Methods for studying human sirtuins with activity-based chemical probes

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Sirtuins are NAD+-dependent enzymes regulating crucial cellular processes like metabolism and transcription.
• Understanding sirtuin activity is vital for deciphering their roles in health and disease.
• Current methods for assessing sirtuin function have limitations in real-time or in situ analysis.

Purpose of the Study:

• To introduce a novel chemical probe technology for activity-based detection of sirtuins.
• To enable visualization of active sirtuin enzymes in diverse biological contexts.
• To establish protocols for applying these probes to recombinant proteins, cell lysates, and in situ labeling.

Main Methods:

• Development of thioacyllysine peptides with photo-cross-linker and bioorthogonal functionalities.
• Utilizing probes to form stalled reaction intermediates at the sirtuin active site.
• Employing photocrosslinking to create covalent adducts with active sirtuins.
• Visualizing labeled sirtuins via click chemistry with fluorescent tags.

Main Results:

• Successful labeling and visualization of active sirtuins using the developed chemical probes.
• Demonstration of probe utility across different sample types: recombinant proteins, whole cell lysates, and in situ.
• Establishment of robust protocols for probe application and detection.

Conclusions:

• The activity-based chemical probes provide a powerful tool for monitoring sirtuin enzymatic activity.
• This technology facilitates the study of sirtuin function in complex biological systems.
• The developed methods offer new avenues for investigating sirtuin-mediated biological processes.