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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Tandem fluorescence imaging of dynamic S-acylation and protein turnover
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Tandem fluorescence imaging of dynamic S-acylation and protein turnover

机译：动态S酰化和蛋白质更新的串联荧光成像

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摘要

The functional significance and regulation of reversible 5-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other 5-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.
机译：由于有效地定量分析棕榈酸酯周转的方法有限，尚不清楚各种蛋白质上可逆的5-酰化作用的功能意义和调控方式尚不清楚。在这里，我们描述了串联标记和检测方法，以同时监测动态S-棕榈酰化和蛋白质更新。通过将S-酰化和共翻译脂肪酸化学报道分子与正交可点击的荧光团相结合，Lck的双脉冲追踪分析揭示了T细胞活化后棕榈酸酯循环加快。随后的Lck棕榈酸酯周转的药理扰动表明，尚未鉴定出的丝氨酸水解酶有助于细胞中的动态S-酰化。除了双重脂肪酰化的蛋白质外，该串联荧光成像方法还可以使用叠氮高丙氨酸作为蛋氨酸替代品推广到其他5种酰化的蛋白质。这种方法的敏感性和效率应有助于细胞因子和调节蛋白S酰化的药物的功能表征。此外，可以使用其他化学报告基因使用这种串联成像方法分析各种蛋白质修饰，以研究蛋白质功能的动态调节。

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