Natasha Vassileff, Jereme G. Spiers, Sarah E. Bamford, Rohan G. T. Lowe, Keshava K. Datta, Paul J. Pigram, Andrew F. Hill
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Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. 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引用次数: 0
摘要
神经炎症是神经退行性疾病的一个基本特征,通常出现在疾病病因的早期。小胶质细胞活化是神经炎症的一个重要诱因,可通过脂多糖(LPS)处理诱导小胶质细胞活化,导致一氧化氮合酶(iNOS)的诱导型表达,从而产生一氧化氮(NO)。一氧化氮通过 S-亚硝基化对半胱氨酸硫醇进行翻译后修饰,从而改变目标蛋白质的功能。此外,将这些经 NO 修饰的蛋白质包装到细胞外囊泡 (EVs) 中,可以在远处发出 NO 信号,从而进一步传播神经炎症表型。尽管如此,活化的小胶质细胞EVs的NO修饰蛋白质组尚未得到研究。本研究旨在确定 NO 信号在神经炎症中诱导的蛋白质翻译后修饰。从经 LPS 处理的小胶质细胞中分离出的 EVs 利用飞行时间-二次离子质谱(ToF-SIMS)进行质谱表面成像,此外还进行了碘标记和比较蛋白质组分析,以确定翻译后的 S-亚硝基化修饰。ToF-SIMS 成像成功鉴定了经 LPS 处理的小胶质细胞衍生 EV 蛋白中通过 NO 信号修饰的半胱氨酸硫醇侧链。此外,碘标记蛋白质组分析表明,经 LPS 处理的小胶质细胞 EVs 含有表明神经炎症的 S-亚硝基化蛋白质。这些蛋白包括已知的氮氧化物修饰蛋白和与 LPS 诱导的小胶质细胞活化相关的蛋白,它们可能在神经炎症交流中发挥重要作用。总之,这些结果表明,活化的小胶质细胞在神经炎症过程中可通过选择性包装 EVs 来产生广泛的 NO 信号变化。
Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular vesicles
Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.
期刊介绍:
The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies.
The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.