Screening of compounds for protecting polyglutamine diseases

聚谷氨酰胺(PQ)病的一个主要特征是形成PQ包涵体。最近，错误折叠被认为是PQ蛋白聚集的主要因素之一，尽管错误折叠的本质以及错误折叠与扩展的PQ蛋白泛素化之间的关系尚不清楚。通过使用SCA3/MJD的缺陷基因产物ataxin-3，我们证明了PQ蛋白的细胞毒性与谷氨酰胺重复序列的长度成正比，与相应蛋白质的大小成反比。PQ的扩增和截断增强了1C2抗体的反应性(对扩增的PQ具有特异性)、伴侣结合和泛素化，这表明PQ的扩增和蛋白质的截断增强了蛋白质的误折叠。聚谷氨酰胺从…内延伸而来当PQ扩张时，分子β片层增多，孵育一定时间后形成分子间β片层和淀粉样纤维。突变型肌红蛋白经PQ扩增后，表面部分展开，不稳定。我们还研究了突变肌红蛋白的早期纤化阶段，其中插入了35或50个谷氨酰胺重复序列。小角X射线散射和电子显微镜研究表明，在蛋白质纤化的最早阶段，PQ扩展到50个重复导致了准聚集体的形成。我们发现准聚集体中的PQ的β片层暴露在表面，并且不是规则取向的，与淀粉样原纤维中的PQ相反。X-射线衍射分析表明，谷氨酰胺重复序列的扩展对原纤维中PQ的β-Sheet结构没有显著影响。由于某些蛋白质中谷氨酸重复序列的扩张是导致PQ疾病的原因，本研究表明，暴露在准聚集体表面的凝聚和无取向的β-Sheet，而不是纤维中规则排列的β-Sheet，密切参与了各种功能蛋白向聚集体的募集，导致了导致PQ疾病的细胞功能障碍。因此，准聚集态也应该成为PQ病治疗策略的靶点。根据这些结构变化，我们寻找了一些具有稳定分子作用的化合物，并发现了一些有效的化合物。较少

A major hallmark of the polyglutamine (pQ) diseases is the formation of pQ inclusions. Recently, misfolding has come to be considered one of the primary factors for pQ protein aggregation, although, the nature of misfolding and the relationship between misfolding and ubiquitination of the expanded pQ protein is not yet known. By using ataxin-3, the defective gene product of SCA3/MJD, we demonstrated that the cellular toxicity of a pQ protein is directly proportional to the length of the glutamine repeats and inversely dependent on the size of the corresponding protein. The pQ expansion and truncation enhance the reactivity of 1C2 antibody (specific to expanded pQ), chaperone association and ubiquitination, suggesting that pQ expansion and protein truncation enhance the protein misfolding.The protein misfolding induced by pQ expansion was further investigated with our molecular model system using mutant myoglobin which is inserted different size of pQ. Polyglutamine stretches form intra … More molecular β-sheets when pQ expanded and they form intermolecular β-sheets and amyloid fibrils after certain incubation time. The surface of the mutant myoglobin with expanded pQ was partially unfolded and destabilized. We also investigated the early phase of fibrillization of the mutant myoglobin in which 35 or 50 glutamine repeats are inserted. Small-angle X-ray scattering and electron microscopic studies revealed that the expansion of pQ to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. We found that β-sheets of pQ in quasi-aggregate were exposed on the surface and were not regularly oriented, as opposed to those in amyloid fibril. X-ray diffraction analysis indicated that the expansion of glutamine repeat did not show substantial effects on the β-sheet structure of pQ in fibril. Since the expansion of glutanine repeat in certain proteins is responsible for pQ diseases, the present study suggests that the condensed and non-oriented β-sheets exposed on the surface of quasi-aggregate, rather than the regularly aligned β-sheets in fibrils, are closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pQ diseases. Thus, the quasi-aggregate form also should be targeted to the therapeutic strategy for pQ diseases.Base on those structural change, we searched for compounds to stabilize the molecule with expanded polyglutamine and found some effective compounds. Less