Molecular Basis of Procofactor Activation

描述（由申请人提供）：通过特异性蛋白水解激活前体蛋白是血液凝固的标志。失活的前辅因子蛋白因子V （FV）不能在任何程度上参与其大分子酶复合体。活性是在蛋白水解后产生的，这表明原因子向FVa的转化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的研究兴趣广泛，但对特定的键切割和b结构域释放如何促进这种转化过程知之甚少。本提案的长期目标是破译这些分子过程，并提供FV如何作为非活性前因子保存的详细见解。这一分子过程无疑起着至关重要的调节作用，由于FVa对IIa的产生有巨大的影响，它进化为维持正常的止血。在第一个目标中，我们将定义维持FV原因子状态所需的结构需求。我们假设在b结构域中存在一些保守区域，这些区域有助于抑制辅因子活性，尽管这个结构域在脊椎动物中保守性很差并且长度不同。在第二个目标中，我们将研究b结构域序列保持原因子状态的机制。我们假设b结构域的离散区域在重链和/或轻链上直接接触，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物种之间保留了共同的抑制模式。我们假设来自脊椎动物物种的b结构域保留了共同的序列，这将保留人类FV前因子状态。在最后的目的中，我们将研究FV B结构域序列对蛋白酶易感性的影响，并研究保持FV不活跃的生物学意义。我们假设b结构域显著影响蛋白酶如活化蛋白C和凝血酶如何参与和作用于FV。这些假设将使用生化、动力学和平衡结合方法进行测试，并采用表征良好的重组蛋白。对导致FVa功能性结合位点表达的分子事件的完整描述不仅将阐明该蛋白的功能，还将增强我们对保存FV作为非活性前因子的生物学相关性的理解。从这一建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.