Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

ABSTRACT: The nucleus controls cell growth and reproduction by well-orchestrated interactions between nuclear proteins and chromatin. Mutations that result in the dysregulation of these chromatin-associated proteins are known to lead to the onset of numerous diseases. Many of these nuclear proteins have remained recalcitrant to traditional non-covalent small-molecule ligand discovery. Covalent ligands can provide a promising approach for targeting proteins such as transcription factors that lack ordered small-molecule binding pockets. Here, we present a chemoproteomic platform that couples proximity labeling (PL) using a Histone-TurboID (His-TID) construct with competitive isoTOP-ABPP to identify ligandable nuclear cysteines. Application of covalent scout fragments, KB02 and KB05, revealed ligandable cysteine sites on diverse proteins involved in transcriptional regulation, spindle assembly, and DNA repair. To identify functional liganding events that alter target-protein association with chromatin, we monitor the effects of KB02 and KB05 on the chromatin-associated proteome. Importantly, we identify proteins that show both increased and decreased association with chromatin in the presence of the covalent fragments. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and association with chromatin upon covalent liganding at Cys106 by KB02. Together, our PL-assisted nuclear-focused chemoproteomic platform provides us insights into nuclear cysteine ligandability and the functional effects of ligand binding on chromatin association, thereby furthering our understanding of the potentially druggability of the nuclear proteome.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Hela Cell

SUBMITTER: Qianni Peng  

LAB HEAD: Eranthie Weerapana

PROVIDER: PXD043732 | Pride | 2024-01-24

REPOSITORIES: Pride