New chemical probe maps stress-responsive protein networks beyond DNA repair

A research team has developed a photo-clickable, chain-terminating nicotinamide adenine dinucleotide analogue, PCCT-NAD⁺, that enables systematic mapping of proteins interacting with poly(ADP-ribose), or PAR. When applied to cells exposed to oxidative stress, the platform identified 1,576 proteins with altered enrichment in the PAR-associated interactome, revealing particularly strong involvement of translation, ribosome organization, and protein quality-control pathways. The findings broaden the established view of PARylation as primarily a DNA-repair mechanism, suggesting that it also helps coordinate protein synthesis and turnover during cellular stress.

PARylation is a reversible post-translational modification in which PARP enzymes use NAD⁺ to assemble PAR chains on proteins. These chains can function as signaling scaffolds that recruit proteins involved in DNA repair, chromatin organization, transcription, and stress responses. Proteomic probes have begun to reveal these interaction networks, but existing approaches often introduce chemical groups at random positions within PAR chains. Such internal modifications can disturb PAR's native structure, obstruct protein-binding surfaces, and reduce the capture of weak or transient interactors. A method that places functional groups specifically at the end of the polymer is therefore needed to preserve PAR architecture while improving the sensitivity and reliability of interactome profiling.

A study (DOI: 10.48130/targetome-0026-0030) published in Targetome on 15 July 2026 by Mengwei Li's & Xiao-Nan Zhang's team, China Pharmaceutical University, reports that terminally functionalized PAR enables efficient protein capture and reveals extensive stress-induced reorganization of translation and proteostasis networks.

The researchers first designed PCCT-NAD⁺ to act as both a photo-crosslinkable probe and a chain terminator during PARP5A-catalyzed PAR synthesis. Biochemical experiments showed that the analogue could be incorporated into an extending PAR chain but prevented additional ADP-ribose units from being added afterward, thereby positioning the functional group at the polymer terminus. The resulting PAR chains, predominantly at least 20 units long, were biotinylated through click chemistry, purified, and immobilized on streptavidin-coated magnetic beads. Exposure to 365-nanometer ultraviolet light then covalently stabilized interactions between bead-bound PAR and associated proteins. Tests using established PAR-recognition domains confirmed specific capture and showed that PCCT-NAD⁺-derived PAR bound the AF1521 macrodomain and WWE domain approximately 2.8 and 2.1 times more strongly, respectively, than PAR produced using a previously reported randomly incorporated probe. The team next treated HeLa cells with 500 micromolar hydrogen peroxide for two hours to induce oxidative stress. Lysates from treated and control cells were incubated with the PAR-coated beads, photo-crosslinked, stringently washed, digested, and analyzed by data-independent acquisition mass spectrometry across three biological replicates. The analysis identified 1,576 differentially enriched proteins. Functional classification highlighted 266 proteins associated with post-translational modification, protein turnover, and chaperones; 132 involved in translation and ribosome biogenesis; and only 33 assigned to DNA replication, recombination, and repair. KEGG analysis identified 72 PAR-associated proteins in the ribosome pathway, while Gene Ontology analysis likewise emphasized cytoplasmic translation and RNA binding. Four representative proteins—RPLP1, FCF1, PRIM2, and EIF2S1—were validated as PAR interactors. RPLP1 showed the strongest decrease: its total protein level fell to 54% of the control level, its PAR-pulldown signal declined to about 4%, and its transcript abundance dropped to approximately 55%, indicating substantial transcriptional suppression during oxidative stress.

Overall, the study establishes PCCT-NAD⁺ as a versatile tool for examining PAR-centered protein networks while preserving the polymer's internal structure. The results position PARylation as a potential bridge between oxidative-stress signaling, translational regulation, and protein homeostasis. Because the platform predominantly produces linear PAR and the analysis did not globally normalize enrichment against protein abundance, further studies are needed to examine branched PAR structures and distinguish changes in protein concentration from altered PAR-binding affinity. Time-resolved and disease-focused studies could also clarify whether these networks influence stress tolerance or sensitivity to PARP inhibition.

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References

DOI

10.48130/targetome-0026-0030

Original Source URL

https://doi.org/10.48130/targetome-0026-0030

Funding information

This work was supported by the National Natural Science Foundation of China (22377146), and the China Pharmaceutical University Start-Up Fund for New Faculty.

About Targetome

Targetome refers to the complete collection of molecular targets (e.g., proteins, RNA or DNA) that interact with and mediate the effect of a specific biomolecule, such as a drug, toxin, metabolites, transcription factor or microRNA, within a biological system. Targetome is an open access journal publishing rigorously peer-reviewed original research articles, reviews, break-through methods, and perspectives that advance our understanding, identification and validation of molecular targets for new drug development.