A Bifunctional NAD⁺ for Profiling Poly-ADP-Ribosylation-Dependent Interacting Proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein poly-ADP-ribosylation (PARylation) is a dynamic post-translational modification involved in numerous biological processes and human diseases.
• Current methods lack the ability to precisely identify PARylation readers and erasers for specific proteins.

Purpose of the Study:

• To develop a novel chemical tool for mapping protein interactions associated with PARylation.
• To facilitate the identification of PARylation-dependent interacting proteins and their roles.

Main Methods:

• Generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) analog with diazirine-modified adenine and clickable ribose.
• Utilizing the NAD+ analog as a substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation.
• Employing photo-cross-linking and enrichment strategies for proteomic identification of interacting proteins.

Main Results:

• The synthesized bifunctional NAD+ analog effectively serves as a substrate for PARP1.
• The analog enables photo-cross-linking and subsequent enrichment of PARylation-dependent interacting proteins.
• This method allows for proteomic identification of proteins within PARylation-centered networks.

Conclusions:

• The developed bifunctional NAD+ analog is a valuable tool for mapping cellular interaction networks involving protein PARylation.
• This tool is essential for elucidating the functions of PARylation in physiological and pathological conditions.
• Advances understanding of post-translational modifications and their disease relevance.