A Click Chemistry-Based Biorthogonal Approach for the Detection and Identification of Protein Lysine Malonylation for Osteoarthritis Research

Lysine malonylation is a post-translational modification
in which
a malonyl group, characterized by a negatively charged carboxylate,
is covalently attached to the ε-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 Caenorhabditis 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 the protein malonylation. Quantitative proteomics identified 1136
proteins corresponding to 8903 peptides, with 429 proteins showing
a 1-fold increase in the labeled group. Sirtuin 5 regulated 374 of
these proteins. Pull down of newly identified proteins, such as β-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 under OA conditions.