METHODS: A mouse HACE model was established by combination treatment with hypobaric hypoxia exposure and lipopolysaccharides (LPS) stimulation. Lactylated-proteomic analysis of microglia was performed to reveal the global profile of protein lactylation. Molecular modeling was applied to evaluate the 3-D modeling structures. A combination of experimental approaches, including western blotting, quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and RNA interference, were used to explore the underlying molecular mechanisms.
RESULTS: We found that hypoxia exposure increased the lactate concentration and lactylation in mouse HACE model. Moreover, hypoxia aggravated the microglial neuroinflammatory response in a lactate-dependent manner. Global profiling of protein lactylation has shown that a large quantity of lysine-lactylated proteins are induced by hypoxia and preferentially occur in protein complexes, such as the NuRD complex, ribosome biogenesis complex, spliceosome complex, and DNA replication complex. The molecular modeling data indicated that lactylation could affect the 3-D theoretical structure and increase the solvent accessible surface area of HDAC1, MTA1 and Gatad2b, the core members of the NuRD complex. Further analysis by knockdown or selectively inhibition indicated that the NuRD complex is involved in hypoxia-mediated aggravation of inflammation.
CONCLUSIONS: These results revealed a comprehensive profile of protein lactylation in microglia and suggested that protein lysine lactylation plays an important role in the regulation of protein function and subsequently contributes to the neuroinflammatory response under hypoxic conditions.
方法:通过低压低氧暴露和脂多糖(LPS)刺激联合治疗建立小鼠HACE模型。对小胶质细胞进行了酰化蛋白质组学分析,以揭示蛋白质酰化的总体概况。分子建模用于评估3-D建模结构。实验方法的结合,包括西方印迹,定量实时逆转录聚合酶链反应(qRT-PCR),和酶联免疫吸附测定(ELISA),共聚焦显微镜和RNA干扰,用于探索潜在的分子机制。
结果:我们发现低氧暴露会增加小鼠HACE模型中的乳酸浓度和乳酸化。此外,缺氧以乳酸依赖性方式加重了小胶质神经炎症反应。蛋白质乳酸化的全局分析表明,大量的赖氨酸-乳酸化蛋白质是由缺氧诱导的,并优先出现在蛋白质复合物中。比如NuRD综合体,核糖体生物合成复合物,剪接体复合体,和DNA复制复合体.分子模型数据表明,乳化会影响HDAC1,MTA1和Gatad2b的3-D理论结构并增加溶剂可及表面积,NuRD综合体的核心成员。通过敲低或选择性抑制的进一步分析表明,NuRD复合物参与缺氧介导的炎症加重。
结论:这些结果揭示了小胶质细胞中蛋白质的全面乳酸化,并提示蛋白质赖氨酸的乳酸化在蛋白质功能的调节中起重要作用,随后在缺氧条件下促进神经炎症反应。