Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins
Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins
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DOI：
10.1091/mbc.e08-06-0635
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发表时间：
2009-01-01
影响因子：
3.3
通讯作者：
Baron, Gerald S.
中科院分区：
文献类型：
--
作者：
Taguchi, Yuzuru;Shi, Zhen-Dan;Baron, Gerald S.
荧光标记是活细胞中蛋白质成像的有力工具。然而，荧光标记所施加的空间效应损害了许多蛋白质的行为。在这里，我们报告了一种新的技术，即时与DTT，EDT，和低温（IDEAL）标记，快速和特异性FlAsH标记的四环素标记的细胞表面蛋白，使用朊病毒蛋白（PrP）和淀粉样前体蛋白（APP）作为模型。在朊病毒感染的细胞中，FlAsH标记的四环素标记的PrP从正常亚型（PrPsen）转化为疾病相关亚型（PrPres），表明标签的空间效应最小。通过荧光凝胶成像对PrP和APP的脉冲追踪分析证明了IDEAL标记在通过鉴定尚未识别的PrPsen的C-末端片段（C3）和通过表征PrPres和APP代谢的动力学来研究蛋白质代谢中的效用。C3代和N-末端截短的PrPres抑制的抗朊病毒化合物E64，半胱氨酸蛋白酶抑制剂。令人惊讶的是，E64不抑制新的PrPres的合成，提供了对E64降低朊病毒感染细胞中稳态PrPres水平的机制的深入了解。为了扩展四半胱氨酸标记的多功能性，我们创建了新的Alexa Fluor和生物素缀合的四半胱氨酸结合分子，其被应用于成像PrP内吞作用和超微结构定位。IDEAL标记将双砷衍生物的使用扩展到细胞外蛋白质和显微镜成像之外。
Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.