments of the protease activated receptors PAR1, PAR3 and PAR4, thrombomodulin and protein C. In the case of activated protein C, we plan to crystallize the enzyme in the E*, E and E:Na+ forms and in complex with fragments of PAR1 and coagulation factor Va. Structures of these proteins and their complexes will produce major advances in our understanding of the moelcular basis of thrombin procoagulant, prothrombotic and anticoagulant activities in the blood, and the way the enzyme is activated from prothrombin via meizothrombin. The structure of W215A/E217A bound to GPIb will reveal the molecular basis of the remarkable antithrombotic effect of this clinically relevant mutant. Structures of activated protein C will advance our understanding of the function of this enzyme in the control of thrombosis and inflammation.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该提案重点关注与抑制剂、底物和效应物复合的生物学相关丝氨酸蛋白酶，目前我们缺乏这些丝氨酸蛋白酶的结构信息。重点主要是凝血酶和活化蛋白 C，这两种关键蛋白酶参与血液凝固的进展（凝血酶）和抑制（活化蛋白 C）。 我们计划解决许多未解决的问题，这些问题将从晶体结构的可用性中受益匪浅。就凝血酶而言，我们计划将该酶与血栓调节蛋白和蛋白 C 复合物结晶，以建立其抗凝血活性的分子基础。我们计划结晶正在进入 I 期临床试验的凝血酶突变体 W215A/E217A，与血小板受体 GPIb 复合，以确定强调这种新发现的相互作用的结合模式。我们计划使凝血酶原结晶，以获得有关凝血酶酶原形式的结构的信息。我们还计划结晶甲凝血酶，它是凝血酶原途径中最相关的中间体，与 GPIb 和蛋白酶激活受体 PAR1、PAR3 和 PAR4 的片段、血栓调节蛋白和蛋白 C 形成复合物。对于激活的蛋白 C，我们计划结晶E*、E 和 E:Na+ 形式的酶与 PAR1 片段和凝血因子 Va 形成复合物。这些蛋白质及其复合物的结构将产生我们对血液中凝血酶促凝、促血栓和抗凝活性的分子基础，以及凝血酶原通过酶促凝血酶激活酶的方式的理解取得了重大进展。 W215A/E217A 与 GPIb 结合的结构将揭示该临床相关突变体显着抗血栓作用的分子基础。活化蛋白 C 的结构将促进我们对这种酶在控制血栓形成和炎症中的功能的理解。