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，天普大学的王荣盛博士将使用化学探针研究翻译后修饰（PTM）擦除器。 PTM erasers是一类重要的细胞酶，对细胞信号传导至关重要。 然而，这些PTM擦除器的功能特性及其与各种细胞事件的关系尚未得到充分研究。 缺乏合适的工具来研究基于赖氨酸的PTM擦除器的实时活性在很大程度上限制了这一方向的研究进展。 该项目旨在通过发明一种创新的化学传感平台来解决这一问题，该平台通常适用于研究细胞中基于赖氨酸的PTM擦除器的活动。 这种新的实验方法将联合收割机在化学合成，生物化学和细胞生物学的跨学科技术，以回答PTM信号的生物学重要问题。 这项工作旨在更深入地了解PTM erasers在人类细胞中的空间和时间分布，并识别相关的位置特异性蛋白质靶点;这是破译人类细胞迁移和激活过程的所有关键信息。 作为这项职业奖的一部分，拟议的项目将研究纳入教学，培训和社区推广，并概述了一项计划，以解决增加科学素养和投资STEM教育的明确社会需求。 将建立一个以学生为基础的同伴指导团队，并由主要研究者领导，以（1）通过初中/高中外展促进STEM兴趣，（2）通过为来自当地高中的学生建立夏季研究计划创造研究机会，他们有不同的背景，（3）通过组织一年一度的Temple化学生物学研讨会来提高STEM学生的赞助机会。蛋白质的PTM调节蛋白质功能，从而影响细胞稳态并调节与细胞活化、增殖和迁移相关的多种细胞信号传导过程。 目前的努力集中在PTM底物和作家的研究，然而，赖氨酸为基础的PTM擦除酶的特性，它们在活细胞中的动态活动，以及它们与特定的细胞信号转导的关系还有待阐明。 本计画的目的是开发一套分子影像工具，并利用其追踪活细胞中赖氨酸PTM擦除器的时空解析度。 具体而言，赖氨酸类似物，由不同的化学功能正在开发模仿PTM擦除器的天然基板，并作为荧光化学探针。 在拟议的项目期间，将采用从化学合成和分子克隆到生物化学测定，细胞生物学实验和荧光分子成像的多学科研究技术。 这些技术将用于优化探针的结构和活性，并展示其在询问PTM擦除器在细胞事件（如迁移和免疫反应）中的作用（动态亚细胞位置和细胞器相关蛋白底物）中的实用性。 这些研究工作将有助于发展一个机制的理解，各种PTM是如何控制的橡皮擦和修改如何影响细胞的states.This奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的智力价值和更广泛的影响审查标准。

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.