Biochemical Classification of Cancer Missense Mutations in the Retinoblastoma Protein

项目总结/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）失调。Rb是一种多功能中枢蛋白， 促进蛋白质-蛋白质相互作用，以执行与细胞生长和染色质相关的各种功能 调控最近的癌症基因组测序数据突出了复发性体细胞和生殖细胞的位点， 错义突变，其中大部分发生在高度保守的口袋结构域。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失调发生在癌症中的机制，以及确定策略的第一步， 治疗性恢复癌症患者中Rb功能的丧失。 本研究的总体目标将通过两个目标实现，这两个目标将共同产生 完整的结构-功能概况的59个复发性临床错义突变的口袋域 的Rb。目的1的目标是研究Rb突变体作为纯化的蛋白质在生化测定中的特征， 哪些蛋白质结合功能受到哪些突变的干扰。为了实现这一目标， 已经开发了一种用于Rb和蛋白质相互作用伴侣的荧光测定法（DSF）， 由于突变而引起的折叠稳定性和蛋白质-蛋白质相互作用的变化。这些测定 将进一步支持使用荧光偏振（FP），等温滴定量热法产生的数据 (ITC)和差示扫描量热法（DSC）。同时，将测量多个生物物理参数 对于每个突变体;包括，蛋白质解链温度，解折叠的吉布斯自由能，和解离 多个蛋白质结合伴侣的常数。目标2的目标是揭示癌症的结构基础 使用蛋白质X射线晶体学检测Rb突变。假设是突变通过以下途径解除Rb的调节： 各种结构机制，包括：直接破坏关键蛋白质结合界面， 蛋白质结合界面的变构调节，以及口袋结构域折叠稳定性的总体变化。 对突变解除调节机制的结构方面的透彻理解 Rb将为恢复其功能的合理方法提供坚实的基础。

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.