Carija Anita, Ventura Salvador, Navarro Susanna
Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona.
Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona;
J Vis Exp. 2018 Jun 23(136):57470. doi: 10.3791/57470.
Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-β peptide variants with their respective intrinsic aggregation propensities.
蛋白质错误折叠并聚集成淀粉样构象与几种神经退行性疾病的发生和发展有关。然而，关于不溶性蛋白质聚集体如何在体内发挥其毒性作用的信息仍然很少。简单的原核和真核模式生物，如细菌和酵母，对我们目前对细胞内淀粉样蛋白形成、聚集体传播和毒性背后机制的理解做出了重大贡献。在本方案中，描述了使用酵母作为模型来剖析蛋白质聚集体的形成与其对细胞氧化应激的影响之间的关系。该方法将淀粉样蛋白生成蛋白的细胞内可溶性/聚集状态的检测与使用流式细胞术（FC）对其表达导致的细胞氧化损伤的定量相结合。这种方法简单、快速且定量。该研究通过将大量淀粉样β肽变体引起的细胞氧化应激与其各自的内在聚集倾向相关联来说明该技术。
