CAREER: Functional Dissection of Ubiquitination Signaling by Quantitative Chemical Proteomics

凭借这一奖项，化学学部生命过程化学项目资助了明尼苏达大学的陈悦博士，他将使用一种名为定量化学蛋白质组学的创新方法来研究一种特定的细胞信号通路。泛素化是一种具有多种生物学功能的蛋白质修饰，包括信号传导。例如，当DNA受损时，泛素化在激活特定反应途径以启动DNA损伤修复中起关键作用。一种新开发的定量化学蛋白质组学方法被应用于揭示这是如何发生的。实验研究有望确定新的泛素化依赖的调节机制，并在DNA损伤反应中发现以前未知的酶活性。在项目期间，通过全面整合的化学蛋白质组学培训计划，对生物化学本科学生进行化学合成和定量蛋白质组学的跨学科技术培训。额外的推广项目通过特别设计的实地学习活动促进高中生的生物化学教育。研究项目将新的同位素编码化学计量学分析方法和化学蛋白质组学策略结合起来，对泛素化信号通路进行功能剖析。与传统的相对定量相比，这种方法能够对泛素化流行率进行特定位点的绝对定量，因此，可以识别在特定生物过程中被化学计量激活的泛素化靶标。这一策略被应用于确定DNA双链断裂过程中泛素化信号的动态激活。时间动力学分析和功能验证揭示了泛素化的关键调控靶点，这些靶点对特定反应途径的激活至关重要。体外和体内定量分析确定了双链断裂和修复中重要泛素化E3连接酶的底物动力学和特异性。总的来说，这些研究为DNA损伤和反应中泛素化介导的信号传导和调控途径提供了机制见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Yue Chen from the University of Minnesota to investigate a specific cellular signaling pathway using an innovative new methodology called, quanititative chemical proteomics. Ubiquitination is a modification of proteins with a number of biological functions including signaling. For example, when DNA is damaged, ubiquitination plays a critical role in activating specific response pathways to initiate DNA damage repair. A newly developed quantitative chemical proteomics approach is applied to reveal just how this takes place. Experimental studies are expected to identify novel ubiquitination-dependent regulatory mechanisms and find previously unknown enzymatic activities in DNA damage response. During the project, undergraduate biochemistry students train with interdisciplinary techniques in chemical synthesis and quantitative proteomics through a fully integrated chemical proteomic training program. Additional outreach programs promote biochemistry education for high school students through specifically designed field learning activities.The research projects integrate the new isotope-encoded stoichiometry analysis method and chemical proteomics strategies to functionally dissect ubiquitination signaling pathways. Compared to the traditional relative quantification, this approach enables site-specific absolute quantification of ubiquitination prevalence and, therefore, allows the identification of ubiquitination targets that are stoichiometrically activated in specific biological processes. This strategy is applied to determine the dynamic activation of ubiquitination signaling during DNA double-strand break. Temporal dynamics analysis and functional validation reveal key regulatory ubiquitination targets that are critical for the activation of specific response pathways. In vitro and in vivo quantitative analysis determine the substrate dynamics and specificities of important ubiquitination E3 ligases in double-strand break and repair. Overall, these studies provide mechanistic insights into the ubiquitination-mediated signaling and regulatory pathways in DNA damage and response.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.