BLOOD COAGULATION PROTEIN METAL ION LIPID INTERACTIONS

这项研究的总体目标是了解参与止血的维生素K依赖蛋白的结构-功能关系，以及编码这些蛋白的基因。这一应用中的一些重点将放在小鼠的蛋白质和基因上，因为大多数靶向基因改变都是在小鼠模型中进行的，这些改变极大地推动了这一领域的发展。在这个一般的类别中包含的特定感兴趣的蛋白质，即因子(F)VII、IX、X和蛋白C(PC)，通过类似的结构域组装，这些结构域包括伽玛-羧谷氨酸(GLA)结构域、疏水延伸、两个连续的表皮生长因子样(EGF)基序、一个激活肽模块和一个丝氨酸蛋白酶结构域。这些区域融合了这些蛋白质的不同性质，似乎是独立发挥作用的。假设通过严格定义这些蛋白质结构域的关键区域及其对其各种功能至关重要的基因，将有可能调节它们的特定功能，并有可能将一种蛋白质的特定功能转移到另一种蛋白质中。为实现这些目标，提出了五个具体目标：(1)通过体外和体内方法，鉴定和评估小鼠基因fVII、Fx、PC和内皮细胞PC受体(EPCR)的转录调控功能；(2)利用合成肽库来确定被FVIIa和FVIIa/Tf复合体、凝血酶和凝血酶/TM复合体以及APC和APC/EPCR复合体最有效地切割的序列。将为每个酶确定的各种优化的肽序列与另一种蛋白质(FIX)中的可比残基交换，以确定这些酶是否如肽研究预测的那样发生切割；(3)表达人PC的第一生长因子样域(EGF1)的野生型和变异型，并鉴定它们的二价阳离子结合蛋白。通过核磁共振确定EGF1-PC在钙离子存在和不存在的情况下的溶液结构和骨架动力学，以及该模块的一个缺乏额外二硫键环序列的变体；(4)化学合成人PC的伽玛-羧谷氨酸(GLA)结构域的变体，并表征它们对钙的依赖性质。用13C-GLA选择性标记的多肽将被用来评估二价阳离子与多肽的结合，这些多肽的突变方式与表现出与钙相关的缺陷的变异蛋白相同；以及(5)研究人PC与EPCR结合的氨基酸决定因素。

The overall objective of this research is to understand the structure- function relationships of the vitamin K-dependent proteins that are involved in hemostasis, and the genes that encode these proteins. Some of the focus in this application will be placed on murine proteins and genes, since most targeted gene alterations, which have considerably advanced this field, are performed in mouse models. Proteins of specific interest that are contained in this general class, viz., factors (f) VII, IX, X, and protein C (PC), are assembled through similar domains, which include the gamma-carboxyglutamic acid (Gla) domain, a hydrophobic stretch, two consecutive epidermal growth factor-like (EGF) motifs, an activation peptide module, and a serine protease domain. These regions incorporate different properties of these proteins, and appear to function independently. It is hypothesized that by closely defining the critical regions of these protein domains, and their genes, that are essential to their various functions, it will be possible to regulate their specific features, and, likely, to transfer specific functions of one protein into another. Five specific aims are proposed to achieve these goals: (1) to identify and assess the functional roles of transcriptional regulatory of the murine genes, fVII, fX, PC, and the endothelial cell PC receptor (EPCR), through in vitro and in vivo methodology; (2) to employ a synthetic peptide library to determine sequences that are most effectively cleaved by fVIIa and the fVIIa/TF complex, by thrombin and the thrombin/TM complex, and by aPC and the aPC/EPCR complex. To exchange various optimized peptide sequences identified for each enzyme with comparable residues in another protein (fIX) to determine whether cleavages by these enzymes occur as predicted from the peptide study; (3) to express wild-type and variants of the first growth factor-like domain (EGF1) of human PC and to characterize their divalent cation binding proteins. TO determine, by NMR, the solution structures and backbone dynamics of EGF1-PC in the presence and absence of Ca2+, along with a variant of this module lacking its extra disulfide loop sequence; (4) to chemically synthesize variants of the gamma-carboxyglutamic acid (Gla) domain of human PC and to characterize their Ca2+-dependent properties. Peptides that are selectively-labeled with 13C-Gla will be emphasized to evaluate divalent cation binding to peptides mutated in the same manners as variant proteins that show defective Ca2+-related properties; and (5) to investigate amino acid determinants of the binding of human PC to EPCR.