A photo-clickable, chain-terminating NAD⁺ analogue enables systematic profiling of the PAR-interacting proteome under oxidative stress

Poly(ADP-ribosyl)ation (PARylation) is a critical post-translational modification that orchestrates diverse cellular processes through the dynamic assembly of PAR-dependent interaction networks. However, systematic profiling of PAR-interacting proteins under physiological and pathological conditions remains technically challenging due to limitations in probe design and interactome capture strategies. Here, we report a photo-clickable, chain-terminating NAD⁺ analogue (PCCT-NAD⁺) that enables the generation of PAR polymers bearing a terminal photo-clickable ADP-ribose. Leveraging this probe, we developed a bead-immobilized, photo-crosslinkable PAR platform for high-efficiency capture of PAR-interacting proteins. Quantitative proteomics under oxidative stress identified 1,576 differentially enriched PAR-binding proteins, revealing extensive remodeling of the PAR interactome. Functional enrichment analysis revealed pronounced reprogramming toward translation and proteostasis pathways, with canonical DNA repair proteins being modestly represented, suggesting a broader role for PARylation in coordinating protein synthesis and turnover beyond its established functions in genome maintenance. Among the candidates, RPLP1, a core component of the ribosomal P complex, exhibited the most pronounced downregulation, which we validated at both protein and transcript levels, establishing oxidative stress-induced transcriptional suppression of RPLP1. Overall, this work provides a versatile chemical proteomics approach for studying PAR-associated networks and supports a role for PARylation in linking stress signaling to translational control.