Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid Modulators
Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid Modulators
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DOI：
10.1021/acsnano.7b01662
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发表时间：
2017-06-01
期刊：
影响因子：
17.1
通讯作者：
Adler-Abramoyich, Lihi
中科院分区：
文献类型：
--
作者：
Brahmachari, Sayanti;Arnon, Zohar A.;Adler-Abramoyich, Lihi
蛋白质聚集成淀粉样纤维的现象与大量病因学无关的重大疾病有关。揭示淀粉样蛋白自组装的机制和确定控制这一过程的治疗方向是至关重要的。这一领域的研究受到了一些挑战的阻碍，包括可重复性，蛋白质纯化产率低，以及淀粉样蛋白固有的聚集倾向，使其非常难以研究。在此，在组装机制的相似性，以及物理，化学和生物学特性的基础上，二苯丙氨酸纳米结构和芳香族氨基酸含有淀粉样蛋白纤维，我们报告了一个简单的，但强大的肽为基础的平台，可用于筛选小分子可能能够干扰的聚集过程和其作用模式的机械探索。该系统进行了验证，使用四个小分子抑制剂，并通过浊度测定，硫磺素T荧光，和电子显微镜检查的效果。在调节剂存在下，二苯丙氨酸的聚集特征与β-淀粉样多肽的聚集特征非常相似。迷迭香酸作为一种非常有效的抑制剂和不稳定剂的聚集体出现。迷迭香酸的化学计量变化的影响，也探讨了使用微流控技术的不稳定的过程。最后，使用质谱法检测二苯丙氨酸和抑制剂的等摩尔复合物的形成。这种方法不仅提供了一个系统的高通量筛选可能的抑制剂分子从较大的调制器库，但也非常有用的理解的机制方面的相互作用，导致抑制的过程。
The phenomenon of protein aggregation into amyloid fibrils is associated with a large number of major diseases of unrelated etiology. Unraveling the mechanism of amyloid self-assembly and identifying therapeutic directions to control this process are of utmost importance. Research in this field has been hampered by several challenges, including reproducibility, low protein purification yields, and the inherent aggregation propensity of amyloidogenic proteins, making them extremely difficult to study. Herein, on the basis of the similarity in the assembly mechanism, as well as the physical, chemical, and biological characteristics, of diphenylalanine nanostructures and aromatic amino acid containing amyloid fibrils, we report a simple, yet robust peptide-based platform that could be used for screening of small molecules potentially capable of interfering with the aggregation process and for mechanistic exploration of their mode of action. The system was validated using four small molecule inhibitors, and the effect was examined via turbidity assay, thioflavin T fluorescence, and electron microscopy. The aggregation profile of diphenylalanine was very similar to that of beta-amyloid polypeptide in the presence of the modulators. Rosmarinic acid emerged as an extremely potent inhibitor and a destabilizer of the aggregates. The effect of stoichiometric variation of rosmarinic acid on the process of destabilization was also probed using a microfluidic technique. Finally, the formation of equimolar complexes of diphenylalanine and inhibitors was detected using mass spectrometry. This approach not only provides a system for high-throughput screening of possible inhibitor molecules from larger libraries of modulators, but is also highly useful for understanding the mechanistic aspects of the interactions leading to the process of inhibition.