A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One‐Step Cleavage of the Dabcyl Quencher. (9th September 2016) - Kawaguchi, Mitsuyasu
Abstract: Sirtuins (SIRTs) are a family of NAD + ‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ϵ‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells. Abstract : Visualizing endogenous intracellular SIRT1 activity : We have developed sirtuin fluorescence probes, which can detect sirtuin activity in a one‐step procedure. We discovered that sirtuin can recognize and hydrolyze lysine residues acylated with Dabcyl quenchers at the ϵ‐amino group. With our sirtuin probes we succeeded in imaging SIRT1 activity in living cells.