Thioflavin T: not an all-rounder, but a trustworthy friend for over 27 years
Thioflavin T: not an all-rounder, but a trustworthy friend for over 27 years
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硫磺素 T：不是多面手，而是超过 27 年值得信赖的朋友
DOI：
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
H. Naiki
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文献类型：
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作者：
H. Naiki
淀粉样原纤维形成的分子机制的详细体外分析对于阐明淀粉样变性的分子发病机制是必不可少的。 1989年，我们基于硫黄素T（ThT）的独特特性，开发了一种新型荧光方法来体外鉴定淀粉样原纤维[1]。利用荧光光谱，我们发现虽然ThT本身在450 nm激发时不发出明显的荧光，但在与淀粉样原纤维结合后，它在490 nm处发出明亮的荧光。自我们的第一份报告以来，ThT 已成为选择性鉴定体内和体外淀粉样原纤维的最广泛使用的“金标准”之一 [2,3]。自从匹兹堡大学研究小组开发了一种亲脂性 ThT 类似物匹兹堡化合物 B (PiB) 作为一种潜在的淀粉样蛋白成像 PET 示踪剂后，PiB 被广泛用于阿尔茨海默病的诊断 [2]。 ThT 测定系统有几个优点。首先，在恒定的 ThT 浓度下，荧光变化在淀粉样原纤维的较宽重量浓度范围内呈相当线性的变化 [1]。基于ThT的这一特性，我们在体外精确研究了小鼠AApoA-II淀粉样原纤维的聚合动力学。在 20 世纪 90 年代的奥斯陆研讨会上，我们提出淀粉样原纤维的形成可以通过一级动力学模型来解释：即淀粉样原纤维的延伸是通过前体蛋白连续结合到现有原纤维的末端来进行的 [4]。其次，ThT 实际上并不影响体外淀粉样原纤维形成的动力学。基于这一特性，ThT 通常包含在反应混合物中，以便使用 ELISA 酶标仪对聚集动力学进行高通量监测。我们还使用全内反射荧光显微镜在单原纤维水平上实时观察种子依赖性淀粉样蛋白原纤维生长的 Ab(1–40) [5]。 ThT 检测系统存在几个缺陷。首先，ThT 本身并不识别淀粉样蛋白原纤维，而是与淀粉样蛋白原纤维表面常见的分子凹槽结合，并由于围绕分子中间单个 C-C 键的旋转而被激活 [3]。实际上，DNA、环糊精、聚合物膜、SDS 胶束和多孔硅也可以激发ThT 荧光[3]。因此，为了在体外鉴定淀粉样原纤维，我们应该小心地将该系统与其他生化方法（例如浊度测量、EM、AFM、CD 和 ATR-FTIR）一起使用。其次，ThT对多种类型淀粉样原纤维的亲和力和单位重量的荧光强度彼此不同[1]。因此，为了比较不同类型淀粉样原纤维之间的 ThT 荧光，我们应该进行广泛的对照实验。第三，ThT 系统无法完全检测原纤维形成途径中的早期聚集体（即寡聚物和原纤维）。一般来说，当淀粉样前体蛋白在体外孵育时，ThT荧光动力学呈现S形曲线。在最初的滞后阶段，形成低聚物、原纤维和原纤维种子。因此，为了精确分析淀粉样原纤维形成的早期聚集途径，应使用各种生化和生物物理方法（例如 SEC、DLS）。最后，当我们评估有机化合物对淀粉样蛋白原纤维形成的影响时，我们应该非常小心，因为有些化合物与 ThT 竞争结合到淀粉样蛋白原纤维的表面，导致 ThT 荧光减弱。在“ThT检测陷阱——方法能否标准化？”（7月7日星期四）会议中，我们深入讨论了如何将ThT检测系统安全地应用于未来的淀粉样蛋白研究。
The detailed in vitro analysis of the molecular mechanisms of amyloid fibril formation is indispensable to elucidate the molecular pathogenesis of amyloidosis. In 1989, we developed a novel fluorometric method to identify amyloid fibrils in vitro based on the unique characteristics of thioflavin T (ThT) [1]. Using fluorescence spectroscopy, we found that although ThT itself emits no significant fluorescence when it is excited at 450 nm, it fluoresces brightly at 490 nm after it binds to amyloid fibrils. Since our first report, ThT has become one of the most widely used ‘‘gold standards’’ for selectively identifying amyloid fibrils both in vivo and in vitro [2,3]. Since the group of University of Pittsburgh developed a lipophilic ThT analog, Pittsburgh Compound B (PiB) as a potential amyloid-imaging PET tracer, PiB is widely used for the diagnosis of Alzheimer’s disease [2]. The ThT assay system has several advantages. First, under a constant ThT concentration, the fluorescence change is quite linear over a wide range of the weight concentration of amyloid fibrils [1]. Based on this property of ThT, we precisely investigated the polymerization kinetics of murine AApoA-II amyloid fibrils in vitro. In the 1990s Oslo symposium, we presented that amyloid fibril formation can be explained by a first-order kinetic model: that is, extension of amyloid fibrils proceeds via the consecutive association of precursor proteins onto the ends of existing fibrils [4]. Second, ThT does not practically affect the kinetics of amyloid fibril formation in vitro. Based on this property, ThT is generally included in the reaction mixture to perform the high-throughput monitoring of aggregation kinetics using an ELISA plate reader. We also visualized seed-dependent amyloid fibril growth of Ab(1–40) in real-time at the single fibril level using total internal reflection fluorescence microscopy [5]. There are several pitfalls of ThT assay system. First, ThT does not recognize amyloid fibrils per se, but binds to the molecular groove found typically on the surface of amyloid fibrils and becomes activated as a result of the rotation around a single C–C bond in the middle of the molecule [3]. Actually, the ThT fluorescence can also be evoked by DNA, cyclodextrin, polymer membranes, SDS micelles and porous silicon [3]. Thus, to identify amyloid fibrils in vitro, we should carefully use this system together with other biochemical methods, e.g., turbidity measurement, EM, AFM, CD and ATR-FTIR. Second, the affinity of ThT for many types of amyloid fibrils and the fluorescence intensity per unit weight are different from each other [1]. Thus, to compare the ThT fluorescence between different types of amyloid fibrils, we should perform extensive control experiments. Third, the ThT system cannot fully detect the early aggregates (i.e., oligomers and protofibrils) in the fibril-forming pathway. Generally, when amyloid precursor proteins are incubated in vitro, ThT fluorescence kinetics shows a sigmoidal curve. During the initial lag phase, oligomers, protofibrils and fibril seeds are formed. Thus, to precisely analyze the early aggregation pathway of amyloid fibril formation, various biochemical and biophysical methods (e.g., SEC, DLS) should be used. Finally, we should be extremely careful when we evaluate the effects of organic compounds on the amyloid fibril formation, because some compounds compete with ThT to bind to the surface of amyloid fibrils, leading to the reduction in ThT fluorescence. In the session of ‘‘ThT assay pitfalls – can the method be standardised?’’ (Thursday 7 July), we thoroughly discussed how to apply the ThT assay system safely to the future amyloid studies.
影响因子：
4.9
作者：
Mathis CA;Mason NS;Lopresti BJ;Klunk WE
通讯作者：
Klunk WE