免疫代谢物蛋白质修饰 S-2-琥珀酰半胱氨酸和 2,3-二羧丙基半胱氨酸的定量。

IF 4.2 2区 医学 Q1 ENDOCRINOLOGY & METABOLISM American journal of physiology. Endocrinology and metabolism Pub Date : 2024-04-01 Epub Date: 2024-02-07 DOI:10.1152/ajpendo.00354.2023
J Hunter Cox, Richard S McCain, Emery Tran, Shoba Swaminathan, Holland H Smith, Gerardo G Piroli, Michael Shtutman, Michael D Walla, William E Cotham, Norma Frizzell
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引用次数: 0

摘要

三羧酸(TCA)循环代谢产物富马酸盐会与氨基酸半胱氨酸发生非酶反应,生成 S-(2-琥珀酰)半胱氨酸(2SC),即蛋白质琥珀酸化。在脂多糖(LPS)刺激巨噬细胞和小胶质细胞的过程中,免疫代谢产物伊塔康酸会累积。衣康酸与半胱氨酸残基发生非酶反应,生成 2,3-二羧丙基半胱氨酸(2,3-DCP),称为蛋白质二羧丙基化。由于富马酸盐和伊他康酸盐的含量在活化的免疫细胞中会发生动态变化,因此 2SC 和 2,3-二氯丙醇的含量反映了这些代谢产物的丰度及其修饰蛋白质硫醇的能力。我们从蛋白质水解物中生成了 2SC 和 2,3-二氯丙醇的乙酯,并使用稳定同位素稀释质谱法测定了它们在 LPS 刺激的高侵袭性增殖不灭(HAPI)小胶质细胞中的丰度。)为了量化琥珀酰化和二羧丙基化的化学计量,用碘乙酸烷基化还原半胱氨酸,形成 S-羧甲基半胱氨酸(CMC),然后将其酯化。在经 LPS 处理的 HAPI 小胶质细胞中,伊塔康酸衍生的 2,3-二氯丙醇(而非富马酸衍生的 2SC)有所增加。化学计量学测量表明,在 LPS 刺激下,2,3-二氯丙醇占半胱氨酸总量的比例从 1.57% 增加到 9.07%。这种同时区分和量化 2SC 和 2,3-二氯丙醇的方法将在代谢性疾病的生理学研究中得到广泛应用。此外,我们发现现有的抗 2SC 抗体也能检测到结构相似的 2,3-二氯丙醇,因此 "琥珀酸分子 "可能更适合描述所识别的抗原。
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Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

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来源期刊
CiteScore
9.80
自引率
0.00%
发文量
98
审稿时长
1 months
期刊介绍: The American Journal of Physiology-Endocrinology and Metabolism publishes original, mechanistic studies on the physiology of endocrine and metabolic systems. Physiological, cellular, and molecular studies in whole animals or humans will be considered. Specific themes include, but are not limited to, mechanisms of hormone and growth factor action; hormonal and nutritional regulation of metabolism, inflammation, microbiome and energy balance; integrative organ cross talk; paracrine and autocrine control of endocrine cells; function and activation of hormone receptors; endocrine or metabolic control of channels, transporters, and membrane function; temporal analysis of hormone secretion and metabolism; and mathematical/kinetic modeling of metabolism. Novel molecular, immunological, or biophysical studies of hormone action are also welcome.
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