Vetri Valeria, Carrotta Rita, Picone Pasquale, Di Carlo Marta, Militello Valeria
Dipartimento di Scienze Fisiche ed Astronomiche, Università di Palermo and CNISM-MeSIAM, 90123 Palermo, Italy.
Biochim Biophys Acta. 2010 Jan;1804(1):173-83. doi: 10.1016/j.bbapap.2009.09.013. Epub 2009 Sep 25.
A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which-as a consequence-turns out to be non-toxic.
已知多种神经退行性疾病都涉及蛋白质聚集。淀粉样蛋白的常见机制和结构特性被认为与聚集相关的细胞毒性有关。在此背景下，我们提出了一项关于伴刀豆球蛋白A（Con A）聚集的实验研究，并将其用作研究细胞毒性与聚集过程之间关系的模型。根据溶液条件，通过静态和动态光散射、硫黄素T发射和傅里叶变换红外吸收来监测Con A的聚集。借助原子力显微镜和共聚焦显微镜验证了不同聚集体种类的形态。在聚集过程中，天然蛋白质构象不稳定，因此，在两种条件下都观察到了构象变化和蛋白质聚集的同时发生。通过MTS测定法测试了在LAN5神经母细胞瘤细胞上细胞外添加天然蛋白质、寡聚体和成熟纤维的效果。结果显示前两种物质具有毒性，而淀粉样纤维的影响可忽略不计。发现天然聚集体和寡聚体聚集体都能够通过半胱天冬酶8激活仅通过外在途径激活细胞凋亡。这些结果表明，细胞毒性机制源于与淀粉样聚集途径中发生的不稳定分子的反应性构象的特定膜相互作用。当天然或预先形成的寡聚体孵育时出现的这些构象无法结合细胞膜蛋白。之所以会这样，是因为它们被募集到成熟的纤维状结构中，结果证明该结构是无毒的。
