Identifying the functional contribution of the defatty-acylase activity of SIRT6

Mammalian sirtuin 6 (SIRT6) exhibits many pivotal functions and multiple enzymatic activities, but the contribution of each activity to the various functions is unclear. We identified a SIRT6 G60A mutant that possesses efficient defatty-acylase activity, but has significantly decreased deacetylase activity in vitro and no detectable deacetylase activity in cells. The G60A mutant has decreased ability to bind NAD(+), but the presence of fatty-acyl lysine peptides restores NAD(+) binding, explaining the retention of the defatty-acylase activity. Using this mutant, we found that SIRT6’s defatty-acylase activity regulates the secretion of numerous proteins. Interestingly, many ribosomal proteins were secreted via exosomes from Sirt6 KO mouse embryonic fibroblasts, and these exosomes increased NIH 3T3 cell proliferation compared with control exosomes. Our data supports that distinct activities of SIRT6 regulate different pathways, and that the G60A mutant is a useful tool to study the contribution of the defatty-acylase activity to SIRT6’s various functions.