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

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.