Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Lysine deacetylases, including histone deacetylases (HDACs) and sirtuins (SIRTs), regulate crucial cellular processes like metabolism, DNA repair, and stress responses.
• Sirtuin isoforms SIRT2 and SIRT3 possess demyristoylase activity, modifying proteins by adding myristoyl groups.
• Existing assays for SIRT demyristoylase activity using myristoylated substrates are often complex or time-consuming.

Purpose of the Study:

• To develop a continuous, fluorescence-based activity assay for sirtuins (SIRTs) that directly measures demyristoylase activity.
• To create a reliable assay system for characterizing SIRT inhibitors, particularly for SIRT2.
• To enable high-throughput screening of SIRT inhibitors using native myristoylated substrates.

Main Methods:

• Developed novel sirtuin substrates with a native myristoyl residue on lysine for direct fluorescence detection.
• Utilized fluorescence changes upon deacylation for continuous monitoring of enzyme activity.
• Incorporated bovine serum albumin to enhance assay dynamic range by quenching substrate fluorescence.
• Validated the assay by determining IC50 and Ki values for SIRT2 inhibitors with high sensitivity.

Main Results:

• The new assay allows for direct, continuous measurement of sirtuin demyristoylase activity.
• The assay exhibits excellent kinetic properties, with low nM K_M values and high specificity constants.
• The developed method accurately determined inhibitor potency (IC50 and K_i values) for SIRT2 using very low enzyme concentrations (50 pM).
• The use of native myristoyl residues avoids artifacts associated with modified fatty acyl groups in other assays.

Conclusions:

• A novel, sensitive, and continuous fluorescence assay for sirtuin demyristoylase activity has been established.
• This assay system provides a significant advancement for studying SIRT2 and SIRT3 activity and for screening inhibitors.
• The assay's efficiency and accuracy facilitate the characterization of SIRT inhibitors in various microtiter plate formats.