Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells
Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells
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DOI：
10.1073/pnas.96.20.11404
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发表时间：
1999-09-28
影响因子：
11.1
通讯作者：
Housman, D
中科院分区：
文献类型：
--
作者：
Kazantsev, A;Preisinger, E;Housman, D
亨廷顿病和相关神经变性疾病中神经元的病理变性与编码延长长度的多聚谷氨酰胺的CAG重复序列的扩增直接相关。延伸的聚谷氨酰胺的物理性质和多聚谷氨酰胺扩增表达的细胞内后果一直是深入研究的对象。我们通过构建编码25-300个谷氨酰胺残基的CAG/CAA重复序列文库，扩展了由重组DNA方法产生的多聚谷氨酰胺的长度范围。我们已经研究了亚细胞定位，与其他含聚谷氨酰胺的多肽的相互作用，以及聚谷氨酰胺在细胞中的聚集形式的物理性质。延伸的polyQ聚集在细胞质中，并且仅当存在强的核定位信号时才被运输到细胞核。低于病理长度的多聚谷氨酰胺可以聚集在一起并运输到异位细胞位置。CREB结合蛋白（CBP），含有19个谷氨酰胺的均聚物延伸，同样被发现以聚谷氨酰胺依赖的方式共聚集，这表明在多聚谷氨酰胺疾病的病理可能是由于细胞耗竭的正常蛋白质含有多聚谷氨酰胺。我们已经观察到一个惊人的去污剂，在病理长度的聚谷氨酰胺产生的聚集体阻力。这一观察结果导致开发了基于荧光的测定法，该测定法利用了聚谷氨酰胺聚集体的去污剂抗性，这应有助于高通量筛选抑制细胞中聚谷氨酰胺聚集的药剂。
Pathological degeneration of neurons in Huntington's disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation, We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a library of CAG/CAA repeats coding for a range of 25-300 glutamine residues. We have investigated the subcellular localization, interaction with other polyglutamine-containing polypeptides, and the physical properties of aggregated forms of polyglutamine in the cell. Extended polyQ aggregated in the cytoplasm and was only transported to the nucleus when a strong nuclear localization signal was present. Polyglutamine below pathological lengths could be captured in aggregates and transported to ectopic cell locations. The CREB-binding protein (CBP), containing a homopolymeric stretch of 19 glutamines, was likewise found to coaggregate in a polyglutamine-dependent manner, suggesting that pathology in polyglutamine disease may result from cellular depletion of normal proteins containing polyglutamine. We have observed a striking detergent,; resistance in aggregates produced from polyglutamine of pathological length. This observation has led to the development of a fluorescence-based assay exploiting the detergent resistance of polyglutamine aggregates that should facilitate high-throughput screening for agents that suppress polyglutamine aggregation in cells.