Molecular Basis of Procofactor Activation

描述（由申请方提供）：通过特异性蛋白水解激活前体蛋白是凝血的标志。无活性的原辅因子蛋白质因子V（FV）不能在其大分子酶复合物中发挥任何重要作用。蛋白水解后产生活性，表明原辅因子转化为FVa必须导致赋予辅因子功能的结构变化。虽然人们对研究这种蛋白质产生了广泛的兴趣，但对特定的键断裂和B结构域释放如何促进这种转化过程知之甚少。这项提案的长期目标是破译这些分子过程，并提供详细的了解如何FV是保存作为一个无活性的原辅因子。这一分子过程无疑起着关键的调节作用，由于FVa对IIa的产生具有巨大的影响，因此进化以维持正常的止血。在第一个目标中，我们将定义维持FV原辅因子状态所需的结构要求。我们推测，有保守的区域在B-域中，是在抑制辅因子活性的工具，尽管事实上，这个域是保守性差，在脊椎动物之间的长度变化。在第二个目标中，我们将研究B结构域序列保持原辅因子状态的机制。我们假设B结构域的离散区域与重链和/或轻链直接接触，从而掩盖了关键的结构决定因素。在第三个目标中，我们将利用脊椎动物FV B-域的多样性，以确定是否有一个共同的抑制模式是跨物种保存。我们假设，B-域从脊椎动物物种保留共同的序列，将保留人类FV原辅因子的状态。在最后的目标中，我们将检查FV B-结构域序列对蛋白酶敏感性的影响，并检查保持FV失活的生物学意义。我们假设B结构域显著影响蛋白酶如活化蛋白C和凝血酶如何参与并作用于FV。这些假设将使用生化，动力学和平衡结合方法，采用充分表征的重组蛋白进行测试。导致FVa上功能结合位点表达的分子事件的全貌将不仅揭示这种蛋白质的功能，而且还将增强我们对保留FV作为无活性前辅因子的生物学相关性的理解。从这一提议中获得的知识也可能揭示以前未被认识到的调节FV/FVa功能的方式。

DESCRIPTION (provided by applicant): Activation of precursor proteins by specific proteolysis is a hallmark of blood coagulation. The inactive procofactor protein factor V (FV) cannot participate to any significant degree in its macromolecular enzyme complex. Activity is generated following proteolysis, indicating that the conversion of the procofactor to FVa must result in structural changes that impart cofactor function. While there has been widespread interest in studying this protein, little is known about how specific bond cleavage and B-domain release facilitate this conversion process. The long-term objective of this proposal is to decipher these molecular processes and provide detailed insight into how FV is preserved as an inactive procofactor. This molecular process undoubtedly plays critical regulatory roles, evolved to maintain normal hemostasis since FVa has a tremendous influence on IIa generation. In the first aim, we will define the structural requirements necessary to maintain the FV procofactor state. We hypothesize that there are conserved regions in the B-domain that are instrumental in suppressing cofactor activity, despite the fact that this domain is poorly conserved and varies in length among vertebrates. In the second aim, we will investigate the mechanism by which B-domain sequences preserve the procofactor state. We hypothesize that discrete regions of the B-domain make direct contacts on the heavy and/or light chain thereby masking critical structural determinants. In the third aim, we will exploit the diversity of vertebrate FV B-domains to determine whether a common mode of inhibition is preserved across species. We hypothesize that B-domains from vertebrate species retain common sequences that will preserve the human FV procofactor state. In the final aim, we will examine the influence of FV B- domain sequences on protease susceptibility and examine the biological significance of keeping FV inactive. We hypothesize that the B-domain significantly affects how proteases such as activated protein C and thrombin engage and act on FV. These hypotheses will be tested using biochemical, kinetic, and equilibrium binding approaches employing well characterized recombinant proteins. A complete picture of the molecular events leading to the expression of functional binding sites on FVa will not only shed light on the function of this protein but will also enhance our understanding of the biological relevance of preserving FV as an inactive procofactor. Knowledge gained from this proposal may also reveal previously unrecognized ways to modulate FV/FVa function.