Genetically encoding ε-N-methacryllysine into proteins in live cells.

Area of Science:

• Biochemistry and Molecular Biology
• Post-Translational Modifications
• Proteomics

Background:

• Lysine acylation is a crucial post-translational modification (PTM) involved in diverse cellular functions.
• While thousands of lysine acylation sites are known, only 27 lysine methacrylation (Kmea) sites have been identified, exclusively in histones.
• Distinguishing Kmea from its isomer, lysine crotonylation (Kcr), poses biochemical challenges.

Purpose of the Study:

• To identify Kmea sites on a non-histone protein, Cyclophilin A (CypA).
• To investigate the functional roles of Kmea in CypA.
• To develop a method for studying Kmea modification and its interactions in live cells.

Main Methods:

• Identification of Kmea sites on Cyclophilin A (CypA).
• Genetic code expansion to incorporate ε-N-Methacryllysine (MeaK), a non-canonical amino acid (ncAA), into target proteins.
• Affinity-purification mass spectrometry (MS) to identify proteins interacting with methacrylated CypA.

Main Results:

• Kmea was identified on the non-histone protein CypA.
• Kmea at CypA site 125 was found to regulate cellular redox homeostasis.
• HDAC1 was identified as a regulator of Kmea on CypA, and genetically encoded Kmea can be further methylated to ε-N-methyl-ε-N-methacrylation (Kmemea) in live cells.

Conclusions:

• This study expands the known landscape of Kmea modifications beyond histones to non-histone proteins like CypA.
• Kmea on CypA plays a significant role in regulating cellular redox balance.
• The developed genetic code expansion approach provides a powerful tool for studying Kmea functions and interactions in vivo.