SIRT1 is an NAD+ dependent deacetylase located in the nucleus and cytoplasm of the cell. Research has shown that SIRT1 has been implicated to be involved in many important cellular functions such as neurodegenerative diseases and aging. SIRT1 has also been studied with resveratrol, a polyphenol, which was shown to modulate activity. While the mechanism of SIRT1 has been studied extensively, exact details are still not known, specifically the allosteric regulation by the N-terminal domain. We want to see if there is a correlation between the activity of SIRT1 and the distance of the N-terminal domain relative to the catalytic core. First, we examined the kinetic parameters (kcat and KM) of SIRT1 with peptide substrates with and without resveratrol. A recent study examined the activity of SIRT1 on peptides of different acetylation sites and found that resveratrol could either induce or not change SIRT1 activity. We will use a continuous enzyme-coupled assay, rather than an endpoint assay, to see if there were changes in either KM or kcat. If KM changes, SIRT1 activity is affected by a change in substrate recognition. If kcat changes, activity is affected by the change in chemistry in the catalytic core. The next step will be to determine the overall conformation change of SIRT1 using Small Angle X-ray Scattering (SAXS) techniques. From this, we could see the movement of the N-terminal domain to the catalytic core. Due to a previous study showing different conformations of SIRT1 with distinct curves, we will use these curves as a model when examining SIRT1 complexed with different combinations of resveratrol and peptide substrate. If resveratrol increases SIRT1 activity, we expect the N-terminal domain to move closer to the catalytic core. If resveratrol decreases SIRT1 activity, we expect the N-terminal domain to move farther away from the catalytic core. Based on the data from SAXS and assays we hope to see a link between the movement of the N-terminal domain and SIRT1 activity. Learning more about the allosteric mechanism of SIRT1 could aid in the design of therapeutic targets in modulating SIRT1 activity in the cell.

Using kinetics and small angle X-ray scattering to elucidate the allosteric regulation of SIRT1

2019 Fall National Meeting

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