Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex

Androgen signaling
in prostate cancer cells involves
multisite
cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7.
The AR modification is read by ADP-ribosyl binding macrodomains in
PARP9, but the reason that multiple cysteines are modified is unknown.
Here, we use synthetic peptides to show that dual ADP-ribosylation
of closely spaced cysteines mediates recognition by the DTX3L/PARP9
complex. Mono and dual ADP-ribosylated cysteine peptides were prepared
using a novel solid-phase synthetic strategy utilizing a key, Boc-protected,
ribofuranosylcysteine building block. This synthetic strategy allowed
us to synthesize fluorescently labeled peptides containing a dual
ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex
recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides
with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9
complex proved crucial for ADP-ribosyl-peptide interaction since a
deletion mutant of the complex that prevents its oligomer formation
dramatically reduced peptide binding. Our data show that features
of the substrate modification and the reader contribute to the efficiency
of the interaction and imply that multivalent interactions are important
for AR-DTX3L/PARP9 assembly.