Protein misfolding and aggregation

描述（由申请人提供）：由于缺乏对其多因素病理的理解，大多数年龄相关神经退行性疾病的有效治疗策略仍然难以捉摸。疾病相关蛋白质的小的可溶性寡聚体已被提议作为几种此类疾病（包括阿尔茨海默病和肌萎缩性侧索硬化症（ALS））中的细胞毒性剂。在后一种情况下，可溶性非天然构象的铜，锌超氧化物歧化酶（SOD 1）出现症状发作前，并参与许多异常的相互作用与细胞成分，支持这些物种在ALS发病机制的主要作用。因此，促进可溶性SOD 1寡聚体形成或有害相互作用的表面补丁的阻断具有治疗潜力，但需要尚未获得的详细结构和机制见解。拟议工作的最终目标是确定ALS中SOD 1错误折叠和聚集的原因，机制和后果，特别是在涉及潜在毒性可溶性错误折叠状态的早期阶段。我们最近的研究结果表明，SOD 1不稳定的氧化翻译后修饰表明，SOD 1的错误折叠和聚集是潜在的有害因素，即使在不稳定的疾病相关的突变。拟议的工作的目的是确定非天然的结构特征获得的SOD 1，因为它错误折叠和聚集，并确定在细胞环境中的决定因素（即氧化翻译后修饰），驱动这些重排。我们假设疾病相关突变和氧化修饰均诱导SOD 1的结构变化，从而促进寡聚化并暴露参与非天然细胞相互作用的表面。基于鉴定野生型和突变型SOD 1的亚稳可溶性寡聚体的初步数据，以及可逆氧化修饰的去稳定作用，（Cys-111谷胱甘肽化），我们将从三个具体目标来检验我们的假设：1）表征可溶性SOD 1寡聚体的结构特征; 2）表征氧化修饰对SOD 1聚集途径的影响;和3）鉴定促进可溶性SOD 1寡聚物形成的非天然相互作用表面。在前两个目标中，我们将使用成熟的生物物理和生物化学技术来评估野生型和突变体SOD 1聚集体的寡聚状态和表面疏水性的变化。我们还将探测亚稳态的非天然寡聚体与最近设计的抗体，特异性地识别在ALS患者中发现的错误折叠的SOD 1，从而识别与潜在的有害物种结构相似的寡聚体。第二个目标将通过将目标1中使用的方法应用于修饰的SOD 1的研究来解决生理上普遍的氧化修饰Cys-111谷胱甘肽化对非天然寡聚化的贡献。在目标3中，我们将利用创新的计算方法来生成非天然SOD 1寡聚体的结构模型。通过鉴定可溶性SOD 1寡聚体中暴露的表位以及稳定它们的非天然相互作用，这些研究可能揭示用于防止这些潜在毒性物质的形成和异常相互作用的靶点。

DESCRIPTION (provided by applicant): Effective therapeutic strategies remain elusive for most age-related neurodegenerative disorders due to deficits in understanding of their multi-factorial pathologies. Small, soluble oligomers of disease-linked proteins have been proposed as cytotoxic agents in several such disorders, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). In the latter case, soluble non-native conformers of Cu, Zn superoxide dismutase (SOD1) appear before symptom onset and participate in numerous aberrant interactions with cellular components, supporting a primary role for these species in ALS pathogenesis. The blocking of surface patches that facilitate formation or deleterious interactions of soluble SOD1 oligomers thus holds therapeutic potential, but requires detailed structural and mechanistic insight that is not yet available. The ultimate goal of the proposed work is the determination of causes, mechanisms, and consequences of SOD1 misfolding and aggregation in ALS, particularly in early stages involving potentially toxic soluble misfolded states. Our recent findings of SOD1 destabilization by oxidative post-translational modifications suggest that SOD1 misfolding and aggregation are potential noxious factors even in the absence of destabilizing disease-associated mutations. The objective of the proposed work is to identify non-native structural features acquired by SOD1 as it misfolds and aggregates, and to identify determinants in the cellular environment (i.e. oxidative post-translational modifications) that drive these rearrangements. We hypothesize that both disease-linked mutations and oxidative modifications induce structural changes in SOD1 that promote oligomerization and expose surfaces that participate in non-native cellular interactions. Based on preliminary data identifying metastable soluble oligomers of wild type and mutant SOD1, as well as the destabilizing effect of a reversible oxidative modification (Cys-111 glutathionylation), we will test our hypothesis in three specific aims: 1) Characterize structural features of soluble SOD1 oligomers; 2) Characterize the impact of oxidative modification on the SOD1 aggregation pathway; and 3) Identify non-native interacting surfaces that facilitate formation of soluble SOD1 oligomers. In the first two aims, we will use proven biophysical and biochemical techniques to assess changes in oligomeric state and surface hydrophobicity as wild type and mutant SOD1 aggregate. We will also probe metastable non-native oligomers with recently designed antibodies that specifically recognize misfolded SOD1 found in ALS patients, thereby identifying oligomers with structural similarity to potentially noxious species. The second aim will ad- dress the contribution of a physiologically prevalent oxidative modification, Cys-111 glutathionylation, to non- native oligomerization by applying approaches used in aim 1 to the study of modified SOD1. In aim 3, we will utilize innovative computational approaches to generate a structural model of a non-native SOD1 oligomer. By identifying exposed epitopes in soluble SOD1 oligomers as well as non-native interactions that stabilize them, these studies may reveal targets for preventing formation and aberrant interactions of these potentially toxic species.