Proteinase Allostery and the Regulation of Blood Coagulation
蛋白酶变构和凝血调节
基本信息
- 批准号:8463606
- 负责人:
- 金额:$ 39.87万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-07-15 至 2015-04-30
- 项目状态:已结题
- 来源:
- 关键词:AccountingActive SitesAddressAffectAnticoagulantsAnticoagulationBindingBloodBlood ClotBlood coagulationCalorimetryCoagulation ProcessCoenzymesColorComplementCrystallographyDevelopmentDiseaseDistantEnzyme PrecursorsEnzymesEquilibriumEyeFaceFibrinFibrinogenFibrinolysisGoalsHeartHeatingHemostatic functionKineticsKnowledgeLifeLigand BindingLigandsLigationLightMeasurementMethodologyN-terminalPathway interactionsPeptide HydrolasesPhysiologicalPlatelet ActivationPlayProcessProductionPropertyProteinsRNAReactionRegulationResearchResolutionRoleSchemeSeriesSerine ProteaseSiteSpecificityStructureTestingTherapeuticThermodynamicsThrombinThrombomodulinThrombusTitrationsTranslatingUrsidae FamilyVariantVascular Diseasesaptamerbasehuman diseaseinsightmeizothrombinnovelnovel strategiesphrasespreventresponsestructural biologytherapeutic target
项目摘要
DESCRIPTION (provided by applicant): Thrombin, the terminal serine proteinase of the blood coagulation cascade, plays a pivotal role in thrombus formation as well as its regulation. These multiple and sometimes opposing roles of thrombin in coagulation are influenced by two exosites (ABE1 and ABE2) on opposite faces of the enzyme and cofactors or ligands that bind to these or other sites to allosterically regulate the proteinase. Major gaps and inconsistencies remain in the mechanistic understanding of how thrombin allostery is achieved and translates into regulated function. We broach the problem with the unexpected finding that thrombin can readily and reversibly interconvert along a continuum of zymogen-like and proteinase-like states depending on the complement of ligands bound to the enzyme. We propose that these interconversions lie at the heart of thrombin allostery. Our studies center on the finding that fragment 1.2 (F12), the authentic protein ligand for ABE2, thermodynamically favors zymogen- like forms. We will now employ titration calorimetry to establish the thermodynamic basis by which thrombomodulin (TM), which binds to ABE1, selectively stabilizes and favors proteinase-like forms to oppose the effects of F12. We also find that meizothrombin (mIIa), produced as an intermediate during thrombin formation, is particularly zymogen-like because of covalent linkage between F12 and the proteinase domain. We will employ a stopped-flow kinetic approach to examine the distribution of mIIa between zymogen-like and proteinase-like forms. Although the F2 region within F12 is expected to contact ABE2, we now propose a novel function for the Ca2+ stabilized structure of the putatively distant F1 in enforcing the ability of F12 to favor zymogen-like forms. Based on these mechanistic studies, we will study the regulation of thrombin function by opposing effects of ABE1 binding by different ligands and F12 binding to ABE2, with an eye to explaining the apparent differences in specificity ascribed to mIIa and various anticoagulant thrombins. We hypothesize that their altered specificity lies in their significantly zymogen-like character that can be variably rescued by the binding of ligands and substrates. These concepts also provide the appropriate framework for the development of novel aptamer probes that can modulate the distribution of thrombin between zymogen-like and proteinase- like states and thereby regulate its specificity with therapeutic potential. Finally, based on our recent successful entry into the structural biology arena, we propose the long term goal of solving a series of novel structures to establish the structural basis for the zymogenizing function of F12. Our strategies bring fresh and unifying concepts to the important problem of thrombin allostery. We anticipate our findings to shed new light on the mechanisms at play in regulating thrombin function in normal hemostasis and in disease states. Our findings have the potential to reveal new strategies for therapeutic targeting of this enzyme in thrombotic and vascular disease.
