Structural Determinants in Cell Growth Control by p21

描述(申请人提供)：结构生物学的一个新兴主题是 无序蛋白质在生物系统中扮演着重要的角色。为 例如，一些无序的蛋白质在重要的信号转导或 监管事件。然而，这些事件的结构性决定因素是 未知，排除了结构和功能的相关性。这个 细胞周期蛋白依赖性激酶(CDK)抑制物p21是两个 重要的肿瘤抑制基因P53和BRCA1。在人类中，依赖于p53的肿瘤 抑制涉及p21的激活，导致细胞周期停滞。我们有 研究表明，p21和一种名为p27的相关蛋白质在 解决方案。尽管如此，p21和p27与细胞周期蛋白/CDK复合体结合， 细胞周期的计时器，具有很高的亲和力和特异性。在过去, P21和p27被认为是CDKs的通用抑制剂；最近的研究表明， 然而，已经证明p21和p27只抑制CDK的一个子集(即 CDK2/细胞周期蛋白A)，它们稳定和激活其他蛋白(即CDK4/细胞周期蛋白 d)。 我们研究的一个重要目的是揭示这种双重现象的物理基础 通过蛋白质结构和动力学研究p21和p27的功能 用核磁共振波谱和等温法研究结合热力学 滴定热法(ITC)。PI的实验室使用核磁共振技术证明了p21 和p27在溶液中具有一个瞬间填充的螺旋？？“连接物螺旋”。 -并表明这一结构特征是 功能。这一假说将通过蛋白质工程学进行验证 稳定和破坏“连接物螺旋”的稳定，然后测定 结合参数和活性。未来对p21同源物的研究将 确定这种结构特征是否在进化上是保守的。另外， 核磁共振研究将扩展到周期蛋白/CDK复合体中的p21和p27，以 揭示CDK抑制与激活的结构决定因素。 最后，ITC和其他技术正被用来阐明结构 以及p21和p27对细胞周期CDKs的特异性的热力学基础。 这项工作很重要，因为p21和p27调节细胞生长停滞。 在人类癌症中最常被破坏的机制，因为 这些蛋白质的结构和功能之间的关系不是很好 明白了。

DESCRIPTION (provided by applicant): An emerging theme in structural biology is that disordered proteins play important roles in biological systems. For example, some disordered proteins adopt structure during important signaling or regulatory events. The structural determinants of these events, however, are unknown, precluding the correlation of structure and function. The cyclin-dependent kinase (Cdk) inhibitor, p21, is a regulatory target of two important tumor suppressors, p53 and BRCA1. In humans, p53-dependent tumor suppression involves activation of p21 that causes cell cycle arrest. We have shown that p21, and a related protein named p27, are dynamically disordered in solution. Despite this, p21 and p27 bind to cyclin/Cdk complexes, the timekeepers of the cell cycle, with high affinity and specificity. In the past, p21 and p27 were considered to be universal inhibitors of Cdks; recent studies, however, have shown that p21 and p27 inhibit only a subset of Cdks (i.e. Cdk2/cyclin A) and that they stabilize and activate others (i.e. Cdk4/cyclin D). One important aim of our studies is to uncover the physical basis for the dual functions of p21 and p27 through studies of protein structure and dynamics using NMR spectroscopy and studies of binding thermodynamics using isothermal titration calorimetry (ITC). The PI's laboratory has shown using NMR that p21 and p27 possess a transiently populated -helix in solution - the "linker helix" - and suggests that this structural feature is an important determinant of function. This hypothesis will be tested using protein engineering to both stabilize and destabilize the "linker helix" followed by the determination of binding parameters and activity. Future studies with p21 homologs will determine whether this structural feature is evolutionarily conserved. Also, NMR studies will be extended to p21 and p27 within cyclin/Cdk complexes to uncover the structural determinants of Cdk inhibition versus activation. Finally, ITC and other techniques are being used to elucidate the structural and thermodynamic basis for the specificity of p21 and p27 for cell cycle Cdks. This work is important because p21 and p27 regulate the cell growth arrest mechanism that is most often disrupted in human cancer and because the relationship between the structure and function of these proteins is not well understood.