Bonanomi Marcella, Visentin Cristina, Natalello Antonino, Spinelli Michela, Vanoni Marco, Airoldi Cristina, Regonesi Maria E, Tortora Paolo
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano (Italy).
Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), UdR Milano-Bicocca, Milano (Italy).
Chemistry. 2015 Dec 7;21(50):18383-93. doi: 10.1002/chem.201503086. Epub 2015 Nov 5.
Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia type 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.
表没食子儿茶素-3-没食子酸酯（EGCG）和四环素是两种已知的淀粉样蛋白聚集抑制剂，能够对抗大多数与神经退行性疾病相关的蛋白质的纤维化。我们最近研究了它们对ataxin-3（AT3）的影响，AT3是一种含多聚谷氨酰胺的蛋白质，是导致3型脊髓小脑共济失调的原因。我们之前表明，EGCG和四环素可以通过不同机制抑制扩展型AT3的聚集过程和毒性。在这里，我们通过仅使用约瑟芬结构域（JD）进行了进一步实验，以进一步阐明这两种化合物的作用机制。通过蛋白质溶解度测定和傅里叶变换红外光谱（FTIR），我们首先观察到，EGCG和四环素对JD聚集的影响基本上与作用于全长扩展型AT3时显示的方式相同。然后，通过饱和转移差异（STD）核磁共振实验，我们表明EGCG能结合单体和寡聚体形式的JD，而四环素只能与寡聚体形式相互作用。表面等离子体共振（SPR）分析证实了仅EGCG能够结合单体JD，尽管其解离常数（KD）值表明这是一种非特异性相互作用。我们的研究提供了关于JD与EGCG和四环素相互作用的新细节，这可以解释这两种化合物降低AT3毒性的不同机制。
