Impact of gamma-glutamyl carboxylase processivity on vitamin K-dependent protein modification and function in human health and disease

γ-谷氨酰羧化酶持续合成能力对维生素 K 依赖性蛋白质修饰和人类健康和疾病功能的影响

• 批准号: 10455606
• 负责人: KATHLEEN Lucile BERKNER
• 金额: $ 52.67万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10455606
• 关键词: Active Sites; Affinity; Animals; Anticoagulant therapy; Anticoagulants; Binding; Binding Proteins; Biological Assay; Blood; Blood Coagulation Factor; Blood coagulation; CRISPR/Cas technology; Calcium Binding; Catalysis; Catalytic Domain; Cells; Coagulation Process; Data; Defect; Disease; Factor IX; Factor X; Genetic Diseases; Glutamic Acid; Goals; Health; Hemorrhage; Hemostatic Agents; Human; Impairment; Individual; Link; Liver; Mammalian Cell; Mass Spectrum Analysis; Mediating; Metabolism; Modification; Monitor; Mus; Mutation; Osteogenesis; Outcome; Oxidoreductase; Patients; Persons; Pharmaceutical Preparations; Phenotype; Physiology; Play; Post-Translational Protein Processing; Production; Proteins; Prothrombin; Pseudoxanthoma Elasticum; Regulation; Role; Skin; Testing; Time; Tissues; Vitamin K; Waran; Warfarin; Work; base; calcification; carboxylate; carboxylation; cofactor; dietary; gamma-glutamyl carboxylase; in vivo; innovation; insight; matrix Gla protein; mutant; novel; protein function; reduced vitamin K; response; soft tissue; virtual

Dietary vitamin K is used by the gamma-glutamyl carboxylase to convert clusters of Glus to gamma- carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins in virtually all tissues of the body. The first VKD proteins identified were coagulation factors; however, the identification of nonhemostatic VKD proteins has revealed additional roles, e.g. the regulation of calcification. Carboxylation activates VKD proteins by generating a calcium-binding module required for their function, and a single gamma-glutamyl carboxylase modifies all VKD proteins. Naturally occurring mutations in the carboxylase cause two diseases: vitamin K clotting factor deficiency 1 that is associated with severe bleeding defects, and pseudoxanthoma elasticum-like (PXE-like) that is associated with mild bleeding but excessive soft tissue calcification. How these carboxylase mutations cause PXE-like was previously unknown. We studied two carboxylase mutations present in a PXE- like patient. Analysis of a VKD clotting factor (factor IX) and a VKD protein that inhibits calcification (Matrix Gla Protein) revealed partial carboxylation due to a defect in carboxylase processivity. Processivity refers to the carboxylase remaining bound to a VKD protein until the multiple Glu residues are carboxylated. We developed a novel assay to monitor processive carboxylation, and found that the wild type carboxylase shields the VKD protein, i.e. limiting access of other VKD proteins into the active site until the VKD protein is extensively carboxylated. In contrast, the PXE-like mutants allowed promiscuous access of VKD protein substrates into the active site, resulting in the production of partially carboxylated VKD proteins. Our studies also revealed that a single wild type carboxylase binds two VKD proteins at the same time. As tissues express multiple VKD proteins thought to have widely different affinities, how full carboxylation of all VKD proteins is achieved is an open question. Our long-term goal is to understand how partial VKD protein carboxylation impacts human physiology. Central questions are whether treatment with the anticoagulant warfarin, which limits VKD protein carboxylation, generates partially carboxylated proteins, and whether warfarin evokes PXE-like phenotypes. We will approach these questions using a combination of protein mapping and activity assays to determine how partial carboxylation by PXE-like carboxylases impacts VKD protein function (Aim 1), determine whether the carboxylation of a VKD protein is impacted by the presence of a different VKD protein (Aim 2), and examine the consequence of warfarin therapy and a PXE-like mutant on VKD protein carboxylation and function in vivo (Aim 3). Results from these studies will provide the first insights that link the extent of protein carboxylation to different phenotypic outcomes.

γ-谷氨酰羧化酶使用饮食维生素K将glus簇转换为γ- 维生素K依赖性（VKD）蛋白几乎所有组织中的维生素K依赖性（VKD）中的羧化glus（GLA）。第一个 鉴定出的VKD蛋白是凝血因子。但是，鉴定非尖端VKD蛋白 揭示了其他角色，例如钙化调节。羧化激活VKD蛋白 产生其功能所需的钙结合模块，并产生一个γ-谷氨酰羧化酶 修饰所有VKD蛋白。羧化酶中天然发生的突变引起两种疾病：维生素K 凝血因子缺乏1与严重的出血缺陷以及弹性瘤样相关 （类似于PXE），与轻度出血但软组织钙化有关。这些羧化酶如何 突变引起类似PXE的样子以前未知。我们研究了PXE-中存在的两个羧化酶突变 喜欢病人。 VKD凝结因子（因子IX）和抑制钙化的VKD蛋白的分析（矩阵GLA 蛋白质）揭示了由于羧化酶加工性缺陷而导致的部分羧化。 加工性是指羧酸酶保持与VKD蛋白结合，直到多个GLU残基为 羧化。我们开发了一种新颖的测定方法来监测加法羧化，并发现野生型 羧化酶屏蔽VKD蛋白，即限制其他VKD蛋白进入活性位点直至VKD 蛋白质是广泛的羧化。相比之下，PXE样突变体允许VKD滥交 蛋白质底物进入活性位点，从而产生部分羧化VKD蛋白。我们的 研究还表明，一种野生型羧化酶同时结合了两种VKD蛋白。作为组织 表达多种VKD蛋白被认为具有截然不同的亲和力，所有VKD的羧化如何 实现蛋白质是一个悬而未决的问题。 我们的长期目标是了解部分VKD蛋白羧化如何影响人类生理。 中心问题是抗凝华法林的治疗是否限制了VKD蛋白 羧化，产生部分羧化蛋白，以及华法林是否唤起了PXE样表型。 我们将使用蛋白质映射和活动测定的结合来解决这些问题，以确定 PXE样羧酸酶的部分羧化如何影响VKD蛋白功能（AIM 1），确定是否是否 VKD蛋白的羧化受到不同VKD蛋白（AIM 2）的影响，并且 检查华法林治疗的后果和对VKD蛋白羧化和PXE样突变体的结果 在体内功能（AIM 3）。这些研究的结果将提供第一批见解，以联系蛋白质的程度 羧化对不同的表型结果。