A Cell-Permeable Biscyclooctyne As a Novel Probe for the Identification of Protein Sulfenic Acids
A Cell-Permeable Biscyclooctyne As a Novel Probe for the Identification of Protein Sulfenic Acids
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DOI：
10.1021/acschembio.6b00742
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发表时间：
2016-12-01
影响因子：
4
通讯作者：
Olson, Michael F.
中科院分区：
文献类型：
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作者：
McGarry, David J.;Shchepinova, Maria M.;Olson, Michael F.
活性氧通过蛋白质硫醇的氧化在细胞信号传导和稳态中充当重要的第二信使。然而，蛋白质氧化的动态性质和现有分子探针缺乏敏感性阻碍了我们对此类反应的理解；因此，需要新的工具来应对这些挑战。我们设计了应变双环[6.1.0]壬炔（BCN-E-BCN）的双功能变体，它能够标记细胞内蛋白质磺酸以进行双正交无铜点击化学。在验证研究中，BCN-E-BCN 结合肌动蛋白切断蛋白肌丝蛋白丝切蛋白的磺酰化形式，而同源半胱氨酸残基的突变则消除了其结合。 BCN-E-BCN 具有细胞渗透性，可与培养细胞中的半胱氨酸磺酸快速反应。使用不同的叠氮标记缀合物，我们证明 BCN-E-BCN 可用于检测磺化蛋白质的各种应用。值得注意的是，叠氮化物标记的荧光团与体内产生的 BCN-E-BCN 标记的蛋白质的环加成可以通过荧光显微镜观察到，以揭示其定位。这些发现证明了一种检测和捕获次磺酸的新颖且多方面的方法。
Reactive oxygen species act as important second messengers in cell signaling and homeostasis through the oxidation of protein thiols. However, the dynamic nature of protein oxidation and the lack of sensitivity of existing molecular probes have hindered our understanding of such reactions; therefore, new tools are required to address these challenges. We designed a bifunctional variant of the strained bicydo[6.1.0]nonyne (BCN-E-BCN) that enables the tagging of intracellular protein sulfenic acids for biorthogonal copper-free click chemistry. In validation studies, BCN-E-BCN binds the sulfenylated form of the actin-severing protein cofilin, while mutation of the cognate cysteine residues abrogates its binding. BCN-E-BCN is cell permeable and reacts rapidly with cysteine sulfenic acids in cultured cells. Using different azide-tagged conjugates, we demonstrate that BCN-E-BCN can be used in various applications for the detection of sulfenylated proteins. Remarkably, cycloaddition of an azide-tagged fluorophore to BCN-E-BCN labeled proteins produced in vivo can be visualized by fluorescence microscopy to reveal their localization. These findings demonstrate a novel and multifaceted approach to the detection and trapping of sulfenic acids.