Biochemical Classification of Cancer Missense Mutations in the Retinoblastoma Protein

项目摘要/摘要 这项研究的总体目标是描述癌症的结构机制 突变使视网膜母细胞瘤肿瘤抑制蛋白(Rb)失控。Rb是一种多功能枢纽蛋白， 促进蛋白质之间的相互作用，以执行与细胞生长和染色质相关的各种功能 监管。最近的癌症基因组测序数据突出了复发的体细胞和生殖系的位置 错义突变，其中大部分发生在高度保守的口袋结构域。一个详细的分子 理解这些突变的直接结构和功能后果是解析 RB在癌症中发生放松调控的机制，以及确定策略的第一步 从治疗上恢复癌症患者失去的Rb功能。 这项研究的总体目标将通过两个目标来实现，这两个目标将共同产生 Pocket结构域59个反复出现的临床错义突变中每一个的完整结构-功能图谱 RB的。目标1的目标是研究Rb突变体在生化分析中作为纯化蛋白的特性 哪些蛋白质结合功能会被哪些突变所干扰。为了实现这一目标，差分扫描 荧光分析(DSF)已经被开发用于Rb和蛋白质相互作用伙伴，它可以诊断 由于突变引起的折叠稳定性和蛋白质-蛋白质相互作用的变化。这些化验 将进一步得到使用荧光偏振(FP)、等温滴定量热法产生的数据的支持 (ITC)和差示扫描量热法(DSC)。我们将一起测量多个生物物理参数 对于每个突变体，包括蛋白质熔化温度、吉布斯展开自由能和解离 多个蛋白质结合伙伴的常量。目标2的目标是揭示癌症的结构基础。 用蛋白质X-射线结晶学方法突变为Rb。假设是这些突变通过以下途径解除对Rb的调控 各种结构机制，包括：关键蛋白结合界面的直接破坏，负性 蛋白结合界面的变构调节，以及口袋结构域折叠的稳定性的整体变化。 彻底了解支持突变放松调控的机制的结构方面 RB将为理性方法恢复其功能提供坚实的基础。

Project Summary/Abstract The overall objective of this research is to characterize the structural mechanisms by which cancer mutations deregulate the retinoblastoma tumor suppressor protein (Rb). Rb is a multifunctional hub protein that facilitates protein-protein interactions in order to carry out various functions related to cell growth and chromatin regulation. Recent cancer genome sequencing data have highlighted sites of recurrent somatic and germline missense mutations, the majority of which occur to the highly-conserved pocket domain. A detailed molecular understanding of the direct structural and functional consequences of these mutations is key to parsing the mechanisms by which Rb deregulation occurs in cancers, as well as a first step toward identifying strategies to therapeutically restore loss of Rb function in cancer patients. The overall objective of this research will be achieved through two aims, which together will generate complete structure-function profiles for each of the 59 recurrent clinical missense mutations to the pocket domain of Rb. The goal of Aim 1 is to study Rb mutants as purified proteins in biochemical assays that characterize which protein-binding functions are perturbed by which mutations. To achieve this goal, a differential scanning fluorimetry (DSF) assay has been developed for use with Rb and protein interaction partners, which diagnoses changes to fold stability and protein-protein interactions that arise as a consequence of mutations. These assays will be further supported by data generated using fluorescence polarization (FP), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC). Together, multiple biophysical parameters will be measured for each mutant; including, protein melting temperatures, Gibbs free energy of unfolding, and dissociation constants for multiple protein binding partners. The goal of Aim 2 is to reveal the structural basis of cancer mutations to Rb using protein x-ray crystallography. The hypothesis is that the mutations deregulate Rb through various structural mechanisms, including: the direct disruption of critical protein binding interfaces, negative allosteric regulation of protein binding interfaces, and overall changes to the stability of the pocket domain fold. A thorough understanding of the structural aspects underpinning the mechanisms by which mutations deregulate Rb will provide a solid basis for rational approaches to restore its function.