Molecular Basis of Procofactor Activation

描述(由申请人提供)：通过特定的蛋白分解激活前体蛋白是血液凝固的标志。失活的原辅因子蛋白因子V(Fv)不能在任何程度上参与其大分子酶复合体。活性是在蛋白分解后产生的，这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。虽然人们对这种蛋白质有广泛的兴趣，但人们对特定的键断裂和B结构域的释放如何促进这一转换过程知之甚少。这项提议的长期目标是破译这些分子过程，并提供关于FV如何作为一种非活性促癌因子保存的详细见解。这一分子过程无疑起着关键的调节作用，由于FVA对IIa的产生有巨大的影响，因此进化成维持正常止血。在第一个目标中，我们将定义维持FV原因子状态所必需的结构要求。我们假设在B结构域中存在有助于抑制辅因子活性的保守区，尽管该结构域保守程度较低，并且在脊椎动物中的长度不同。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结构域的多样性来确定是否在不同物种之间保留了共同的抑制模式。我们假设脊椎动物物种的B结构域保留了共同的序列，这将保留人类FV原因子状态。最后，我们将研究Fv B结构域序列对蛋白水解酶敏感性的影响，并研究保持Fv失活的生物学意义。我们假设B结构域显著影响激活的蛋白C和凝血酶等蛋白水解酶如何参与并作用于FV。这些假说将使用生化、动力学和平衡结合的方法进行验证，这些方法使用具有良好特性的重组蛋白。完整地了解导致FVA功能结合位点表达的分子事件不仅有助于阐明该蛋白的功能，还将增强我们对将FV作为非活性原因子保存的生物学意义的理解。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功能的方法。

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.