Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.
Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.
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DOI：
10.1021/acs.jproteome.8b00002
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发表时间：
2018-05-04
影响因子：
4.4
通讯作者：
Hang HC
中科院分区：
文献类型：
--
作者：
Thinon E;Fernandez JP;Molina H;Hang HC
S脂肪酰化是指长链脂肪酸，主要是棕榈酸酯(C16：0，S棕榈酰化)，通过蛋白质上的硫酯键与半胱氨酸残基的共价结合。这种翻译后和可逆的脂质修饰调节真核生物中蛋白质的功能和定位，在哺乳动物生理学和人类疾病中具有重要意义。虽然化学标记方法已经改进了S脂肪酰化蛋白的检测和富集化，但修饰位点的定位和内源性结合脂肪酸的特征仍然具有挑战性。在这里，我们描述了羟胺介导的S脂肪酰化蛋白的脂肪酸化学报告标记和修饰半胱氨酸残基的直接标记选择性地定位脂质修饰位点的整合和优化。与以前描述的方法相比，这提供了更好的丰富和直接鉴定许多蛋白质S-脂肪酰化位点。值得注意的是，我们直接确定了IFITM3的S脂肪酰化位点，IFITM3是一种重要的干扰素刺激的病毒进入抑制物，并进一步证明了高度保守的半胱氨酸残基主要被棕榈酸修饰。本文描述的方法应该有助于直接分析对哺乳动物生理和疾病重要的不同细胞类型和激活状态中的蛋白质S-脂肪酰化位点及其内源附着的脂肪酸。
S-fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins, and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.
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影响因子：
4.8
作者：
Gottlieb, Colin D.;Zhang, Sheng;Linder, Maurine E.
通讯作者：
Linder, Maurine E.
影响因子：
4.1
作者：
KOEGL, M;ZLATKINE, P;MAGEE, AI
通讯作者：
MAGEE, AI
影响因子：
30.3
作者：
Jones ML;Collins MO;Goulding D;Choudhary JS;Rayner JC
通讯作者：
Rayner JC
影响因子：
25
作者：
Cremona, M. Laura;Matthies, Heinrich J. G.;Pau, Kelvin;Bowton, Erica;Speed, Nicole;Lute, Brandon J.;Anderson, Monique;Sen, Namita;Robertson, Sabrina D.;Vaughan, Roxanne A.;Rothman, James E.;Galli, Aurelio;Javitch, Jonathan A.;Yamamoto, Ai
通讯作者：
Yamamoto, Ai
影响因子：
4.6
作者：
Collins MO;Woodley KT;Choudhary JS
通讯作者：
Choudhary JS