Kokona Bashkim, Cunningham Nicole R, Quinn Jeanne M, Jacobsen Danielle R, Garcia F Jay, Galindo Sierra M, Petrucelli Leonard, Stafford Walter F, Laue Thomas M, Fairman Robert
Department of Biology, Haverford College, 370 Lancaster Ave, Haverford, PA, 19041, USA.
Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA; Neurobiology of Disease Graduate Program, Mayo Graduate School, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA.
Anal Biochem. 2025 Feb;697:115720. doi: 10.1016/j.ab.2024.115720. Epub 2024 Nov 22.
Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large GC hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro), (Gly-Ala), and (Gly-Arg) fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala) in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP) is largely monomeric. In contrast, (GA) forms both intermediate and large-scale aggregates. (GR) is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.
利用配备荧光检测装置的分析型超速离心机的沉降速度以及电泳方法，来研究转基因动物模型系统中蛋白质的聚集情况。我们之前的工作验证了这种方法在分析突变型亨廷顿蛋白聚集方面的有效性。我们证明，这种方法可应用于另一种神经退行性疾病，即研究由含有大GC六核苷酸重复序列的突变型c9orf72异常翻译产生的三种二肽重复序列（DPRs）的聚集情况。这些重复序列扩增是家族性肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）最常见的病因。我们使用分析型超速离心荧光检测法（AU-FDS）和十二烷基硫酸钠变性凝胶电泳（SDD-AGE），分析了在从黑腹果蝇制备的样品中与荧光蛋白融合的（甘氨酸-脯氨酸）、（甘氨酸-丙氨酸）和（甘氨酸-精氨酸），以及秀丽隐杆线虫中的（甘氨酸-丙氨酸）的聚集模式。结果表明，（甘氨酸-脯氨酸）主要以单体形式存在。相比之下，（甘氨酸-丙氨酸）形成中等规模和大规模的聚集体。（甘氨酸-精氨酸）部分以单体形式存在，在SDD-AGE分析中观察到有一些聚集现象。这种DPR的聚集可能代表与DNA和/或RNA的共聚集状态。这些方法的强大之处在于能够在动物模型系统中收集有关聚集模式和特征的数据，然后可用于通过基因或分子治疗干预来解释聚集的缓解情况。
