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.

赖氨酸丙二酰化（lysine malonylation）是一种翻译后修饰（post-translational modification），即带有负电羧酸根特征的丙二酰基（malonyl group）共价结合于赖氨酸的ε-氨基侧链，进而影响蛋白质的结构与功能。本课题组在衰老与肥胖状态下的软骨组织中发现了Mak的表达上调，该事件与骨关节炎（OA）的发生发展密切相关。当前基于抗体的检测方法在鉴定Mak靶点时存在明显局限。在此，我们研发了一种炔基功能化探针MA-diyne，该探针可经代谢途径整合进入蛋白质，能够借助一价铜离子催化的点击化学反应，使标记后的蛋白质与叠氮基荧光染料或亲和纯化标签结合。凝胶内荧光成像实验证实，MA-diyne可在多种细胞类型与物种的蛋白质中实现整合，包括小鼠软骨细胞、脂肪细胞、人胚胎肾细胞HEK293T以及秀丽隐杆线虫（Caenorhabditis elegans）。下拉实验（pull-down）成功鉴定出已知的Mak靶蛋白，如甘油醛-3-磷酸脱氢酶（GAPDH）与醛缩酶（Aldolase）。在沉默信息调节因子5（Sirtuin 5）缺陷细胞中，MA-diyne的修饰程度更高，提示这些被修饰的蛋白质为Sirtuin 5的底物。脉冲追踪实验（pulse-chase）证实了蛋白质丙二酰化的动态特性。定量蛋白质组学分析共鉴定出1136种蛋白质，对应8903条肽段，其中429种蛋白质在标记组中的修饰水平提升1倍。Sirtuin 5可调控其中374种蛋白质的修饰状态。我们还对新鉴定出的蛋白质如β-肌动蛋白（β-actin）与信号转导与转录激活因子3（Stat3）开展了下拉验证实验。本研究证实，MA-diyne是一种功能强大的化学工具，可用于探究骨关节炎条件下赖氨酸丙二酰化的分子靶点及其生物学功能。