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Chemical tools for profiling the intracellular ADP-ribosylated proteome
Simeon D Draganov1, Michael J Gruet1,2, Daniel Conole1
1Department of Chemistry, Molecular Sciences Research Hub, Imperial College London London W12 0BZ UK e.tate@imperial.ac.uk.
Abstract:
The post-translational modification (PTM) ADP-ribosylation plays an important role in cell signalling and regulating protein function and has been implicated in the development of multiple diseases, including breast and ovarian cancers. Studying the underlying mechanisms through which this PTM contributes towards disease development, however, has been hampered by the lack of appropriate tools for reliable identification of physiologically relevant ADP-ribosylated proteins in a live-cell environment. Herein, we explore the application of an alkyne-tagged proprobe, 6Yn-ProTide-Ad (6Yn-Pro) as a chemical tool for the identification of intracellular ADP-ribosylated proteins through metabolic labelling. We applied targeted metabolomics and chemical proteomics in HEK293T cells treated with 6Yn-Pro to demonstrate intracellular metabolic conversion of the probe into ADP-ribosylation cofactor 6Yn-NAD+, and subsequent labelling and enrichment of PARP1 and multiple known ADP-ribosylated proteins in cells under hydrogen peroxide-induced stress. We anticipate that the approach and methodology described here will be useful for future identification of novel intracellular ADP-ribosylated proteins.
Insights
Researchers developed a new chemical probe, 6Yn-Pro, to identify ADP-ribosylated proteins in live cells. This tool aids in understanding how ADP-ribosylation contributes to diseases like cancer.
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.
