Dissecting the Functions of the Multiple Enzymatic Activities of SirT2 in Cancer

SirT2 is a member of a class of enzymes known as sirtuins that are NAD-dependent protein lysine deacylases.  SirT2 plays critical roles in many biological processes such as cell cycle regulation, stress response, and metabolism that are significant in human disease. SirT2 has traditionally been considered a robust deacetylase, and its known biological functions are attributed to this deacetylase activity. Recently, SirT2 has been found to remove fatty-acyl groups from lysine residues with a catalytic efficiency comparable to that of removing an acetyl group. However, it remains unclear whether the novel enzymatic activity has any physiological relevance and whether it contributes to the known biological functions of SirT2. To dissect the roles of the deacetylase and defatty-acylase activities in cancer, we aim to discover SirT2 mutants with only one enzymatic activity and probe the contribution of each activity of SirT2 in cancer. By mutating key residues in the substrate-binding pocket, we have isolated SirT2 mutants with selective defatty-acylase activity based on the in vitro SirT2 activity assay. With these mutant enzymes, we further performed kinetic measurements and activity assays using known SirT2 deacetylase and defatty-acylase target proteins as substrates. The results suggest that the SirT2 mutants with selective defatty-acylase activity could be potentially employed as a tool to study the specific function and physiological roles of SirT2 defatty-acylase activity. Overall, we will use these enzyme mutants to understand the various roles of SirT2 and thus to understand the potential of SirT2 modulators as anti-cancer therapies.