Targeting Fluorescent Sensors to Endoplasmic Reticulum Membranes Enables Detection of Peroxynitrite During Cellular Phagocytosis.
Targeting Fluorescent Sensors to Endoplasmic Reticulum Membranes Enables Detection of Peroxynitrite During Cellular Phagocytosis.
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DOI：
10.1021/acschembio.8b00535
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发表时间：
2018-09-21
影响因子：
4
通讯作者：
Peterson BR
中科院分区：
文献类型：
--
作者：
Knewtson KE;Rane D;Peterson BR
过氧亚硝酸盐是一种源自超氧化物和一氧化氮的高活性氧化剂。在正常的脊椎动物生理学中，一些吞噬细胞利用这种氧化剂作为对抗外来病原体的细胞毒素。为了提供检测内源性细胞过氧亚硝酸盐的新方法，我们合成了针对内质网（ER）膜的荧光传感器。这些膜的表面积非常高，大约比细胞质膜大 30 倍，被设想为一个巨大的细胞内平台，用于显示瞬态活性物质的传感器。通过将内质网靶向前荧光团与反应性酚连接起来，传感器被设计成可被过氧亚硝酸盐裂解并释放出高荧光内质网相关的对甲酚。水性缓冲液中的动力学研究揭示了线性自由能关系，其中供电子取代基加速了该反应。然而，在活细胞中，内源性细胞过亚硝酸盐的检测效率与内质网膜的结合成正比。通过掺入 2,6-二甲基苯酚来加速反应并增强这种亚细胞靶向，在添加抗体调理的触手微球后，可以轻松检测到活体 RAW 264.7 巨噬细胞中的内源性过氧化亚硝酸盐，而无需额外的刺激，这是其他已知荧光传感器无法检测到的过程。这种方法为研究活哺乳动物细胞中的瞬时活性物质提供了独特的敏感工具。
Peroxynitrite is a highly reactive oxidant derived from superoxide and nitric oxide. In normal vertebrate physiology, some phagocytes deploy this oxidant as a cytotoxin against foreign pathogens. To provide a new approach for detection of endogenous cellular peroxynitrite, we synthesized fluorescent sensors targeted to membranes of the endoplasmic reticulum (ER). The very high surface area of these membranes, approximately 30 times greater than the cellular plasma membrane, was envisioned as a vast intracellular platform for the display of sensors to transient reactive species. By linking an ER-targeted profluorophore to reactive phenols, sensors were designed to be cleaved by peroxynitrite and release a highly fluorescent ER-associated rhodol. Studies of kinetics in aqueous buffer revealed a linear free energy relationship where electron-donating substituents accelerate this reaction. However, in living cells, the efficiency of detection of endogenous cellular peroxynitrite was directly proportional to association with ER membranes. By incorporating a 2,6-dimethylphenol to accelerate the reaction and enhance this subcellular targeting, endogenous peroxynitrite in living RAW 264.7 macrophage cells could be readily detected after addition of antibody-opsonized tentagel microspheres, without additional stimulation, a process undetectable with other known fluorescent sensors. This approach provides uniquely sensitive tools for studies of transient reactive species in living mammalian cells.
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