Assigning functionality to cysteines by base editing of cancer dependency genes

Abstract

Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We further demonstrate how measurements of reactivity in native versus denatured proteomes can discriminate essential cysteines amenable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

“…Here we envisioned combining proximity labeling via the ultra-fast biotin ligase TurboID with cysteine chemoproteomics 11,21,[24][25][26][27][28][29][30]57,58 to enable fractionation-free capture of the subcellular cysteinome, for both residue identification and quantification of cysteine oxidation. We were inspired by recent reports of two-step capture for subcellular phosphoproteomics, in which proteins biotinylated by TurboID were first enriched on avidin resin followed by peptide-level capture of phosphopeptides 59 .…”

“…Application of these methods have pinpointed cysteines differentially oxidized in association with high levels of ROS and RNS, such as those of TRX 13,14 , GAPDH 15,16 and HBB 17 . Given the recent advent of cysteine-reactive small molecules as precision therapies for the treatments of cancers and immune disorders [18][19][20] , cysteine chemoproteomic methods have also emerged as enabling technology for pinpointing ligandable or potentially 'druggable' residues proteome-wide [21][22][23][24][25][26][27][28][29][30] . A central remaining challenge for these studies is the lack of a priori knowledge about the functional impact of covalent modification.…”

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics

“…Looking beyond the current study, we expect that Cys-Surf analysis should synergize with genetic 144 and proteogenomic 145 approaches aimed at pinpointing functional and therapeutically relevant cysteines, including those impacted by genetic variation. Given the seeming ubiquity of reduction-labile disulfide bonds identified by Cys-Surf, we expect that a subset of these cysteines should be susceptible to covalent inhibitor development efforts, including a subset of the >500 ligandable cysteines identified here.…”

Activity‐Based Protein Profiling – Finding General Solutions to Specific Problems

“…We and others are attempting to develop more global ways to relate liganding events to functional outcomes, including, for instance, effects on protein-protein interactions [55] and cell or organismal growth. [92,93] But, I suspect these efforts are only scratching the surface of the types of function-first assays that might be combined with the ligandability maps generated by ABPP, especially those evaluating physicochemically matched active and inactive compounds, [51][52][53]55] to accelerate our understanding of small-molecule effects on protein activities in cells.…”