An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation
- Cell. 2020 Nov 12;183(4):1086-1102.e23. doi: 10.1016/j.cell.2020.09.055.
- 1. Research Group of Proteomics and ADP-ribosylation Signaling, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany.
- 2. Protein Expression and Purification Core Facility, EMBL Heidelberg, 69126 Heidelberg, Germany.
- 3. Research Group of Proteomics and ADP-ribosylation Signaling, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany; Cologne Excellence Cluster for Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany. Electronic address: [email protected].
Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.