Protein misfolding and aggregation

描述（由申请人提供）：由于对大多数与年龄相关的神经退行性疾病的多因素病理认识不足，有效的治疗策略仍然难以捉摸。小的、可溶的疾病相关蛋白低聚物已被认为是几种疾病的细胞毒性药物，包括阿尔茨海默病和肌萎缩侧索硬化症（ALS）。在后一种情况下，Cu， Zn超氧化物歧化酶（SOD1）的可溶性非天然构象在症状出现之前出现，并参与与细胞成分的许多异常相互作用，支持这些物种在ALS发病机制中的主要作用。因此，阻断可促进可溶性SOD1寡聚物形成或有害相互作用的表面斑块具有治疗潜力，但需要详细的结构和机制了解，目前尚无法获得。这项工作的最终目标是确定ALS中SOD1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。我们最近通过氧化翻译后修饰发现SOD1不稳定，这表明即使没有不稳定的疾病相关突变，SOD1错误折叠和聚集也是潜在的有害因素。这项工作的目的是确定SOD1在错误折叠和聚集时获得的非天然结构特征，并确定细胞环境中驱动这些重排的决定因素（即氧化翻译后修饰）。我们假设疾病相关突变和氧化修饰都会诱导SOD1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SOD1亚稳可溶性低聚物的初步数据，以及可逆氧化修饰（Cys-111谷胱甘肽化）的不稳定作用，我们将在三个具体目标上验证我们的假设：1)表征可溶性SOD1低聚物的结构特征；2)表征氧化修饰对SOD1聚集途径的影响；3)鉴定促进可溶性SOD1低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化技术来评估野生型和突变型SOD1聚集时寡聚物状态和表面疏水性的变化。我们还将用最近设计的抗体来探测亚稳态非天然低聚物，这些抗体专门识别在ALS患者中发现的错误折叠的SOD1，从而识别与潜在有害物种结构相似的低聚物。第二个目标将通过将目标1中使用的方法应用于改性SOD1的研究，解决生理上普遍存在的氧化修饰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SOD1低聚物的结构模型。通过鉴定可溶性SOD1寡聚物中暴露的表位以及稳定它们的非天然相互作用，这些研究可能揭示防止这些潜在毒性物种形成和异常相互作用的靶标。

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.