WHAT IS THE STRUCTURAL CORRELATE OF INTRACELLULAR POLYGLUTAMINE TOXICITY?

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 长度大于36-40个谷氨酰胺的扩展多聚谷氨酰胺（polyQ）是导致神经退行性疾病的12种不相关蛋白质的唯一共同特征。有证据表明，异常长的polyQ片段赋予新的毒性功能，这可能与含有polyQ蛋白的可溶性单体或寡聚体形式的存在有关。虽然已知这些形式最终聚集，但并不知道哪些可溶性物质是有毒的。这些有毒物质的鉴定将是理解这些神经退行性疾病生物学基础的一个重大突破。我们计划使用SAXS来确定与结合polyQ的Fab复合的含polyQ蛋白的分子包膜和单体或寡聚体状态。为了鉴定有毒物质，将来自SAXS实验的信息与抗体染色预测细胞培养实验中神经元变性的能力相关联。我们用3B 5 H10 Fab（一种特异于polyQ的疾病相关片段的Fab）和具有39个谷氨酰胺重复序列的亨廷顿蛋白的N-末端片段的复合物进行的初步实验表明，纯化的复合物在1.5- 5 mg/ml的浓度下在溶液中表现良好，没有聚集的迹象。本研究的数据用于获得复合物中亨廷顿蛋白片段的单体构象的分子包膜。我们计划通过在亨廷顿蛋白片段中使用更长的polyQ重复序列以及具有可变聚谷氨酰胺重复序列的共济失调蛋白-3构建体来扩展这些实验。此外，将研究可促进细胞培养物中的多聚谷氨酰胺毒性的Fab，如MW 1 Fab。将研究在预测聚谷氨酰胺毒性方面无效的聚Q结合Fab作为对照。

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.