Seraj Zahra, Seyedarabi Arefeh, Saboury Ali Akbar, Habibi-Rezaei Mehran, Ahmadian Shahin, Ghasemi Atiyeh
Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
School of Biology, University College of Science, University of Tehran, Tehran, Iran.
PLoS One. 2018 Jan 22;13(1):e0189754. doi: 10.1371/journal.pone.0189754. eCollection 2018.
This study investigated for the first time the molecular effectiveness of 'aroma' from three small molecules including a phenol (phenyl ethyl alcohol; PEA) and an aldehyde (cinnamaldehyde; Cin) both containing an aromatic ring, and a diamine (N,N,N,N'- Tetramethylethylenediamine; TEMED) at two different amounts (small; S and large; L) in preventing hen egg white lysozyme (HEWL) amyloid fibril formation using Thioflavin T and Nile red fluorescence assays, circular dichroism spectroscopy, SDS-polyacrylamide gel electrophoresis, atomic force microscopy, dynamic light scattering and HEWL activity test. Interestingly, the results revealed that (1) the aroma of PEA, identified as an active constituent of Rosa damascena, prevented fibril formation since PEA-L was able to trap the oligomeric form of HEWL in contrast to PEA-S where protofibrils but not mature fibrils were formed; (2) Cin, previously shown to prevent fibril formation in the liquid form, was also shown to do so in the aroma form by producing protofibrils and not mature fibrils in both Cin- L and Cin-S aroma forms and (3) the aroma of TEMED-L was able to retain HEWL's native structure completely and prevented both aggregation and fibril formation, while TEMED-S prevented HEWL fibril formation and instead directed the pathway towards amorphous aggregate formation. Furthermore, the ability to trap oligomeric species (by PEA-L aroma) is of great importance for further research as it provides routes for preventing the formation of toxic oligomeric intermediates along the fibrillation pathway. Last but not least, the novelty of this in vitro study on the effect of aroma at the molecular level with a unique experimental set-up using HEWL as a model protein in assessing amyloid fibril formation paves the way for more and detailed studies on the importance of aroma producing molecules and their effects.
本研究首次使用硫黄素T和尼罗红荧光测定法、圆二色光谱法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法、原子力显微镜法、动态光散射法以及蛋清溶菌酶（HEWL）活性测试，研究了三种小分子“香气”在两种不同用量（少量；S和大量；L）下预防HEWL淀粉样原纤维形成的分子效果。这三种小分子包括一种含有芳环的酚（苯乙醇；PEA）、一种醛（肉桂醛；Cin）以及一种二胺（N,N,N,N'-四甲基乙二胺；TEMED）。有趣的是，结果表明：（1）被鉴定为大马士革玫瑰活性成分的PEA香气可预防原纤维形成，因为与形成原纤维而非成熟原纤维的PEA-S相比，PEA-L能够捕获HEWL的寡聚体形式；（2）之前已证明以液体形式存在的Cin可预防原纤维形成，研究还表明，在香气形式下，Cin-L和Cin-S两种香气形式均通过产生原纤维而非成熟原纤维来预防原纤维形成；（3）TEMED-L的香气能够完全保留HEWL的天然结构，预防聚集和原纤维形成，而TEMED-S则可预防HEWL原纤维形成，而是将途径导向无定形聚集体形成。此外，捕获寡聚体物种（通过PEA-L香气）的能力对进一步研究非常重要，因为它为预防沿纤维化途径形成有毒寡聚中间体提供了途径。最后但同样重要的是，这项体外研究以独特的实验设置在分子水平上研究香气对以HEWL作为模型蛋白评估淀粉样原纤维形成的影响，为更详细地研究产生香气的分子的重要性及其影响铺平了道路。
