Detection of ADP-Ribosylation of the Androgen Receptor Using the Recombinant Macrodomain AF1521 from Archaeoglobus fulgidus.

ADP-ribosylation is a posttranslational modification generated by members of the superfamily of ADP-ribosyltransferases, known as the Parp enzymes. Depending on the superfamily member, Parp enzymes can mono- or poly-ADP-ribosylate a protein substrate. Parp superfamily members confer regulation to a variety of biological processes that include cell signaling, DNA repair, transcription, and stress responses. Here, we describe biochemical methods for detection of ADP-ribose conjugated to the androgen receptor (AR) using the archaeal macrodomain, AF1521, from Archaeoglobus fulgidus. The utility of AF1521 is based on its highly selective recognition of ADP-ribose conjugated to protein. AF1521 immobilized on beads can be used to enrich for ADP-ribosylated proteins, which in our application results in recovery of ADP-ribosylated AR from prostate cancer cell extracts. We engineered tandem AF1521 macrodomains and found this improves the recovery of ADP-ribosylated AR under native conditions, and it enabled development of an assay for detection of ADP-ribosylation on blots. Thus, AF1521 can be used to query ADP-ribosylation of protein under both native and denaturing conditions. Our assays should prove useful for understanding how ADP-ribosylation regulates AR function.