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bioRxiv · 10.1101/2025.05.25.655943

Ketone Bodies-mediated Cysteine Modifications Discovered by Chemical Proteomics

Zhou, Y.-F.·Zhang, L.·Niu, Z. L.·WANG, X.·Hunt, R.·Zhao, Y.·Sharifi, N.·Wang, Z. A.
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Abstract

All the studies of ketone body-dependent post-translational modifications (PTMs), notably those mediated by ketone bodies, {beta}-hydroxybutyrate (Bhb) and acetoacetate (Acac), have focused on lysine acylations. However, given the chemically diverse and reactive nature of metabolites generated, it remains unclear whether non-lysine modifications can also happen. Here, we report the synthesis of an acetoacetate-alkyne (Acac-alkyne) chemical probe that enables efficient metabolic labeling, robust fluorescent visualization, and mass spectrometry-based identification of Acac-modified proteins. By combining chemical proteomics with open-search strategy, we showed that Acac will induce previously uncharacterized cysteine modifications in mammalian cells. Notably, cysteine S-crotonation (Ccr) is validated by employing both probe-based and standard peptide-based co-elution assays. Metabolic pathway tracing further identifies BDH1 and ECHS1 as key enzymes that generate Ccr formation. Together, these findings establish ketone metabolism as a novel source of cysteine modifications and provide an alternative mechanistic pathway to explain the profound biological effects of ketone body.

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Zhou, Y.-F., Zhang, L., Niu, Z. L., WANG, X., Hunt, R., Zhao, Y., Sharifi, N., Wang, Z. A.. 2025-05-28. Ketone Bodies-mediated Cysteine Modifications Discovered by Chemical Proteomics. https://doi.org/10.1101/2025.05.25.655943

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