Crystallization of Ataxin-3

描述(申请人提供)：聚谷氨酰胺疾病是影响全球数十万人的不可治愈的神经退行性疾病；它们是由蛋白质中正常出现的聚谷氨酰胺束扩张引起的。患者的运动和认知能力不断恶化，随后死亡。这项研究的目的是研究多谷氨酰胺病的结构基础，特别是ataxin-3蛋白。在正常人群中，蛋白质中的聚谷氨酰胺束的长度各不相同，但在健康个体中永远不会超过35-40个连续的谷氨酰胺。将这些残基的数量扩大到40个以上的突变会导致疾病。具有扩展的多谷氨酰胺区域的蛋白质错误折叠并聚集成淀粉样纤维；聚集和纤维形成与细胞功能障碍和死亡密不可分。扩张的谷氨酰胺重复序列破坏了天然的蛋白质结构，有利于错误折叠状态的形成，错误折叠状态可以组装成有毒的聚集体。要了解这种异常的折叠过程，需要对蛋白质的自然和病理状态有透彻的结构知识。纤维结构的表征已经取得了进展，但目前还没有任何聚谷氨酰胺蛋白的三维结构可用。这种结构信息的缺乏极大地阻碍了理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。Aaxin-3是马查多-约瑟夫病的病原体，马查多-约瑟夫病是最常见的多谷氨酰胺病。中国科学院S实验室正在开发ataxin-3作为研究多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于X射线衍射研究的ataxin-3晶体。因此，我们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。提出了三个具体的目标，即鉴定和结晶ataxin-3的分离结构域，使用合理的诱变来提高我们生长衍射级晶体的能力，以及使用抗体片段来制备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的治疗设计的先决条件，这些治疗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.