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Published on: February 7, 2021
A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One-Step Cleavage of the Dabcyl Quencher
Mitsuyasu Kawaguchi1, Shohei Ikegawa1, Naoya Ieda1
1Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan.
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.
Insights
Researchers developed novel probes to image sirtuin (SIRT) activity in living cells. A new probe, KST-F, specifically visualizes endogenous SIRT1 activity, overcoming limitations of current screening methods for therapeutic target development.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Sirtuins (SIRTs) are NAD+-dependent histone deacetylases implicated in metabolic diseases and cancer.
- Dysfunctional SIRTs present therapeutic challenges, but current screening methods are limited.
- A need exists for tools to visualize endogenous SIRT activity in living cells.
Purpose of the Study:
- To develop novel, sensitive probes for detecting sirtuin activity.
- To create tools for imaging endogenous SIRT activity in real-time within living cells.
- To identify specific probes for key sirtuin family members.
Main Methods:
- Design and synthesis of novel fluorescent probes based on FRET technology.
- Utilizing a histone H3-derived peptide substrate with a cleavable quencher.
- Testing probe activity against various SIRT isoforms and optimizing for cellular applications.
Main Results:
- Developed probes SFP3 and KST-F for detecting SIRT activity.
- SFP3 detected activities of SIRT1, -2, -3, and -6.
- KST-F demonstrated specificity for SIRT1 and enabled visualization of endogenous SIRT1 activity in living cells.
Conclusions:
- Novel fluorescent probes provide sensitive detection of SIRT activity.
- KST-F is a valuable tool for visualizing endogenous SIRT1 activity in live cells.
- These probes offer improved methods for studying SIRTs and developing therapeutic strategies.
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