PARprolink: a photoaffinity probe for identifying poly(ADP-ribose)-binding proteins

Abstract

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite its clinical significance, a molecular understanding of PAR function, including its binding partners, remains incomplete. In this work, we synthesize a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel binders involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer over 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteome-wide view of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

First body of evidence suggesting a role of a tankyrase-binding motif (TBM) of vinculin (VCL) in epithelial cells

“…Gagné et al (Gagné et al, 2008;Gagné et al, 2012) detected hVCL (but not E-cadherin or β-catenin) in fractions immunoprecipitated with anti-PAR antibodies followed by mass spectrometry (Gagné et al, 2008;Gagné et al, 2012). Moreover, VCL was recently identified in a pool of octomeric PAR (~8-mer PAR) binding proteins but not among~40-mer PAR binders, using photoaffinity probes of defined lengths (Dasovich et al, 2020). As TNKS synthesizes short chain PAR, it is not surprising that VCL has been recovered among~8-mer PAR binders.…”