Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Poly(ADP-ribose) (PAR) is crucial for DNA damage response and is targeted by cancer therapies.
• The molecular functions and binding partners of PAR remain incompletely understood.
• Identifying PAR-binding proteins is essential for elucidating PAR signaling pathways.

Purpose of the Study:

• To develop a method for identifying endogenous poly(ADP-ribose) (PAR) binding proteins.
• To create a comprehensive census of PAR-binding proteins, including novel candidates.
• To investigate the role of PAR polymer length in protein binding and signaling.

Main Methods:

• Synthesis of a novel PAR photoaffinity probe.
• Affinity capture and isolation of endogenous PAR-binding proteins.
• Confirmation of PAR binding using pull-down and electrophoretic mobility shift assays.
• Analysis of protein binding selectivity using PAR probes of defined lengths.

Main Results:

• Identification of dozens of known and hundreds of novel PAR-binding proteins.
• Confirmation of PAR binding for eight novel candidate proteins.
• Demonstration of length-dependent PAR binding, with proteins showing preference for longer PAR chains (40-mer vs. 8-mer).
• Association of PAR binding with RNA metabolism and biomolecular condensate formation.

Conclusions:

• The study provides the first comprehensive census of PAR-binding proteins.
• PAR polymer length is a critical regulator of PAR-protein interactions and signaling.
• Newly identified PAR-binding proteins offer potential therapeutic targets in cancer and other diseases.