SIRT1 is an NAD+-dependent protein deacetylase that plays an important role in pathological and physiological events. This enzyme is involved in neurodegeneration, cancer, obesity, and the aging process. The purpose of this lab is to study how the unstructured N-terminus domain of SIRT1 regulates its catalytic activity in a substrate-specific manner. To have a better understanding, we will observe how resveratrol, a small molecule, modulates SIRT1 activity. Resveratrol is a natural phenol product from plants that has been shown to modulate SIRT1 activity by interacting with the N-terminus domain of SIRT1. Recent studies have shown that resveratrol has different effects on SIRT1 activity based on the different acetylated peptide substrates used, including increase, decrease and no change in deacetylation activity. Our investigation focuses on the detailed kinetics information on how resveratrol changes the activity of SIRT1 in a substrate-specific manner. The aim of this study is to investigate how the N-terminal domain of SIRT1 regulates catalytic activity, in which we hypothesize that the conformational change of the N-terminal domain affects the ability of SIRT1 to recognize its substrates. To test this hypothesis, peptides are selected based on three categories where SIRT1 deacetylation activity was activated, deactivated, or unchanged by resveratrol. SIRT1 activity towards these peptides will then be examined with a coupling assay to obtain their kcat and KM values. A change in KM would suggest resveratrol modulates the substrate recognition of SIRT1, likely through conformational change of the N-terminus domain, while a change in kcat would suggest an effect on the chemical reaction. These can determine the role that the N-terminus plays in SIRT1 deacetylation activity. Our results reveal how resveratrol contributes to affecting the N-terminus domain of SIRT1 to modulate its enzyme activity. Collectively, the understanding of the substrate-specific regulation of SIRT1 by resveratrol may serve as further information for designing target specific drugs for therapeutic purpose.

Analyzing resveratrol effects on SIRT1 with warying peptide substrates using an enzyme-coupled assay

2019 Fall National Meeting

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