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bioRxiv · 10.1101/2024.12.12.628274

A click chemistry-based biorthogonal approach for the detection and identification of protein lysine malonylation for osteoarthritis research

Binoy, A.·Nanjan, P.·Chellamuthu, K.·Liu, H.·Zhu, S.
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Abstract

Lysine malonylation is a post-translational modification where a malonyl group, characterized by a negatively charged carboxylate, is covalently attached to the {varepsilon}-amino side chain of lysine, influencing protein structure and function. Our laboratory identified Mak upregulation in cartilage under aging and obesity, contributing to osteoarthritis (OA). Current antibody-based detection methods face limitations in identifying Mak targets. Here, we introduce an alkyne-functionalized probe, MA-diyne, which metabolically incorporates into proteins, enabling copper(I) ion-catalyzed click reactions to conjugate labeled proteins with azide-based fluorescent dyes or affinity purification tags. In-gel fluorescence confirms MA-diyne incorporation into proteins across various cell types and species, including mouse chondrocytes, adipocytes, Hek293T cells, and C. elegans. Pull-down experiments identified known Mak proteins such as GAPDH and Aldolase. The extent of MA-diyne modification was higher in Sirtuin 5-deficient cells suggesting these modified proteins are Sirtuin 5 substrates. Pulse-chase experiments confirmed the dynamic nature of protein malonylation. Quantitative proteomics identified 1136 proteins corresponding to 8903 peptides with 429 proteins showing 1-fold increase in labeled group. Sirtuin 5 regulated 374 of these proteins. Pull down of newly identified proteins such as {beta}-actin and Stat3 was also done. This study highlights MA-diyne as a powerful chemical tool to investigate the molecular targets and functions of lysine malonylation in OA conditions.

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Binoy, A., Nanjan, P., Chellamuthu, K., Liu, H., Zhu, S.. 2024-12-13. A click chemistry-based biorthogonal approach for the detection and identification of protein lysine malonylation for osteoarthritis research. https://doi.org/10.1101/2024.12.12.628274

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