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Updated: May 8, 2026
Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates
Published on: February 27, 2016
Generating mammalian sirtuin tools for protein-interaction analysis
Kathleen A Hershberger1, Jonathan Motley, Matthew D Hirschey
1Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, NC, USA.
Abstract:
The sirtuins are a family of NAD(+)-dependent deacylases with important effects on aging, cancer, and metabolism. Sirtuins exert their biological effects by catalyzing deacetylation and/or deacylation reactions in which Acyl groups are removed from lysine residues of specific proteins. A current challenge is to identify specific sirtuin target proteins against the high background of acetylated proteins recently identified by proteomic surveys. New evidence indicates that bona fide sirtuin substrate proteins form stable physical associations with their sirtuin regulator. Therefore, identification of sirtuin interacting proteins could be a useful aid in focusing the search for substrates. Described here is a method for identifying sirtuin protein interactors. Employing basic techniques of molecular cloning and immunochemistry, the method describes the generation of mammalian sirtuin protein expression plasmids and their use to overexpress and immunoprecipitate sirtuins with their interacting partners. Also described is the use of the Database for Annotation, Visualization, and Integrated Discovery for interpreting the sirtuin protein-interaction data obtained.
Insights
Researchers developed a new method to identify sirtuin protein interactors. This technique helps pinpoint sirtuin targets, aiding research into aging, cancer, and metabolism.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Sirtuins are NAD(+)-dependent deacylases impacting aging, cancer, and metabolism.
- Identifying specific sirtuin targets is challenging due to numerous acetylated proteins.
- Sirtuin substrates are known to physically associate with their regulators.
Purpose of the Study:
- To describe a novel method for identifying sirtuin protein interactors.
- To facilitate the discovery of bona fide sirtuin substrates.
- To aid in understanding sirtuin functions in biological processes.
Main Methods:
- Utilizing molecular cloning to create mammalian sirtuin protein expression plasmids.
- Employing immunochemistry techniques for overexpression and immunoprecipitation of sirtuins and interacting partners.
- Applying the Database for Annotation, Visualization, and Integrated Discovery (DAVID) for data interpretation.
Main Results:
- Successfully generated expression plasmids for sirtuin proteins.
- Established a method to co-immunoprecipitate sirtuins with their interacting proteins.
- Obtained data on sirtuin-protein interactions for further analysis.
Conclusions:
- The described method effectively identifies sirtuin protein interactors.
- This approach can aid in discovering novel sirtuin substrates.
- The findings support the strategy of identifying interacting proteins to find sirtuin targets.
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