CAREER: Chemical Probes to Interrogate Real-Time Cellular Activity of Lysine-based Post-translational Modification Erasers

该奖项的部分资金来自《2021年美国救援计划法案》(公法117-2)。在化学系生命过程化学项目的支持下，天普大学的王荣生(Ross)博士将使用化学探针研究翻译后修饰(PTM)橡皮。PTM激光是一类重要的细胞酶，在细胞信号转导中起着关键作用。然而，这些PTM擦除器的功能特征及其与各种细胞事件的关系还没有得到充分的研究。缺乏合适的工具来研究基于赖氨酸的PTM橡皮擦的实时活性，在很大程度上限制了这一方向的研究进展。该项目试图通过发明一种创新的化学传感平台来解决这个问题，该平台通常适用于研究基于赖氨酸的PTM激光在细胞中的活动。这种新的实验方法将结合化学合成、生物化学和细胞生物学的交叉学科技术来回答PTM信号转导的生物学重要问题。这项工作旨在更深入地了解PTM激光在人类细胞中的时空分布，以及识别相关的、位置特定的蛋白质靶标；这些都是破译人类细胞迁移和激活过程的关键信息。作为这一职业奖的一部分，拟议的项目将研究纳入教学、培训和社区宣传，并概述了一项计划，以满足增加科学素养和对STEM教育投资的明确社会需求。将建立一个以学生为基础的同伴指导小组，并由首席调查员领导，以(1)通过初中/高中的外展活动促进STEM的兴趣，(2)通过为来自不同背景的当地高中的学生建立暑期研究计划来创造研究机会，以及(3)通过组织每年一次的Temple化学生物学研讨会来改善STEM学生的赞助机会。蛋白质的PTM调节蛋白质的功能，从而影响细胞的动态平衡，调节与细胞激活、增殖和迁移相关的各种细胞信号过程。目前的研究主要集中在PTM底物和编写体的研究上，然而，基于赖氨酸的PTM擦除酶的特性、它们在活细胞中的动态活性以及它们与特定细胞信号的关系还没有被阐明。该项目旨在发明一种分子成像工具包，并利用它以时空分辨率跟踪活细胞中赖氨酸PTM擦除器的活动。具体地说，人们正在开发由各种化学功能组成的赖氨酸类似物，以模拟PTM橡皮擦的天然基质，并用作荧光化学探针。在拟议的项目期内，将使用多种跨学科研究技术，从化学合成和分子克隆到生化分析、细胞生物学实验和荧光分子成像。这些技术将被用来优化探针的结构和活性，并展示它们在询问PTM激光在细胞事件中的作用(动态亚细胞定位和细胞器相关蛋白底物)中的作用，如迁移和免疫反应。这些研究工作将有助于从机械上理解各种PTM是如何由橡皮擦控制的，以及这些修改如何影响细胞状态。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in part under the American Rescue Plan Act of 2021 (Public Law 117-2). With the support of the Chemistry of Life Processes Program in the Chemistry Division, Dr. Rongsheng (Ross) Wang from Temple University will investigate post-translational modification (PTM) erasers using chemical probes. PTM erasers are a class of important cellular enzymes that are pivotal to cell signaling. Yet, the functional characterization of these PTM erasers and their relationship to various cellular events have not been fully explored. A lack of suitable tools to study the real-time activity of lysine-based PTM erasers largely limits research progress in this direction. This project seeks to address this problem by inventing an innovative chemical sensing platform that is generally applicable to studying activities of lysine-based PTM erasers in cells. This new experimental approach will combine interdisciplinary techniques in chemical synthesis, biochemistry, and cell biology to answer the biologically important questions of PTM signaling. This work is directed at achieving a deeper understanding of the spatial and temporal distribution of PTM erasers in human cells and the identification of related, location-specific, protein targets; this is all critical information to decipher the processes of human cell migration and activation. As part of this CAREER award, the proposed project integrates research into teaching, training, and community outreach and outlines a plan to address the clear social need for increased scientific literacy and investment in STEM education. A student-based peer mentorship team will be established and led by the principal investigator to (1) promote STEM interest through middle/high school outreach, (2) create research opportunities through the establishment of a summer research program for students from local high schools who have diverse backgrounds, and (3) improve sponsorship opportunities for STEM students by organizing an annual Temple chemical biology symposium. PTMs of proteins modulate protein function, thereby affecting cellular homeostasis and regulating diverse cellular signaling processes related to cell activation, proliferation, and migration. Current efforts have focused on the study of PTM substrates and writers; however, the characterization of lysine-based PTM eraser enzymes, their dynamic activities in living cells, and their relationships with specific cellular signaling are yet to be elucidated. This project aims to invent a molecular imaging toolkit and utilize it to track lysine PTM eraser activities in living cells with spatiotemporal resolution. Specifically, lysine analogues that consist of varied chemical functionalities are being developed to mimic the natural substrates of PTM erasers and to serve as fluorogenic chemical probes. Over the proposed project period, multiple interdisciplinary research techniques that range from chemical synthesis and molecular cloning to biochemical assays, cell biology experiments, and fluorescence molecular imaging will be employed. These techniques will be used to optimize the structure and activities of probes and to showcase their utility in interrogating the roles (dynamic subcellular locations and organelle-associated protein substrates) of PTM erasers in cellular events such as migration and immune responses. These research efforts will help develop a mechanistic understanding of how various PTMs are controlled by erasers and how the modifications affect cell states.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.