Vilasi Silvia, Iannuzzi Clara, Portaccio Marianna, Irace Gaetano, Sirangelo Ivana
Dipartimenti di Biochimica e Biofisica and Medicina Sperimentale, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy.
Biochemistry. 2008 Feb 12;47(6):1789-96. doi: 10.1021/bi701530w. Epub 2008 Jan 19.
Trehalose, a disaccharide present in many nonmammalian species, protects cells against various environmental stresses. Trehalose has recently been shown to decrease aggregate formation and toxicity in cell models and to alleviate amyloid-induced diseases. The aim of our study was to use two amyloid-forming proteins, i.e., W7FW14F apomyoglobin and insulin, as model systems to elucidate the molecular mechanism by which trehalose affects the amyloid aggregation process and to investigate further its therapeutic potential. Protein aggregation was examined by far-UV circular dichroism, UV absorption, thioflavin T fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, atomic force microscopy, and Fourier transform infrared spectroscopy. Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. We found that trehalose does not inhibit protein aggregation but acts at different stages of the fibrillization process depending on the protein model used. In fact, trehalose dose-dependently inhibited fibril formation in the W7FW14F apomyoglobin model and increased the lag phase in the insulin model. In both cases, trehalose caused accumulation of toxic oligomeric species. The results suggest that trehalose may favor or inhibit the formation of "on-pathway" or "off-pathway" oligomeric intermediates depending on the nature of the aggregating protein.
海藻糖是一种存在于许多非哺乳动物物种中的二糖，可保护细胞免受各种环境压力的影响。最近的研究表明，海藻糖能减少细胞模型中的聚集体形成和毒性，并缓解淀粉样蛋白诱导的疾病。我们研究的目的是使用两种形成淀粉样蛋白的蛋白质，即W7FW14F脱辅基肌红蛋白和胰岛素，作为模型系统来阐明海藻糖影响淀粉样蛋白聚集过程的分子机制，并进一步研究其治疗潜力。通过远紫外圆二色性、紫外吸收、硫黄素T荧光、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、原子力显微镜和傅里叶变换红外光谱来检测蛋白质聚集。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐还原试验来研究细胞活力。我们发现海藻糖并不抑制蛋白质聚集，而是根据所使用的蛋白质模型在纤维化过程的不同阶段发挥作用。事实上，海藻糖在W7FW14F脱辅基肌红蛋白模型中剂量依赖性地抑制纤维形成，并在胰岛素模型中延长延迟期。在这两种情况下，海藻糖都会导致有毒寡聚体物种的积累。结果表明，根据聚集蛋白的性质，海藻糖可能有利于或抑制“正常途径”或“非正常途径”寡聚中间体的形成。
