The enzyme sirtuin 6, or SIRT6, serves many key biological functions in regulating genome stability, DNA repair, metabolism and longevity, but how its multiple enzyme activities relate to its various functions is poorly understood.

A team of Cornell researchers, led by Hening Lin, professor of chemistry and chemical biology, has devised a method for isolating one specific enzyme activity to determine its contribution and lead to better overall understanding of SIRT6.

Their work, “Identifying the functional contribution of the defatty-acylase activity of SIRT6,” was published June 20 in Nature Chemical Biology. Xiaoyu Zhang, graduate student in chemistry and chemical biology and a member of the Lin Group, was lead author.

Their method involves a mutant form of SIRT6, a protein called G60A, which displays one of SIRT6’s enzyme activities (defatty-acylation) with no detectable activity in another (deacetylation) in cells.

Lin – who recently published a paper on the strong connection between another of the seven mammalian sirtuins, SIRT5 and healthy heart function – said Zhang’s work in identifying this mutant was “not very easy.”

“Normally when you want to understand a gene’s function or a protein’s function, the way you do it is delete that gene in cells and look at the effect,” he said. “In this case, you cannot do that because if you knock out or knock down SIRT6, you get rid of both [defatty-acylation and deacetylation].”

Zhang tested four potential SIRT6 mutant candidates before identifying G60A, then performed a battery of tests to confirm its defatty-acylase activity and lack of deacetylase activity. The experiments were replicated three times to confirm the results.

One of SIRT6’s functions as a result of defatty-acylation is the regulation of 50 proteins, including 41 ribosomal proteins, that are essential in the process of translation (the creation of proteins). These ribosomal proteins are secreted through exosomes – cell-derived delivery vesicles. The group’s data suggest SIRT6 inhibits ribosomal and other protein sorting to the exosomes, control of which has implications for developing novel therapeutics, Lin said.

“This offers a way to investigate how a protein is delivered to the exosome for secretion,” he said. “If you can really understand this, you could control what proteins are loading into the exosome and thus generating therapeutically useful exosomes.”

Other contributors included Richard Cerione, the Goldwin Smith Professor of Chemistry and Chemical Biology; Marc Antonyak, senior research associate in the College of Veterinary Medicine; former postdoctoral researcher Hong Jiang; and graduate students Saba Khan, Xiao Chen, Nicole Spiegelman and Jonathan Shrimp.

This work was supported by grants from the National Institutes of Health. Imaging was done in the Cornell Biotechnology Resource Center, which is supported by a grant from New York State Stem Cell Science.