描述(申请人提供):凝血酶,凝血级联的末端丝氨酸蛋白酶,在血栓形成及其调节中起着关键作用。凝血酶在凝血中的这些多重的、有时是相反的作用受到酶相反面上的两个外切酶(ABE1和ABE2)以及结合到这些或其他位置以变构调节蛋白酶的辅因子或配体的影响。在凝血酶变构是如何实现并转化为调节功能的机械性理解中仍然存在主要的差距和不一致。我们提出了一个出人意料的发现,凝血酶可以很容易地和可逆地沿着酶原和蛋白酶样状态的连续体相互转换,这取决于与酶结合的配体的补充。我们认为这些相互转换是凝血酶变构的核心。我们的研究集中在发现片段1.2(F12),ABE2的真正蛋白质配体,热力学上有利于酶原样形式。我们现在将使用滴定量热法来建立热力学基础,通过该基础,与ABE1结合的血栓调节蛋白(TM)选择性地稳定和有利于类似于酶的形式,以对抗F12的影响。我们还发现,在凝血酶形成过程中作为中间体产生的甲硫凝血酶(MIIA),由于F12和蛋白酶域之间的共价连接,具有特别的酶原样活性。我们将使用停流动力学方法来检查MIIA在酶原样和蛋白酶样形式之间的分布。虽然F12内的F2区预计会与ABE2接触,但我们现在提出了一种新的功能,即距离较远的F1的钙稳定结构在增强F12支持酶原样形式的能力方面。在这些机制研究的基础上,我们将通过不同配体与ABE1结合和F12与ABE2结合的相反作用来研究凝血酶功能的调节,以期解释MIIA和各种抗凝血酶在特异性上的明显差异。我们假设它们改变的特异性在于它们显著的酶原样特性,这种特性可以通过配体和底物的结合而可变地挽救。这些概念也为新型适体探针的开发提供了合适的框架,该探针可以调节凝血酶在酶原样态和蛋白酶样状态之间的分布,从而调节其特异性和治疗潜力。最后,在我们最近成功进入结构生物学领域的基础上,我们提出了解决一系列新结构的长期目标,为F12的产酶功能奠定结构基础。我们的策略为凝血酶变构这一重要问题带来了新鲜而统一的概念。我们期待我们的发现能为正常止血和疾病状态下调节凝血酶功能的机制提供新的线索。我们的发现有可能揭示这种酶在血栓和血管疾病中的治疗靶向的新策略。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Sriram Krishnaswamy其他文献
Sriram Krishnaswamy的其他文献
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{{ truncateString('Sriram Krishnaswamy', 18)}}的其他基金
Hemostasis and Thrombosis: Chemistry, Biology and Physiology
止血和血栓形成:化学、生物学和生理学
- 批准号:
9982398 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
Hemostasis and Thrombosis: Chemistry, Biology and Physiology
止血和血栓形成:化学、生物学和生理学
- 批准号:
9769852 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
Hemostasis and Thrombosis: Chemistry, Biology and Physiology
止血和血栓形成:化学、生物学和生理学
- 批准号:
10439604 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
Hemostasis and Thrombosis: Chemistry, Biology and Physiology
止血和血栓形成:化学、生物学和生理学
- 批准号:
10175000 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
Core B: Molecular Biology, Protein Expression and Structural Biology
核心 B:分子生物学、蛋白质表达和结构生物学
- 批准号:
10175002 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
Core B: Molecular Biology, Protein Expression and Structural Biology
核心 B:分子生物学、蛋白质表达和结构生物学
- 批准号:
10439607 - 财政年份:2018
- 资助金额:
$ 39.87万 - 项目类别:
2012 Hemostasis Gordon Research Conference and Gordon Research Seminar
2012止血戈登研究会议暨戈登研究研讨会
- 批准号:
8388646 - 财政年份:2012
- 资助金额:
$ 39.87万 - 项目类别:
Proteinase Allostery and the Regulation of Blood Coagulation
蛋白酶变构和凝血调节
- 批准号:
8299448 - 财政年份:2011
- 资助金额:
$ 39.87万 - 项目类别:
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