BLOOD COAGULATION PROTEIN-METAL ION-LIPID INTERACTIONS

凝血蛋白-金属离子-脂质相互作用

• 批准号: 2215306
• 负责人: FRANCIS J CASTELLINO
• 金额: $ 25.37万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-12-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2215306
• 关键词: activation product; blood coagulation; calcium; calcium binding protein; chimeric proteins; coagulation factor IX; coagulation factor V; coagulation factor VII; coagulation factor VIII; conformation; intermolecular interaction; laboratory mouse; laboratory rabbit; phospholipids; protein C; protein S; protein sequence; protein structure function; receptor binding; site directed mutagenesis; synthetic peptide; thrombin; thrombomodulin; vitamin K; zymogens

The overall objective of our research is to understand the structure- function relationships of proteins that are involved in blood coagulation and anticoagulation. We are focusing herein on the structural biochemistry of human protein C (PC) and activated protein C (APC), an anticoagulant zymogen and enzyme, respectively. Our overall hypothesis is that by understanding the structure-function relationships of individual regions of these proteins we can employ this knowledge to effectively up- regulate or downregulate certain functions, and to incorporate features of unrelated proteins. Proteins of the class of PC and APC contain areas classified as domains, which include the gamma-carboxyglutamic acid (Gla) domain (GD), a helical stretch (HS), two consecutive epidermal growth factor-like (EGF) domains, and a serine protease (PD) homology domain. It is hypothesized that these regions incorporate different functions of these proteins. We propose to rigorously define the roles of these individual domains and to assess whether they can function independently. Five specific aims are proposed: (l) to synthesize polypeptides containing amino acid sequences present in the [GD]-[HS], [HS]-[EGF1] and [PD] domains of PC and APC and to utilize these polypeptides to define details of their Ca2+-related properties; (2) to express cDNAs containing domain combinations of regions of PC and APC, particularly the [GD]-]HS]-[EGF1], [GD]-[HS]-[EGF1]-[EGF2], [HS]-[EGF1]- [EGF2]-[PD], [EGF2]-[PD] and [PD] domains, and to determine whether these constructs possess or inhibit functional Ca2+-dependent properties of the intact proteins; (3) to evaluate the importance of specific amino acid residues in the Ca2+-dependent structural and functional properties of r- PC and r-APC by site-directed mutagenesis strategies; (4) to construct and express r-chimeric PC, thrombomodulin (TM), fvII, and fIX mutants in which [GD] (where relevant) and/or [EGF]-like domains have been exchanged between these proteins, and to determine whether the structural and functional properties of both the chimeric protein and the newly incorporated domain have been gained or lost; and (5) to perform charge- to-alanine replacements of residues within [EGF1], [EGF2], and [PD] of PC in order to reveal locations within these modules that are involved in activation of PC by thrombin and thrombin/TM, and the loci of APC that are responsible for inactivation of fVa and fVIII. The significance of this work lies in development of a rigorous basic understanding of the functions of domains of proteins and, as a result of this knowledge, to potentially incorporate desired features of one protein into another in a rational and predictable manner. This will allow strategic molecular drug design to occur which could result in therapeutic benefits in patients with coagulation abnormalities.

我们研究的总体目标是了解结构- 参与血液凝固的蛋白质的功能关系 和抗凝剂。 我们在此重点关注的是 人蛋白C（PC）和活化蛋白C（APC）的生物化学， 抗凝酶原和酶。我们的总体假设是 通过理解个体的结构-功能关系 我们可以利用这些知识来有效地提高这些蛋白质的区域， 调节或下调某些功能，并纳入 无关蛋白质 PC和APC类蛋白含有被分类为结构域的区域， 其中包括γ-羧基谷氨酸（Gla）结构域（GD），一个螺旋结构域 伸展（HS），两个连续表皮生长因子样（EGF）结构域， 和丝氨酸蛋白酶（PD）同源结构域。据推测，这些 区域包含这些蛋白质的不同功能。我们建议 严格界定这些领域的作用，并评估 它们是否能独立运作。提出了五个具体目标： (l)合成含有存在于 PC和APC的[GD]-[HS]、[HS]-[EGF 1]和[PD]结构域，并利用 这些多肽以定义其Ca 2+相关性质的细节;（2） 为了表达含有PC和APC区域的结构域组合的cDNA， 特别是[GD]-[HS]-[EGF 1]、[GD]-[HS]-[EGF 1]-[EGF 2]、[HS]-[EGF 1]- [EGF 2]-[PD]、[EGF 2]-[PD]和[PD]结构域，并确定这些结构域是否 构建体具有或抑制细胞的功能性Ca 2+依赖性性质， 完整蛋白质;（3）评估特定氨基酸的重要性 残基的Ca 2+依赖的结构和功能特性的r- （4）构建重组人PC和r-APC基因， 表达r-嵌合PC、血栓调节蛋白（TM）、fvII和fIX突变体，其中 [GD]（如相关）和/或[EGF]样结构域已交换 这些蛋白质之间，并确定是否结构和 嵌合蛋白和新的嵌合蛋白的功能特性 （五）有下列情形之一的，应当立即通知当事人： PC的[EGF 1]、[EGF 2]和[PD]内残基的丙氨酸取代 为了揭示这些模块中涉及的位置， 凝血酶和凝血酶/TM对PC的激活，以及 负责灭活fVa和fVIII。 这项工作的意义在于发展一个严格的基本 了解蛋白质结构域的功能， 这种知识，潜在地整合一种蛋白质的所需特征， 以理性和可预测的方式进入另一个世界。这将允许 战略分子药物设计的发生，这可能导致治疗， 对凝血异常患者的益处。