WHAT IS THE STRUCTURAL CORRELATE OF INTRACELLULAR POLYGLUTAMINE TOXICITY?

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在导致神经退行性疾病的12种无关蛋白质中，长度超过36-40个谷氨酸的膨体聚谷氨酰胺(PolyQ)是唯一的共同特征。证据表明，异常长的多聚Q延伸赋予新的毒性功能，这可能与含有多Q的蛋白质的可溶性单体或低聚形式的存在有关。虽然我们知道这些形式最终会聚集在一起，但不知道哪种可溶物质是有毒的。这些有毒物种的鉴定将是理解这些神经退行性疾病的生物学基础的重大突破。我们计划使用SAXS来确定在与Fabs结合多Q的络合物中含有多Q的蛋白质的分子包膜和单体或寡聚状态。为了识别有毒物种，SAXS实验的信息将与抗体染色在细胞培养实验中预测神经元退化的能力相关联。我们用3B5H10 Fab(一种针对疾病相关的PolyQ片段的Fab)和亨廷顿蛋白的N端片段与39个谷氨酰胺重复序列的复合体进行的初步实验表明，纯化的复合体在浓度为1.5-5 mg/ml的溶液中表现良好，没有聚集迹象。本研究的数据被用来获得亨廷顿蛋白片段在复合体中的单体构象的分子包膜。我们计划扩大这些实验，在Huntingtin片段中使用更长的PolyQ重复序列，以及构建具有可变多谷氨酰胺重复序列的ataxin-3。此外，还将研究可能促进细胞培养中多谷氨酰胺毒性的Fab，如MW1 Fab。一种在预测多谷氨酰胺毒性方面无效的多Q结合Fab将作为对照进行研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Expanded polyglutamine (polyQ) stretches greater than 36-40 glutamines in length are the only common feature in twelve unrelated proteins that cause neurodegenerative disease. Evidence indicates that abnormally long polyQ stretches confer new toxic functions that may be associated with the presence of soluble monomeric or oligomeric forms of the polyQ-containing protein. Although it is known that these forms eventually aggregate, it is not which of the soluble species are toxic. Identification of these toxic species will be a major breakthough in understanding the biological basis of these neurodegenerative diseases. We plan to use SAXS to determine the molecular envelope and monomeric or oligomeric state of polyQ-containing proteins in complexes with Fabs that bind polyQ. To identify toxic species, the information from the SAXS experiments will be correlated with the ability of the antibody staining to predict degeneration of neurons in cell culture experiments. Our preliminary experiments with complexes of the 3B5H10 Fab, an Fab specific for disease-associated stretches of polyQ, and an N-terminal fragment of the huntingtin protein with a 39 glutamine repeat indicated that the purified complex was well-behaved in solution at concentrations of 1.5- 5 mg/ml with no signs of aggregation. Data from this study was used to obtain of the molecular envelope of the monomeric conformation of the huntingtin fragment in the complex. We plan to extend these experiments by using longer polyQ repeats in the huntingtin fragment as well as constructs of ataxin-3 with variable polyglutamine repeats. In addition, Fabs such as the MW1 Fab which may promote polyglutamine toxicity in cell culture will be studied. A polyQbinding Fab that is ineffective at predicting polyglutamine toxicity will be studied as a control.