Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports...

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Lysine lactylation is a crucial post-translational modification involved in immunity, cancer, and neurodegenerative diseases.
• Genetic code expansion (GCE) enables site-specific protein lactylation but is limited by inefficient cellular uptake of lactyllysine (Klac).

Purpose of the Study:

• To develop a method for enhanced cellular uptake of lactyllysine (Klac) for site-specific protein lactylation.
• To investigate the functional consequences of protein lactylation using improved yields of lactylated proteins.

Main Methods:

• Design and synthesis of esterified Klac derivatives, such as ethylated Klac (KlacOEt), to increase lipophilicity and cellular uptake.
• Application of GCE with KlacOEt for site-specific lactylation of proteins of interest (POIs) in *Escherichia coli* and HEK293T cells.
• Utilized immunoprecipitation mass spectrometry and Target Responsive Accessibility Profiling to analyze lactylated proteins.

Main Results:

• KlacOEt significantly enhanced cellular uptake, doubling the expression of site-specifically lactylated POIs.
• Verified increased yield of precisely lactylated fructose-bisphosphate aldolase A (ALDOA) using KlacOEt.
• Demonstrated that lactylation at ALDOA-K147 alters protein conformation, potentially reducing enzyme activity.

Conclusions:

• Esterified Klac derivatives effectively improve the yield of lactylated proteins via GCE.
• This enhanced approach enables detailed investigation of lactylation's effects on protein interactions, conformation, and activity.
• Provides a powerful tool for dissecting the biological roles of lysine lactylation in various cellular processes and diseases.