Burke Kathleen A, Legleiter Justin
The C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, USA.
Methods Mol Biol. 2013;1017:21-40. doi: 10.1007/978-1-62703-438-8_2.
Mutations which cause an expansion of CAG triplet repeats encoding polyglutamine (polyQ) are responsible for the subsequent misfolding of specific proteins that contribute directly to the pathogenesis of at least nine neurodegenerative disorders, including Huntington's disease (HD) and the spinocerebellar ataxias (SCAs). Expansion of polyQ tracts results in the aggregation of these proteins, potentially through a variety of precursor aggregates, into fibrillar structures. There may also be a variety of aggregates formed that are off-pathway to the formation of fibrils. Here, detailed protocols for analyzing the aggregation of mutant huntingtin (htt) fragments (associated with HD) and synthetic polyQ peptides with atomic force microscopy (AFM) are described. Ex situ AFM can be used to characterize htt aggregate formation and morphology. In situ AFM allows for tracking the formation and fate of individual polyQ peptide aggregates on surfaces. The interaction of htt with a variety of surfaces, including lipid bilayers, can also be investigated. Furthermore, the mechanical impact of htt on lipid surfaces can be studied using specialized AFM techniques. Methods to analyze AFM images of htt aggregates are also presented.
导致编码聚谷氨酰胺（polyQ）的CAG三联体重复序列扩增的突变，是特定蛋白质随后错误折叠的原因，这些蛋白质直接导致至少九种神经退行性疾病的发病机制，包括亨廷顿舞蹈症（HD）和脊髓小脑共济失调（SCA）。聚谷氨酰胺链的扩增导致这些蛋白质聚集，可能通过多种前体聚集体形成纤维状结构。也可能形成各种偏离纤维形成途径的聚集体。本文描述了用原子力显微镜（AFM）分析突变型亨廷顿蛋白（htt）片段（与HD相关）和合成聚谷氨酰胺肽聚集的详细方案。异位AFM可用于表征htt聚集体的形成和形态。原位AFM允许追踪单个聚谷氨酰胺肽聚集体在表面的形成和命运。还可以研究htt与包括脂质双层在内的各种表面的相互作用。此外，可以使用专门的AFM技术研究htt对脂质表面的机械影响。本文还介绍了分析htt聚集体AFM图像的方法。
