ENHANCED VITAMIN K DEPENDENT PROTEINS

增强维生素 K 依赖性蛋白质

• 批准号: 2686448
• 负责人: Gary L Nelsestuen
• 金额: $ 30.24万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2686448
• 关键词: activation product; active sites; aminoacid; antigen presenting cell; biophysics; blood coagulation; calcium; calorimetry; coagulation factor VII; cofactor; enzyme activity; enzyme substrate; fluorescence spectrometry; gamma carboxyglutamate; membrane activity; membrane proteins; membrane structure; mutant; nuclear magnetic resonance spectroscopy; protein C; protein folding; protein structure function; tissue /cell culture; vitamin K; zymogens

DESCRIPTION: (Investigator's abstract) This study will examine vitamin K-dependent proteins that display improved membrane affinity. The vitamin K-dependent plasma proteins function in both pro- and anti-coagulation pathways. Membrane contact is important for nearly all of the steps of these reactions. Calcium and membrane binding by vitamin K-dependent proteins depend on several gamma-carboxyglutamic acid (Gla) residues in amino acids 1-45 of these proteins. Gla is synthesized in the liver in a vitamin K-dependent reaction. Administration of vitamin K antagonists, which lower the level of Gla-containing proteins in the circulation, is a common biomedical application for persons subject to thrombosis. In addition, administration of intact proteins is important to treatment for some bleeding disorders. A major goal of this project is to establish the membrane contact mechanisms of the vitamin K-dependent proteins and to use that knowledge to generate proteins with improved function. This proposal presents a new hypothesis for membrane contact. Seven specific aims include: 1. Design mutants of protein C and factor VII that have improved membrane contact sites. 2. Determine the relationship between amino acids in the Gla domain and membrane binding affinity. 3. Characterize the relationship between membrane affinity and the ability of a zymogen protein to function as substrate for activation by other membrane-bound enzymes. 4. Determine the impact of membrane affinity (specific aim 1) on the enzyme activity of the activated forms of these proteins (factor VIIa and activated protein C, APC), including the impact of cofactor proteins, membrane content, and other properties. 5. Determine the mechanisms of membrane contact by vitamin K proteins. 6. Determine the role of the hydrophobic residues in the Gla domain. 7. Determine the structures responsible for greatest folding stability of the Gla domain. Methods used include expression of mutant proteins and their characterization by enzyme and coagulation assays, spectroscopic assays by fluorescence, and biophysical characterization with NMR and calorimetry. The use of carefully prepared membrane vesicles is required in most areas of this project. This study should reveal structure/function relationships of gamma-carboxyglutamic acid, and its ultimate function in creating a membrane-contact site. It will increase knowledge of the role that membranes play in enzymatic events of coagulation and in biological systems. It may ultimately provide new and/or improved materials for biomedical applications.

描述：（研究者摘要）本研究将检查维生素 K-依赖性蛋白质，表现出改善的膜亲和力。 维生素 K依赖性血浆蛋白在促凝和抗凝中均起作用 途径。 膜接触对于制备的几乎所有步骤都是重要的。 这些反应。 维生素K依赖性钙和膜结合 蛋白质依赖于几个γ-羧基谷氨酸（Gla）残基， 这些蛋白质的氨基酸1-45。 Gla在肝脏中合成， 维生素K依赖性反应 给予维生素K拮抗剂， 降低循环中含Gla蛋白的水平，是一种 用于血栓形成患者的常见生物医学应用。 在 此外，施用完整蛋白质对于治疗 一些出血性疾病 该项目的一个主要目标是建立 维生素K依赖性蛋白的膜接触机制及其应用 利用这些知识来生产功能更好的蛋白质。 这项建议 提出了膜接触的新假设。 七个具体目标 有：1. 设计蛋白C和因子VII的突变体， 膜接触部位。 2. 确定氨基酸之间的关系 Gla结构域和膜结合亲和力。 3. 表征 膜亲和性与酶原蛋白能力之间的关系 以作为其他膜结合酶激活的底物。 4. 确定膜亲和力（具体目标1）对酶的影响 这些蛋白质的活化形式（因子VIIa和活化的 蛋白C，APC），包括辅因子蛋白，膜 内容和其他属性。 5. 确定膜的机制 接触维生素K蛋白。 6. 确定疏水作用 Gla结构域中的残基。 7. 确定负责以下方面的结构： Gla结构域的最大折叠稳定性。 使用的方法包括 突变体蛋白的表达及其通过酶和 凝血测定、荧光光谱测定和生物物理测定 通过NMR和量热法进行表征。 使用精心准备的 膜囊泡在该项目的大部分区域中是必需的。 本研究 应该揭示γ-羧基谷氨酸的结构/功能关系 酸，以及它在创造膜接触位点中的最终功能。 它 将增加对膜在酶促反应中所起作用的认识 和生物系统中的作用。 它最终可能会提供新的 和/或用于生物医学应用的改进材料。