Taguchi Yuzuru, Shi Zhen-Dan, Ruddy Brian, Dorward David W, Greene Lois, Baron Gerald S
Laboratory of Persistent Viral Diseases and Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
Mol Biol Cell. 2009 Jan;20(1):233-44. doi: 10.1091/mbc.e08-06-0635. Epub 2008 Nov 5.
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.
荧光标记是对活细胞中的蛋白质进行成像的一种强大工具。然而，荧光标签所带来的空间效应会损害许多蛋白质的行为。在此，我们报告了一种新技术，即利用二硫苏糖醇（DTT）、乙二硫醇（EDT）和低温（IDEAL）进行即时标记，以朊病毒蛋白（PrP）和淀粉样前体蛋白（APP）为模型，对带有四半胱氨酸标签的细胞表面蛋白进行快速且特异性的荧光素砷络合物（FlAsH）标记。在朊病毒感染的细胞中，FlAsH标记的带有四半胱氨酸标签的PrP从正常异构体（PrPsen）转变为疾病相关异构体（PrPres），这表明该标签的空间效应最小。通过荧光凝胶成像对PrP和APP进行脉冲追踪分析，证明了IDEAL标记在研究蛋白质代谢中的实用性，即通过鉴定PrPsen一个尚未被识别的C端片段（C3）以及表征PrPres和APP代谢的动力学。抗朊病毒化合物E64（一种半胱氨酸蛋白酶抑制剂）可抑制C3的产生和PrPres的N端截短。令人惊讶的是，E64并不抑制新PrPres的合成，这为E64降低朊病毒感染细胞中PrPres稳态水平的机制提供了见解。为了扩展四半胱氨酸标签的通用性，我们创建了新的与Alexa Fluor和生物素偶联的四半胱氨酸结合分子，并将其应用于PrP内吞作用成像和超微结构定位。IDEAL标记将双砷衍生物的应用扩展到细胞外蛋白质以及微观成像之外。
