蛋白质聚集
体外
生物化学
化学
脊髓小脑共济失调
淀粉样蛋白(真菌学)
纤维
融合蛋白
生物
细胞生物学
基因
重组DNA
无机化学
作者
Erich E. Wanker,Eberhard Scherzinger,Volker Heiser,Annie Sittler,Antonio Leyva,Hans Lehrach
出处
期刊:Methods in Enzymology
日期:1999-01-01
卷期号:: 375-386
被引量:228
标识
DOI:10.1016/s0076-6879(99)09026-6
摘要
The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1, 3, and 7. Furthermore, it has been shown in vitro that the proteolytic cleavage of fusion proteins of glutathione S-transferase (GST) and the polyglutamine-containing huntingtin peptide coded for by the first exon of the HD gene s leads to the formation of insoluble high molecular weight protein aggregates with a fibrillar or ribbonlike morphology reminiscent of β-amyloid fibrils in Alzheimer's disease and scrapie prion rods. This chapter demonstrates that the cellulose acetate filter retardation assay can be a useful tool for the identification, structural characterization, and quantification of SDS-insoluble polyglutamine-containing protein aggregates formed in vitro and in vivo. In addition to the histochemical identification of amyloids, it useful in detecting insoluble protein aggregates in all types of human and animal amyloidoses, including the polyglutamine diseases, and also in screening compound libraries for potential aggregation inhibitors. Currently, attempts to develop a microtiter plate-based high-throughput filter retardation assay to identify chemical compounds that slow down the rate of formation of polyglutamine-containing fibrils in vitro are in progress. The amyloid-binding agents arising from this screen then will be tested in a HD cell culture model system and in the HD animal model for their therapeutic potential.
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