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-谷氨酰羧化酶利用膳食维生素 K 将 Glus 簇转化为 γ- 身体几乎所有组织中维生素 K 依赖性 (VKD) 蛋白中的羧化谷氨酸 (Glas) 均存在。第一个 鉴定出的 VKD 蛋白是凝血因子；然而，非止血性 VKD 蛋白的鉴定 还透露了其他角色，例如钙化的调节。羧化通过以下方式激活 VKD 蛋白 产生其功能所需的钙结合模块，以及单个γ-谷氨酰羧化酶 修饰所有 VKD 蛋白。羧化酶自然发生的突变会导致两种疾病： 维生素 K 凝血因子缺乏 1 与严重出血缺陷和弹性假黄瘤样相关 （PXE 样）与轻度出血但软组织钙化过度相关。这些羧化酶如何 导致 PXE 样突变的原因以前是未知的。我们研究了 PXE- 中存在的两种羧化酶突变 像病人一样。分析 VKD 凝血因子（因子 IX）和抑制钙化的 VKD 蛋白 (Matrix Gla 由于羧化酶持续合成能力的缺陷，蛋白质）显示出部分羧化。 持续合成能力是指羧化酶保持与 VKD 蛋白结合，直到多个 Glu 残基被结合。 羧化的。我们开发了一种新的检测方法来监测持续的羧化作用，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，直到 VKD 蛋白质被广泛羧化。相比之下，类 PXE 突变体允许 VKD 的混杂访问 蛋白质底物进入活性位点，从而产生部分羧化的 VKD 蛋白质。我们的 研究还表明，单个野生型羧化酶可同时结合两个 VKD 蛋白。作为纸巾 表达多种被认为具有广泛不同亲和力的 VKD 蛋白，所有 VKD 的完全羧化如何 蛋白质的实现是一个悬而未决的问题。 我们的长期目标是了解 VKD 蛋白部分羧化如何影响人体生理学。 核心问题是抗凝剂华法林治疗是否会限制 VKD 蛋白 羧化、产生部分羧化的蛋白质，以及华法林是否会引起 PXE 样表型。 我们将结合蛋白质图谱和活性测定来解决这些问题，以确定 PXE 样羧化酶的部分羧化如何影响 VKD 蛋白功能（目标 1），确定是否 VKD 蛋白的羧化受到不同 VKD 蛋白存在的影响（目标 2），并且 检查华法林治疗和 PXE 样突变体对 VKD 蛋白羧化的影响， 体内功能（目标 3）。这些研究的结果将提供将蛋白质的程度联系起来的第一个见解 羧化作用导致不同的表型结果。