STRUCTURAL AND FUNCTIONAL BASIS OF THE VITAMIN K CYCLE
维生素 K 循环的结构和功能基础
基本信息
- 批准号:9047306
- 负责人:
- 金额:$ 38.13万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2014
- 资助国家:美国
- 起止时间:2014-05-15 至 2019-04-30
- 项目状态:已结题
- 来源:
- 关键词:Active SitesAddressAnticoagulantsBacteriaBindingBiochemicalBiochemistryBiological AssayBlood Coagulation DisordersBlood Coagulation FactorBlood coagulationCalciumCatalysisCellsCellular biologyComplexCoupledCultured CellsCysteineDataDeep Vein ThrombosisDiseaseElectron TransportElectronsEnzymesEpoxy CompoundsGlutamatesGoalsHealthHomologous GeneHumanHydrophobicityHydroquinonesIn VitroInjuryKnowledgeLaboratoriesMammalian CellMediatingMembraneMembrane ProteinsMethodsModificationMolecular ConformationMotionMutationMycobacterium tuberculosisMyocardial InfarctionNatural regenerationOralOrganismOxidoreductasePharmaceutical PreparationsPilot ProjectsProcessProductionProteinsPublic HealthPulmonary EmbolismQuinonesReactionRecombinantsResearchResistanceResolutionSiteStrokeStructureTestingTherapeutic UsesThrombosisVitamin KWarfarinanalogbasecarboxylatecarboxylationdesigndisulfide bondimprovedin vitro activityinhibitor/antagonistinsightnovelnovel therapeuticspreventprotein foldingreconstitutionreduced vitamin Kresearch studystructural biologyvitamin K epoxide reductasevitamin K1 oxide
项目摘要
DESCRIPTION (provided by applicant): Vitamin K epoxide reductase (VKOR) is a membrane-embedded enzyme and the target of warfarin, the most commonly used oral anticoagulant. Due to limited structural knowledge, there is a fundamental gap in understanding the mechanism of VKOR catalysis and warfarin inhibition. Filling this knowledge gap will provide the basis to produce clotting factors for therapeutic use and to design novel drugs regulating blood clotting. Our long-term goal is to understand the entire vitamin K cycle that supports blood coagulation. The cycle begins with the carboxylation of designated glutamate residues in coagulation factors, a modification required for calcium-dependent activation at sites of injury. The carboxylation reaction consumes vitamin K hydroquinone, which is regenerated by VKOR's reduction of vitamin K epoxide. The reducing equivalents may come from VKOR's partners that transfer electrons to VKOR to maintain its activity. The objective of this application is to use structural biology, biochemistry, and cell biology approaches to elucidate the mechanisms of VKOR catalysis, partner interaction, and warfarin inhibition. Our hypotheses are 1) VKOR uses distinctive catalytic mechanisms for vitamin K reduction and electron transfer, 2) the VKOR-partner interaction is important to support blood coagulation, and 3) unique features at the active site of eukaryotic VKORs confer the epoxide-reductase activity and the warfarin sensitivity. These hypotheses are based on preliminary data produced in the applicant's laboratory. 1) We have determined a new 2.8Ao structure of a VKOR homolog. The structure reveals a hydrophobic pocket at the active site that may increase the reactivity of a catalytic cysteine, and electron transfer is facilitated by the unwinding motion of a designated helix. 2) Using a cell-based assay, we have identified partner proteins that can stimulate the production of carboxylated coagulation factor. 3) We have determined a low-resolution structure of a VKOR homolog in complex with a warfarin analog. In addition, our biochemical experiments have provided a new explanation for warfarin resistance. The hypotheses will be tested by three specific aims. 1) We will determine crystal structures of the VKOR homolog captured in different conformational states during the electron transfer. The structural knowledge will guide our functional studies in human VKOR to elucidate the mechanisms controlling its catalytic efficiency and electron flux. 2) We will assess the ability of partner proteins to support the in vvo and in vitro activity of human VKOR. 3) We will determine the structures of eukaryotic VKORs, and the structures of VKORs in complex with warfarin analogs. We will also elucidate the mechanism of warfarin inhibition and resistance by biochemical studies and by other physical methods to probe the drug binding.
