Rationale-The sirtuins are a class of NAD+ dependent deacetylase enzymes critical to mediating the beneficial effect of calorie restriction on age-related disease and lifespan. Identification of proteins targeted for deacetylation by the sirtuins remains an important challenge for the field. Unfortunately, the transient nature of interactions between sirtuins and their substrates means that traditional pull-down approaches used to screen for protein binding partners are unsuitable to these enzymes.
Objectives- We describe a novel strategy for identifying Sirtuin substrates using a modified biotin switch method.
Methods and Results- In our strategy, cell lysates are subjected to chemical acetylation, causing modification of amines on all free lysines. Recombinant sirtuin is then added to cell lysates in the presence of excess NAD+, and substrate proteins are deacetylated, exposing free amines on deacetylated lysines. Sulfo-NHS-biotin is added to selectively biotinylate deacetylated lysines, and biotinylated proteins are isolated by streptavidin pull-down and identified via mass spectrometry. This technique is suited not just to identification of deacetylation substrates of enzymes such as SIRT1, but as we show here is amenable to other post-translational modifications, such as protein desuccinylation carried out by SIRT5.
Conclusion-This strategy is a stable and powerful method that can be used for the isolation and identification of sirtuin substrates.