Genetically Encoded Bioorthogonal Chemistry in Living Systems

Talk given by Peng Chen (Peking University, China) as part of the International GCE Webinar series. Live talk given on March 21st, 2024.
Employing small molecules or other chemical means to modulate the function of an intracellular protein of interest, particularly in a gain-of-function fashion, remains highly desired but challenging. In this talk, I will introduce a “genetically encoded chemical decaging” strategy that relies on our recently developed bioorthogonal cleavage reactions to control protein activation in living systems. These reactions exhibit high efficiency and low toxicity for decaging the chemically “masked” lysine or tyrosine residues on intracellular proteins, allowing the gain-of-function study of individual enzymes within living cells and mice. Most recently, with the assistance of computer-based design and screening, we further expanded our method from “precise decaging” of enzyme active-sites to “proximal decaging” of enzyme pockets. This new method, termed Computationally Aided and Genetically Encoded Proximal Decaging” (CAGE-prox) (CAGE-prox), showed general applicability for switching on the activity of a broad range of proteins under living conditions. I will end by showcasing exciting applications of our CAGE-prox technique on: i) constructing orthogonal and mutually exclusive kinase signaling cascades; ii) temporal caspase activation for time-resolved profiling of proteolytic events upon apoptosis; and iii) on-demand activation of bacterial effectors as potential protein prodrugs for cancer therapy. Finally, by coupling with the proximity-labeling enzymes that have been used for subcellular targeting, we further developed a spatial-temporal resolved proteomics strategy for subcellular proteome profiling in living cells.