Precision Chemistry and Activity-Based Proteomics for Posttranslational Control of Protein Function

Lysine acylations are ubiquitous and structurally diverse posttranslational modifications that vastly expand the functional heterogeneity of the human proteome. Targeted acylation of lysine residues has thus emerged as a strategic approach to exert biomimetic control over protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents, and lack methods to quantify the magnitude of specific acylations on a global level. Here, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific induction and proteome-wide mapping of posttranslational lysine acylations in cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify a paralog-selective chemical probe that acetylates a conserved lysine within an interferon-stimulated antiviral RNA-binding protein, generating a de novo proteoform with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy to induce and quantify biologically relevant posttranslational lysine acylations on a global scale, without relying on genetic intervention, endogenous acylation machinery, cellular acyl pools, or being limited to proteins with preexisting ligands. This approach not only expands the chemical and proteomics toolbox for interrogating the biological processes orchestrated by posttranslational lysine acylations but also lays the foundation for a viable therapeutic modality.