Labeling lysine acetyltransferase substrates with engineered enzymes and functionalized cofactor surrogates

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Protein lysine acetyltransferases (KATs) play crucial roles in biological and pathological processes.
• Understanding KAT function requires knowledge of their substrate localization within the cellular proteome.
• Current methods for identifying KAT substrates are limited.

Purpose of the Study:

• To design and apply novel chemical probes for labeling and detecting substrates of major human KAT enzymes.
• To develop a versatile chemical biology strategy for proteomic-level profiling of KAT targets.

Main Methods:

• Engineered KAT enzymes were combined with synthetic acetyl-CoA surrogates.
• These synthetic surrogates effectively labeled KAT substrates, overcoming competition from endogenous acetyl-CoA.
• Labeled substrates were detected using fluorescent reporters via copper-catalyzed azide-alkyne cycloaddition.

Main Results:

• A facile method for labeling and detecting substrates of three major human KAT enzymes was established.
• The approach enabled effective labeling even in the presence of native acetyl-CoA.
• Proteomic-level profiling of KAT targets was achieved using this strategy.

Conclusions:

• The developed chemical probes and synthetic cofactor system offer a versatile strategy for studying KATs.
• This method facilitates the elucidation of biological and pathological functions of KATs by identifying their targets.
• The approach provides a powerful tool for chemical biology research in proteomics.