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.

饮食维生素K被γ-谷氨酰羧化酶用于将Glus簇转化为γ-谷氨酰羧化酶。 羧化Glus（Glas）在身体的几乎所有组织中的维生素K依赖性（VKD）蛋白中。第一 鉴定的VKD蛋白是凝血因子;然而，非止血VKD蛋白的鉴定 已经揭示了其他的作用，例如钙化的调节。羧基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，和单个γ-谷氨酰羧化酶 修饰所有VKD蛋白。羧化酶的自然突变导致两种疾病：维生素K 凝血因子缺乏症1，与严重出血缺陷和弹性假黄瘤样 （类PXE）与轻度出血但软组织过度钙化相关。这些羧化酶 突变导致PXE样是以前未知的。我们研究了存在于PXE中的两种羧化酶突变- 像个病人VKD凝血因子（因子IX）和抑制钙化的VKD蛋白（Matrix Gla）的分析 蛋白质）显示部分羧化，由于羧化酶持续合成能力的缺陷。 持续加工性是指羧化酶保持与VKD蛋白结合，直到多个Glu残基被去除。 羧化的我们开发了一种新的检测方法来监测进行性羧化，并发现野生型 羧化酶保护VKD蛋白，即限制其他VKD蛋白进入活性位点，直到VKD蛋白被羧化。 蛋白质被广泛羧化。相反，PXE样突变体允许VKD混杂进入， 蛋白底物进入活性位点，导致产生部分羧化的VKD蛋白。我们 研究还揭示了单个野生型羧化酶同时结合两种VKD蛋白。如组织 表达多种被认为具有广泛不同亲和力的VKD蛋白质，所有VKD如何完全羧化 蛋白质是一个开放的问题。 我们的长期目标是了解部分VKD蛋白羧化如何影响人体生理学。 中心问题是是否使用抗凝剂华法林治疗，限制VKD蛋白 羧化，产生部分羧化的蛋白质，以及华法林是否引起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， PXE样羧化酶的部分羧化如何影响VKD蛋白功能（目的1），确定是否 VKD蛋白的羧化受到不同VKD蛋白的存在的影响（目的2），和 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（目的3）。这些研究的结果将提供第一个见解，将蛋白质的程度 羧化对不同表型结果的影响。