Chemical tools for profiling the intracellular ADP-ribosylated proteome

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• ADP-ribosylation is a critical post-translational modification involved in cell signaling and protein function.
• Dysregulation of ADP-ribosylation is implicated in various diseases, including breast and ovarian cancers.
• Existing tools for identifying ADP-ribosylated proteins in live cells are limited, hindering mechanistic studies.

Purpose of the Study:

• To introduce and validate a novel chemical probe, 6Yn-Pro, for the identification of intracellular ADP-ribosylated proteins.
• To demonstrate the utility of metabolic labeling with 6Yn-Pro for studying ADP-ribosylation in a live-cell context.

Main Methods:

• Application of an alkyne-tagged probe (6Yn-ProTide-Ad, 6Yn-Pro) for metabolic labeling.
• Utilized targeted metabolomics and chemical proteomics in HEK293T cells.
• Induced cellular stress using hydrogen peroxide.

Main Results:

• Demonstrated intracellular metabolic conversion of 6Yn-Pro to 6Yn-NAD+.
• Successfully labeled and enriched PARP1 and other known ADP-ribosylated proteins.
• Identified ADP-ribosylated proteins in live cells under oxidative stress.

Conclusions:

• The 6Yn-Pro probe serves as an effective chemical tool for identifying intracellular ADP-ribosylated proteins.
• This methodology facilitates the study of ADP-ribosylation in live-cell environments.
• The approach is expected to aid in the discovery of novel ADP-ribosylated proteins and their roles in disease.