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

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Androgen receptor (AR) signaling is vital in prostate cancer.
• PARP7 modifies AR via cysteine ADP-ribosylation, but the role of multiple modifications is unclear.
• PARP9's macrodomains bind these modifications, suggesting a regulatory mechanism.

Purpose of the Study:

• To investigate the structural basis for DTX3L/PARP9 recognition of ADP-ribosylated AR.
• To elucidate the role of dual ADP-ribosylation in mediating protein-protein interactions.
• To understand the contribution of substrate modification and reader complex oligomerization to binding affinity.

Main Methods:

• Novel solid-phase synthesis of mono- and dual ADP-ribosylated cysteine peptides.
• Preparation of fluorescently labeled peptides containing dual ADP-ribosylation motifs.
• Biochemical assays using wild-type and mutant DTX3L/PARP9 complexes to determine binding affinities (e.g., K_d).

Main Results:

• DTX3L/PARP9 specifically recognizes dual ADP-ribosylated AR peptides with high affinity (K_d = 80.5 nM).
• Dual ADP-ribosylation confers significantly higher binding affinity compared to single ADP-ribosylation.
• Oligomerization of the DTX3L/PARP9 complex is essential for efficient ADP-ribosyl-peptide interaction, as demonstrated by a deletion mutant.

Conclusions:

• Dual ADP-ribosylation of closely spaced cysteines is a key recognition motif for the DTX3L/PARP9 complex.
• The interaction relies on both substrate modification features and the oligomeric state of the reader complex.
• Multivalent interactions are critical for the assembly of the AR-DTX3L/PARP9 complex, impacting androgen signaling in prostate cancer.