Peptide arrays identify isoform-selective substrates for profiling endogenous lysine deacetylase activity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Lysine deacetylases (KDACs) play crucial roles in cellular processes.
• Understanding the specific functions of individual KDAC isoforms is challenging.
• Existing assays for KDAC activity can perturb enzyme function.

Purpose of the Study:

• To develop isoform-selective peptide substrates for measuring endogenous KDAC activities.
• To establish a label-free method for profiling KDAC activity in complex biological samples.
• To investigate the role of KDACs in cell cycle regulation.

Main Methods:

• Peptide arrays were synthesized and immobilized on gold surfaces.
• Substrate specificity of KDAC isoforms (KDAC2, KDAC3, KDAC8, SIRT1) was determined.
• Self-assembled monolayers for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (SAMDI-MS) was used for analysis.
• Nuclear extracts from various cell types were used to measure endogenous KDAC activities.

Main Results:

• Novel isoform-selective peptide substrates for KDACs were identified.
• SAMDI-MS enabled label-free quantification of endogenous KDAC activities.
• KDAC activity was profiled throughout the HeLa cell cycle.
• A KDAC3-selective peptide's activity profile correlated with H4 histone acetylation during the cell cycle.

Conclusions:

• The developed peptide substrates and SAMDI-MS method provide a powerful tool for studying KDACs.
• This approach allows for the investigation of differential KDAC isoform functions in biological processes.
• Findings suggest a role for KDAC3 in regulating cell cycle progression via histone acetylation.