Yu Dayoung, Gao Yue, Luzarowski Marcin, Seebach Elisabeth, Heitkamp Thomas, Börsch Michael, Ruppert Thomas, Kubatzky Katharina F
Medical Faculty Heidelberg, Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany.
Department of Infectious Diseases, University Hospital Heidelberg, Germany.
FEBS J. 2025 Jul;292(14):3736-3754. doi: 10.1111/febs.70090. Epub 2025 Apr 2.
Tumour necrosis factor ligand superfamily member 11 (TNFSF11; RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into osteoclasts. This process is characterised by changes in metabolic activity that support energy-consuming processes. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) through increased expression of the enzyme aconitate decarboxylase (ACOD1), which results in an upregulation of intracellular succinate levels. Succinate then causes post-translational succinylation of lysine residues. ACOD1 as an inducer of protein succinylation and the desuccinylase NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) are regulated differentially, and the initially high expression of ACOD1 decreases towards the end of differentiation, whereas SIRT5 levels increase. To mimic the effect of protein succinylation, diethyl succinate or a SIRT5 inhibitor was added during differentiation, which reduced the formation of large osteoclasts, showing its relevance for osteoclastogenesis. To identify succinylated proteins, we used an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Most lysine succinylated proteins were mitochondrial metabolic enzymes. Citrate synthase (CS), the enzyme catalysing the first reaction of the TCA cycle, showed a notable difference in succinylation levels before and after RANKL stimulation, with succinylation detected exclusively in stimulated cells. Immunoprecipitation assays confirmed CS succinylation. Using whole cell extracts, we observed that RANKL treatment decreased CS activity in a concentration-dependent manner. This suggests that CS could be critical in the context of energy production during osteoclastogenesis and that protein succinylation modulates the differentiation program of osteoclasts.
肿瘤坏死因子配体超家族成员11（TNFSF11；RANKL）和巨噬细胞集落刺激因子1受体（M-CSF）可将巨噬细胞分化为破骨细胞。这一过程的特征是代谢活动发生变化，以支持耗能过程。用RANKL处理会引发一种加速代谢的表型，糖酵解增强，并且通过乌头酸脱羧酶（ACOD1）表达增加导致三羧酸循环（TCA）最初受到破坏，从而使细胞内琥珀酸水平上调。然后，琥珀酸会导致赖氨酸残基的翻译后琥珀酰化。作为蛋白质琥珀酰化诱导剂的ACOD1和去琥珀酰化酶NAD依赖性蛋白质脱酰基酶线粒体沉默调节蛋白5（SIRT5）受到不同调节，ACOD1最初的高表达在分化末期下降，而SIRT5水平升高。为模拟蛋白质琥珀酰化的作用，在分化过程中添加了琥珀酸二乙酯或SIRT5抑制剂，这减少了大型破骨细胞的形成，表明其与破骨细胞生成相关。为鉴定琥珀酰化蛋白，我们采用了基于免疫亲和的液相色谱-串联质谱（LC-MS/MS）方法。大多数赖氨酸琥珀酰化蛋白是线粒体代谢酶。柠檬酸合酶（CS）是催化TCA循环第一步反应的酶，在RANKL刺激前后，其琥珀酰化水平存在显著差异，仅在受刺激细胞中检测到琥珀酰化。免疫沉淀试验证实了CS的琥珀酰化。使用全细胞提取物，我们观察到RANKL处理以浓度依赖性方式降低了CS活性。这表明CS在破骨细胞生成过程中的能量产生方面可能至关重要，并且蛋白质琥珀酰化调节破骨细胞的分化程序。
