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: 10315102
• 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/10315102
• 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; 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.

膳食维生素K被γ -谷氨酰羧化酶用于将Glus簇转化为γ -