Structure and conformational diversity of amyloid oligomers

描述（由申请人提供）：本研究项目的目的是探索淀粉样蛋白聚集体的结构异质性以及这种构象变异与淀粉样蛋白寡聚体毒性或致病活性的关系。几种不同类型的淀粉样蛋白沉积物在疾病脑中积累，目前的证据表明可溶性低聚体形式的Ass可能在发病机制中起主要作用。最近的研究结果表明，构象依赖的单克隆抗体可以区分不同类型的可溶性Ass寡聚体。这些抗体也区分其他构象的屁股，包括单体，原纤维和天然折叠的APP。我们最近制备了两个额外的构象依赖性抗血清，识别与淀粉样蛋白原纤维和孔样环状原纤维，从许多不同类型的淀粉样蛋白形成的通用表位（见初步数据，下面）。这些抗体与抗寡聚体抗体A11互补，并识别对原纤维和环状原纤维聚集体具有特异性的表位。我们假设这些构象不同的装配状态的屁股是差异与AD的发病机制。我们预计这些结果将有助于澄清一些明显的不一致和相互矛盾的数据，例如观察到总的Ass淀粉样蛋白沉积与疾病相关性很差，有些人有大量的淀粉样蛋白并且认知正常，而其他大脑样本几乎没有可观察到的淀粉样蛋白沉积与认知功能障碍有关。我们假设不同类型的可溶性寡聚体或环状原纤维形式的Ass具有不同的毒性。构象依赖性抗体具有识别和区分特定组装状态的潜力，因为它们仅识别特定的错误折叠状态，并且不与天然折叠的前体蛋白反应。这个项目的具体目标将解决以下问题：什么是淀粉样蛋白聚集体的构象多样性？构象依赖性抗体特异性和淀粉样蛋白寡聚体结构的结构基础是什么？淀粉样蛋白不同构象状态之间的关系是什么？不同淀粉样蛋白构象状态的病理意义是什么？这些问题的答案应该提供洞察的范围内的淀粉样蛋白低聚物构象多样性和单克隆抗体试剂，区分不同构象的低聚物。这可能为低聚物的分类提供更合理的结构基础，并为不同实验室报告的低聚物制剂的变异性提供见解。确定与淀粉样蛋白寡聚体结合的单克隆Fab的三维结构可以提供对淀粉样蛋白寡聚体的结构和特异性抗体识别机制的前所未有的了解，这可能有助于开发靶向寡聚体形成或防止其与细胞靶标相互作用的免疫治疗剂。特异性抑制不同类型低聚物形成的小分子的鉴定应有助于澄清低聚物是否是形成更高级结构（如原纤维）的中间体，或者它们是否代表稳定的替代终产物。该目的还可以提供特异性抑制淀粉样蛋白寡聚体形成的小分子先导化合物用于治疗开发。在人类和转基因小鼠脑中，哪种类型的淀粉样蛋白低聚物与发病机制更密切相关的表征可能有助于确定治疗开发的靶点。PHS 398/2590（Rev. 04/06） 公共卫生相关性：淀粉样蛋白聚集体被认为在神经退行性疾病如阿尔茨海默病、帕金森病、亨廷顿病和朊病毒病的发展中起核心作用。该建议的目标是确定存在多少不同类型的淀粉样蛋白聚集体，并确定淀粉样蛋白聚集体的精确三维结构。该提案还将研究这些聚集体中哪一种对神经元更具毒性，与疾病关系最密切。另一个好处是，该提案可能会产生单克隆抗体和药物，专门针对这些淀粉样蛋白聚集体的治疗发展。

DESCRIPTION (provided by applicant): The goal of this research project is to explore the structural heterogeneity of amyloid aggregates and the relationships of this conformational variation to the toxicity or pathogenic activities of amyloid oligomers. Several distinct types of amyloid deposits accumulate in disease brain and current evidence suggests that soluble, oligomeric forms of Ass may play primary role in pathogenesis. Recent results indicate that conformation-dependent monoclonal antibodies can distinguish between different types soluble Ass oligomers. These antibodies also distinguish other conformations of Ass, including monomers, fibrils and natively-folded APP. We have recently prepared two additional conformation-dependent antisera that recognize generic epitopes associated with amyloid fibrils and pore-like annular protofibrils that are formed from many different types of amyloids (see Preliminary Data, below). These antibodies are complementary to the anti-oligomer antibody, A11 and recognize epitopes that are specific to fibrils and annular protofibril aggregates. We hypothesize that these conformationally distinct assembly states of Ass are differentially associated with AD pathogenesis. We anticipate that these results will help clarify some apparent inconsistencies and conflicting data, such as the observations that the total Ass amyloid deposited correlates poorly with disease and some people have large amounts of amyloid and are cognitive normal, while other brain samples that have little observable amyloid deposits are associated with cognitive dysfunction. We hypothesize that the distinct types of soluble oligomeric or annular protofibril forms of Ass have distinct toxicities. Conformation-dependent antibodies hold the potential of identifying and distinguishing specific assembly states because they only recognize a specific misfolded state and do not react with the natively folded precursor protein. The specific aims of this project will address the following questions: What is the conformational diversity of amyloid aggregates? What is the structural basis of conformation dependent antibody specificity and amyloid oligomer structural? What are the relationships between different conformational states of amyloids? What is the pathological significance of the different amyloid conformational states? The answers to these questions should provide insight into the range of amyloid oligomer conformational diversity and monoclonal antibody reagents that distinguish different conformations of oligomers. This may provide a more rational structural basis for the classification of oligomers and provide insight into the variability in oligomer preparations reported by different laboratories. Determining the 3 dimensional structure of the monoclonal Fabs bound to amyloid oligomers may provide unprecedented insight into the structure of amyloid oligomers and the mechanism of specific antibody recognition that may be useful for development of immunological therapeutics that target oligomer formation or prevent their interaction with cellular targets. The identification of small molecules that specifically inhibit the formation of different types of oligomers should help to clarify whether the oligomers are intermediates in the formation of higher order structures, like fibrils or whether they represent stable alternative end products. This aim may also provide small molecule lead compounds that specifically inhibit amyloid oligomer formation for therapeutic development. The characterization of which types of amyloid oligomers are more closely related to pathogenesis in human and transgenic mouse brain may help to identify targets for therapeutic development. PHS 398/2590 (Rev. 04/06) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Amyloid aggregates are believed to play a central role in the development of neurodegenerative diseases, like Alzheimer, Parkinson, Huntington and prion diseases. The goal of this proposal is to determine how many different types of amyloid aggregates exist and to determine the precise 3 dimensional structure of the amyloid aggregates. The proposal will also examine which of these aggregates is more toxic to neurons and most closely related to disease. An additional benefit is that this proposal may produce monoclonal antibodies and drugs that specifically target these amyloid aggregates for therapeutic development.