A one-step specific assay for continuous detection of sirtuin 2 activity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Sirtuins (SIRTs) are NAD+-dependent deacetylases with critical physiological roles.
• Developing efficient assays is crucial for screening SIRT modulators.

Purpose of the Study:

• To design and validate novel fluorescent probes for detecting sirtuin 2 (SIRT2) activity.
• To establish convenient assays for screening SIRT2 modulators in vitro and in cell lysates.

Main Methods:

• Synthesis of fluorescent nonapeptide probes based on SIRT substrates.
• Activity-based assessment of probes with SIRT1-SIRT3 and SIRT6.
• Optimization of probes for SIRT2 specificity and stability.
• Evaluation of probe performance in vitro and in cell lysates.

Main Results:

• Developed two specific and efficient fluorescent probes for SIRT2: ne-D9 and ne-K4a.
• Identified ne-K4a as suitable for in vitro modulator screening.
• Confirmed ne-D9 stability in cell lysate for detecting endogenous SIRT2 activity.
• Demonstrated the system's suitability for SIRT2 activity detection.

Conclusions:

• Presented a novel strategy for detecting SIRT2 activity.
• Developed specific fluorescent probes (ne-D9 and ne-K4a) for versatile SIRT2 detection.
• The assays facilitate efficient screening of SIRT2 modulators in various biological contexts.