WHAT IS THE STRUCTURAL CORRELATE OF INTRACELLULAR POLYGLUTAMINE TOXICITY?

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 扩展的聚谷氨酰胺 (polyQ) 长度超过 36-40 个谷氨酰胺，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延伸赋予了新的毒性功能，这可能与含有polyQ的蛋白质的可溶性单体或寡聚形式的存在有关。尽管已知这些形式最终会聚集，但并不是哪种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的生物学基础的重大突破。我们计划使用 SAXS 来确定与结合 PolyQ 的 Fab 形成的复合物中含有 PolyQ 的蛋白质的分子包膜和单体或寡聚状态。为了识别有毒物质，来自 SAXS 实验的信息将与抗体染色的能力相关联，以预测细胞培养实验中神经元的退化。我们对 3B5H10 Fab（一种对疾病相关的 PolyQ 片段具有特异性的 Fab）和具有 39 个谷氨酰胺重复序列的亨廷顿蛋白 N 末端片段的复合物进行的初步实验表明，纯化的复合物在浓度为 1.5-5 mg/ml 的溶液中表现良好，没有聚集迹象。该研究的数据用于获得复合物中亨廷顿片段单体构象的分子包膜。我们计划通过在亨廷顿片段中使用更长的polyQ重复序列以及具有可变多聚谷氨酰胺重复序列的ataxin-3构建体来扩展这些实验。此外，还将研究可能在细胞培养中促进聚谷氨酰胺毒性的Fab，例如MW1 Fab。对预测聚谷氨酰胺毒性无效的聚 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.