描述(由申请方提供):维生素K环氧化物还原酶(VKOR)是一种膜包埋酶,是最常用的口服抗凝剂华法林的靶点。由于有限的结构知识,在理解VKOR催化和华法林抑制的机制方面存在根本性的差距。填补这一知识空白将为生产用于治疗的凝血因子和设计调节凝血的新药提供基础。我们的长期目标是了解支持血液凝固的整个维生素K循环。该循环始于凝血因子中指定谷氨酸残基的羧化,这是损伤部位钙依赖性活化所需的修饰。羧化反应消耗维生素K对苯二酚,其通过VKOR还原维生素K环氧化物而再生。还原当量可能来自VKOR的伙伴,它们将电子转移到VKOR以维持其活性。本申请的目的是使用结构生物学、生物化学和细胞生物学方法来阐明VKOR催化、伴侣相互作用和华法林抑制的机制。我们的假设是:1)VKOR使用独特的催化机制进行维生素K还原和电子转移,2)VKOR-伴侣相互作用对支持血液凝固很重要,3)真核VKOR活性位点的独特特征赋予环氧化物还原酶活性和华法林敏感性。这些假设基于申请人实验室产生的初步数据。1)我们确定了一个新的2.8Ao结构的VKOR同系物。该结构揭示了在活性位点处的疏水口袋,其可以增加催化半胱氨酸的反应性,并且通过指定螺旋的解旋运动促进电子转移。2)使用基于细胞的测定,我们已经确定了伴侣蛋白,可以刺激生产的羧化凝血因子。3)我们已经确定了一个低分辨率结构的VKOR同系物在复杂的华法林类似物。此外,我们的生化实验为华法林耐药性提供了新的解释。这些假设将通过三个具体目标进行检验。1)我们将确定在电子转移过程中在不同构象状态下捕获的VKOR同系物的晶体结构。结构知识将指导我们在人类VKOR的功能研究,以阐明其催化效率和电子通量的控制机制。2)我们将评估伴侣蛋白支持人VKOR的体内和体外活性的能力。3)我们将确定真核VKORs的结构,以及与华法林类似物复合的VKORs的结构。我们还将通过生物化学研究和其他物理方法来探索药物结合,阐明华法林抑制和耐药的机制。
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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Weikai Li其他文献
Weikai Li的其他文献
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定义四跨膜蛋白 CD53 在调节 B 细胞运输中的伙伴相互作用网络
- 批准号:
10660927 - 财政年份:2022
- 资助金额:
$ 38.13万 - 项目类别:
Defining the partner interaction network of the tetraspanin CD53 in regulating B cell trafficking
定义四跨膜蛋白 CD53 在调节 B 细胞运输中的伙伴相互作用网络
- 批准号:
10364239 - 财政年份:2022
- 资助金额:
$ 38.13万 - 项目类别:
Structural and Functional Basis of the Vitamin K Cycle
维生素 K 循环的结构和功能基础
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10163694 - 财政年份:2014
- 资助金额:
$ 38.13万 - 项目类别:
Structural and Functional Basis of the Vitamin K Cycle
维生素 K 循环的结构和功能基础
- 批准号:
10400676 - 财政年份:2014
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$ 38.13万 - 项目类别:
Structural and Biochemical Basis of the Vitamin K cycle
维生素 K 循环的结构和生化基础
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8166297 - 财政年份:2011
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$ 38.13万 - 项目类别:
Structural and Biochemical Basis of the Vitamin K cycle
维生素 K 循环的结构和生化基础
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8228021 - 财政年份:2011
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$ 38.13万 - 项目类别:
Structural and Biochemical Basis of the Vitamin K cycle
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$ 38.13万 - 项目类别:
Structural and Biochemical Basis of the Vitamin K cycle
维生素 K 循环的结构和生化基础
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