Testing the p53 code for Gene Regulation with Chemical Protein Synthesis

在化学部生命过程化学项目和分子与细胞生物科学部遗传机制集群的共同支持下，来自华盛顿大学的Champak Chatterjee博士将研究蛋白质p53的修饰如何控制人类基因的开启或关闭。p53蛋白控制DNA中的遗传信息如何被重写或转录为m-RNA形式的中间信息。已知p53转录因子的突变会导致人类癌症。p53控制基因的能力本身是由其他小蛋白可逆地添加到该转录因子中来控制的。该项目将使用新的合成化学方法在p53的特定位置可逆地添加称为小泛素相关修饰物（SUMO）的蛋白质变体。研究旨在揭示SUMO修饰如何影响p53调节人类基因表达的能力。本计画将训练研究生与本科生蛋白质合成技术，沿着进行p53功能的生物物理与生化分析。此外，一项推广计划将向高中学生介绍转录因子突变引起的遗传疾病，并为高中教师提供研究培训。本项目将构建特异性转录后修饰的p53蛋白，并利用先进的生物物理和生物化学技术定量表征这些修饰对该转录因子调控功能的作用。已知与SUMO的同种型及其在p53的无序C-末端区域上的乙酰化形式的共价缀合调节基因表达。该项目提供了一种断裂内含肽介导的半合成策略来构建由不同的SUMO异构体修饰的p53，这是不容易从生物学方法获得的。p53类小泛素化的亚型特异性效应与细胞中的转录激活和抑制有关，将通过生物化学测定仔细研究。这项研究产生的基础知识可能会提供新的见解调控的主基因调节在人类可逆的翻译后modifications.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

With joint support from the Chemistry of Life Processes program in the Chemistry Division and the Genetic Mechanisms cluster in the Molecular and Cellular Biosciences Division, Dr. Champak Chatterjee from the University of Washington will investigate how modifications to the protein p53 control how human genes are turned on or off. The p53 protein controls how the genetic information in DNA is rewritten or transcribed to the intermediate message in the form of m-RNA. Mutations in the p53 transcription factor are known to cause human cancers. The ability of p53 to control genes is itself controlled by the reversible addition of other small proteins to this transcription factor. This project will use new synthetic chemistry to reversibly add variants of the protein known as a small ubiquitin-related modifier (SUMO) at specific positions of p53. Studies are designed to reveal how the SUMO modifications affect the ability of p53 to regulate the expression of human genes. This project will train graduate and undergraduate students in protein synthesis techniques, along with biophysical and biochemical assays of p53 function. In addition, an outreach program will introduce high school students to genetic disorders arising from mutations in transcription factors and provide research training to high school teachers. This project will construct specific post-translationally modified p53 proteins and use advanced biophysical and biochemical techniques to quantitatively characterize the roles of these modifications on the regulatory function of this transcription factor. Covalent conjugation with isoforms of SUMO and their acetylated forms on the disordered C-terminal region of p53 are known to regulate gene expression. This project provides a split-intein-mediated semisynthetic strategy to construct p53 modified by distinct SUMO isoforms, which are not readily available from biological approaches. Isoform-specific effects of p53 sumoylation that are confoundingly associated with both transcription activation and repression in cells will be carefully investigated through biochemical assays. Fundamental knowledge generated by this study will likely provide new insights into regulation of the master gene regulator in humans by reversible post-translational modifications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.