Detection of vimentin serine phosphorylation by multicolor Quenchbodies
Detection of vimentin serine phosphorylation by multicolor Quenchbodies
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DOI：
10.1016/j.bios.2012.06.030
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发表时间：
2013-02-15
影响因子：
12.6
通讯作者：
Ueda, Hiroshi
中科院分区：
文献类型：
--
作者：
Jeong, Hee-Jin;Ohmuro-Matsuyama, Yuki;Ueda, Hiroshi
蛋白质磷酸化是细胞内信号转导的关键环节，针对靶蛋白磷酸化的荧光生物传感器被认为是细胞生物学和药物筛选的重要工具。波形蛋白是最丰富的中间丝蛋白，其特定的丝氨酸（Ser）残基以细胞周期依赖的方式被磷酸化。它的结构和功能特征通过磷酸化修饰，从而影响细胞的生物学特性。在这里，我们提出了检测波形蛋白Ser 71磷酸化（PS 71）和波形蛋白Ser 82磷酸化（PS 82）使用一种新的荧光生物传感器Quenchbody，它的工作原理是抗原依赖性的去除猝灭效应的内在色氨酸残基上的羧基四甲基罗丹明（塔姆拉）染料纳入的N-末端区域的单链抗体可变区。首先，我们发现若丹明6 G（R6 G）标记的淬体显示出比TAMRA标记的更上级的响应。接下来，我们制作了几个Quenchbodies来检测PS 71和PS 82。优化反应条件后，在两个位置用R6 G标记的V-H-V-L型PS 71 Quenchbody的荧光强度以抗原依赖性方式增加至4.0倍。此外，双重R6 G标记的V-L-V-H型PS 82 Quenchbody的荧光强度在加入抗原肽后立即增加至6.7倍，也表明由于染料之间的H-二聚体形成而更深的猝灭。由于其结构简单，基于猝灭体的磷酸化生物传感器将以快速、简单和高灵敏度的方式广泛应用于体外诊断、药物筛选和成像。(c)2012 Elsevier B. V.保留所有权利。
Protein phosphorylation is a key event in intracellular signal transduction, and fluorescent biosensor for the specific phosphorylation event in a target protein is considered highly useful as a tool of cellular biology and drug screening. Vimentin, the most abundant intermediate filament protein, is phosphorylated at its specific serine (Ser) residues in a cell cycle dependent manner. Its structural and functional characteristics are modified by the phosphorylation, which affects biological properties of the cell. Here we present the detection of the vimentin Ser71 phosphorylation (PS71) and the vimentin Ser82 phosphorylation (PS82) using a novel fluorescent biosensor Quenchbody, which works on the principle of antigen-dependent removal of a quenching effect by intrinsic tryptophan residues on a carboxytetramethylrhodamine (TAMRA) dye incorporated at the N-terminal region of single chain antibody variable region. First, we found that rhodamine 6G (R6G)-labeled Quenchbody shows superior response than TAMRA-labeled one. Next, we made several Quenchbodies to detect PS71 and PS82. After optimization of reaction conditions, the fluorescence intensity of V-H-V-L type PS71 Quenchbody labeled with R6G at two positions was increased to 4.0-fold in an antigen dependent manner. Furthermore, the fluorescence intensity of doubly R6G-labeled V-L-V-H type PS82 Quenchbody was increased to 6.7-fold immediately after adding antigen peptide, also suggesting deeper quenching due to H-dimer formation between the dyes. Due to its simplicity, the Quenchbody-based phosphorylation biosensors will be widely applicable to in vitro diagnostics, drug screening and imaging in a rapid, simple and high-sensitive manner. (c) 2012 Elsevier B.V. All rights reserved.