Chemical Proteomics Identifies Ketogenesis-Mediated Cysteine Modifications Regulating Redox Function.

Area of Science:

• Biochemistry
• Molecular Biology
• Metabolomics

Background:

• Ketogenesis produces ketone bodies (Bhb, Acac) that mediate lysine acylations.
• The potential for non-lysine post-translational modifications (PTMs) by reactive metabolites remains unexplored.

Purpose of the Study:

• To investigate novel PTMs induced by acetoacetate.
• To identify the specific proteins and amino acid residues modified by acetoacetate.
• To elucidate the functional consequences of these new modifications.

Main Methods:

• Development of an acetoacetate-alkyne (Acac-alkyne) chemical probe for metabolic labeling.
• Application of chemical proteomics with an open-search strategy.
• Validation using probe-based and peptide-based co-elution assays.
• Metabolic pathway tracing to identify key enzymes.

Main Results:

• Acetoacetate induces previously uncharacterized cysteine modifications in mammalian cells.
• Cysteine crotonation (Ccr) was identified and validated as a novel PTM.
• BDH1 and ECHS1 were identified as key enzymes in Ccr formation.
• Ccr at PRDX3 C229 impairs protein dimerization and redox activity.

Conclusions:

• Ketone metabolism is a novel source of cysteine modifications.
• Cysteine crotonation provides a new mechanistic link between ketone bodies and cellular processes.
• This discovery offers insights into the regulation of reactive oxygen species by ketone bodies.