Biochemical Classification of Cancer Missense Mutations in the Retinoblastoma Protein

视网膜母细胞瘤蛋白癌症错义突变的生化分类

基本信息

批准号：
10377583
负责人：
Jason Burke
金额：
$ 11.03万
依托单位：
CALIFORNIA STATE UNIV SAN BERNARDINO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-05 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10377583
关键词：
Allosteric Regulation Binding Binding Proteins Binding Sites Biochemical Biochemistry Biological Assay Biological Process Biopsy California Calorimetry Cancer Patient Cell Cycle Cell Proliferation Cell division Cells Chromatin Clinical Crystallization Crystallography Data Data Analyses Data Collection Data Set Diagnosis Differential Scanning Calorimetry Dissociation Docking E2F transcription factors Event Evolution Family member Fluorescence Polarization Free Energy Future Genes Genetic Goals Growth Hydrogen Bonding Induced Mutation Knowledge Label Libraries Ligand Binding Domain Ligands MAP Kinase Gene Malignant Neoplasms Maps Measures Mediating Missense Mutation Modeling Molecular Molecular Conformation Mutation Non-Small-Cell Lung Carcinoma Pattern Peptides Proteins Recurrence Regulation Research Research Proposals Resolution Resource Sharing Retinoblastoma Retinoblastoma Protein Roentgen Rays Role S phase Scanning Signal Transduction Site Solid Somatic Mutation Spatial Distribution Structure Temperature Therapeutic Time Tissues Titrations Transactivation Tumor Suppressor Proteins Universities Variant X-Ray Crystallography activating transcription factor base biophysical properties cancer classification cancer genome cancer therapy cancer type carcinogenicity cell growth clinical research site exome sequencing experimental study functional loss functional outcomes genome sequencing loss of function melting mutant novel therapeutics osteosarcoma professor protein protein interaction response retinoblastoma tumor suppressor structural biology three dimensional structure trend

项目摘要

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.

项目概要/摘要这项研究的总体目标是表征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 是一种多功能中心蛋白促进蛋白质-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规定。最近的癌症基因组测序数据强调了体细胞和种系复发的位点错义突变，其中大部分发生在高度保守的口袋结构域。详细的分子了解这些突变的直接结构和功能后果是解析这些突变的关键癌症中 Rb 失调的机制，以及确定治疗策略的第一步治疗性恢复癌症患者 Rb 功能的丧失。本研究的总体目标将通过两个目标来实现，这两个目标共同将产生口袋结构域 59 个复发性临床错义突变中每一个的完整结构功能概况铷。目标 1 的目标是在生化测定中研究 Rb 突变体作为纯化蛋白质，表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，差示扫描荧光测定法 (DSF) 已开发用于 Rb 和蛋白质相互作用伙伴，可诊断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。这些化验使用荧光偏振 (FP)、等温滴定量热法生成的数据将进一步支持 (ITC) 和差示扫描量热法 (DSC)。将同时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、展开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 射线晶体学分析 Rb 的突变。假设突变通过以下方式解除了 Rb 的调节：各种结构机制，包括：直接破坏关键的蛋白质结合界面，负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整体变化。全面了解突变解除管制机制的结构方面 Rb将为合理恢复其功能的方法提供坚实的基础。