Crystallization of Ataxin-3

描述（由申请人提供）：多谷氨酰胺病是一种无法治愈的神经退行性疾病，影响全球数十万人；它们是由蛋白质中正常发生的聚谷氨酰胺束的扩张引起的。患者的运动和认知能力持续恶化，随后死亡。本研究的目的是探讨多谷氨酰胺病的结构基础，特别关注ataxin-3蛋白。蛋白质中多谷氨酰胺束的长度在正常人群中有所不同，但在健康个体中从未连续超过35- 40个谷氨酰胺。将这些残基数量增加到40个以上的突变会导致疾病。具有扩大的多谷氨酰胺区域的蛋白质错误折叠并聚集成淀粉样原纤维；聚集和纤维的形成与细胞功能障碍和死亡有着密不可分的联系。扩大的谷氨酰胺重复序列破坏了天然蛋白质结构，有利于形成错误折叠状态，这些状态可以组装成有毒的聚集体。要了解这种异常折叠过程，需要对蛋白质的天然和病理状态都有透彻的结构知识。在纤维结构的表征方面已经取得了进展，但目前还没有任何聚谷氨酰胺蛋白的三维结构。这种结构信息的缺乏极大地阻碍了了解聚谷氨酰胺疾病的分子基础和开发合理治疗方法的进展。Ataxin-3是最常见的多谷氨酰胺疾病Machado-Joseph病的病原体。p.i。他的实验室正在开发ataxin-3，作为研究聚谷氨酰胺蛋白错误折叠和聚集的模型系统。这项工作的结构方面需要适合x射线衍射研究的ataxin-3晶体。因此，我们已经开发了一种系统和全面的方法来获得全长和截断形式的ataxin-3晶体。提出了三个具体目标，即ataxin-3分离结构域的鉴定和结晶，利用合理的诱变提高我们生长衍射质量晶体的能力，以及利用抗体片段制备Fab-ataxin-3共晶体。从这项工作中获得的对ataxin-3的分子理解是任何合理设计治疗方法的前提，这些治疗方法将干扰蛋白质的异常折叠和聚集。

DESCRIPTION (provided by applicant): The polyglutamine diseases are incurable neurodegenerative disorders affecting hundreds of thousands of people worldwide; they are caused by the expansion of normally occurring polyglutamine tracts in proteins. Patients suffer a steady deterioration in motor and cognitive abilities, followed by death. The Aim of this research is to investigate the structural basis of polyglutamine disease, focusing specifically on the ataxin-3 protein. The lengths of polyglutamine tracts in proteins vary within the normal population, but never exceed 35- 40 consecutive glutamines in healthy individuals. Mutations that expand the number of these residues to greater than 40 cause disease. Proteins with expanded polyglutamine regions misfold and aggregate into amyloid fibrils; the aggregation and fibril formation are inextricably linked with cellular dysfunction and death. Expanded glutamine repeats disrupt native protein structure and favor the formation of misfolded states, which can assemble into toxic aggregates. An understanding of this aberrant folding process requires a thorough structural knowledge of both the native and pathological states of the protein. Advances have been made in the characterization of fibril architecture, but no three-dimensional structure is currently available for any polyglutamine protein. This lack of structural information drastically hinders progress toward understanding the molecular basis of polyglutamine disease and developing rational therapies. Ataxin-3 is the causative agent of Machado-Joseph disease, the most common polyglutamine disease. The P. I.'s laboratory is developing ataxin-3 as a model system for studying the misfolding and aggregation of polyglutamine proteins. The structural aspects of this work require crystals of ataxin-3 suitable for X-ray diffraction studies. We therefore have developed a systematic and comprehensive approach to obtaining crystals of full-length and truncated forms of ataxin-3. Three Specific Aims are proposed, namely the identification and crystallization of isolated domains of ataxin-3, the use of rational mutagenesis to improve our ability to grow diffraction-quality crystals, and the use of antibody fragments to prepare Fab-ataxin-3 cocrystals. The molecular understanding of ataxin-3 that will be obtained from this work is a prerequisite for any rational design of therapeutics that will interfere with the protein's aberrant folding and aggregation.