Structure and conformational diversity of amyloid oligomers

描述(申请人提供)：本研究项目的目标是探索淀粉样聚集体的结构异质性，以及这种构象变化与淀粉样寡聚体的毒性或致病活性的关系。几种不同类型的淀粉样蛋白沉积在疾病的大脑中，目前的证据表明，可溶性的寡聚体形式的天冬氨酸可能在发病机制中发挥主要作用。最近的研究结果表明，构象依赖的单抗可以区分不同类型的可溶性Ass寡聚体。这些抗体还区分了Ass的其他构象，包括单体、纤维和天然折叠的APP。我们最近又制备了两种构象依赖的抗血清，它们识别与淀粉样蛋白纤维和由许多不同类型的淀粉样蛋白形成的孔状环形原纤维相关的通用表位(见下文的初步数据)。这些抗体是对抗低聚物抗体A11的补充，并识别针对纤维和环状原纤维聚集体的表位。我们假设这些不同构象的Ass组装状态与AD的发病机制有不同的关联。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的相关性很差，一些人有大量的淀粉样蛋白且认知正常，而其他大脑样本中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，不同类型的可溶性低聚物或环状原纤维形式的ASS具有不同的毒性。构象依赖抗体具有识别和区分特定组装状态的潜力，因为它们只识别特定的错误折叠状态，而不与天然折叠的前体蛋白发生反应。该项目的具体目标将解决以下问题：淀粉样聚集体的构象多样性是什么？构象依赖性抗体特异性和淀粉样寡聚体结构的结构基础是什么？淀粉样蛋白的不同构象状态之间有什么关系？淀粉样蛋白不同构象状态的病理意义是什么？这些问题的答案应该有助于深入了解淀粉样寡聚体的构象多样性和区分不同构象的寡聚体的单抗试剂的范围。这可能为低聚物的分类提供更合理的结构基础，并提供对不同实验室报告的低聚物制剂的差异性的洞察。确定与淀粉样寡聚体结合的单克隆纤维的三维结构可能提供对淀粉样低聚体的结构和特异性抗体识别机制的前所未有的深入了解，这可能有助于开发针对寡聚体形成或阻止其与细胞靶点相互作用的免疫疗法。识别专门抑制不同类型低聚物形成的小分子应该有助于澄清低聚物是否是形成更高级结构(如纤维)的中间体，或者它们是否代表稳定的替代最终产品。这一目标还可能提供小分子先导化合物，专门抑制淀粉样蛋白低聚物的形成，用于治疗开发。在人类和转基因小鼠大脑中，哪些类型的淀粉样寡聚体与发病机制更密切相关的特征可能有助于确定治疗开发的靶点。PHS 398/2590(版本04/06)页面续格式页面

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