Protein Misfolding and Aggregation

蛋白质错误折叠和聚集

• 批准号: 7818210
• 负责人: NIKOLAY DOKHOLYAN
• 金额: $ 28.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7818210
• 关键词: Acetylation; Affect; Affinity; Amyotrophic Lateral Sclerosis; Antioxidants; Binding; Binding Sites; Biochemical; Biological Assay; Cell Death; Cell physiology; Complex; Cuprozinc Superoxide Dismutase; Dependence; Disease; Dissociation; Engineering; Enzymes; Erythrocytes; Escherichia coli; Event; Familial Amyotrophic Lateral Sclerosis; Feedback; Fluorescence; GTP Binding; Glutathione; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Hydrogen Peroxide; Hyperactive behavior; Inflammatory; Knowledge; Laboratories; Lead; Life; Link; Maps; Measures; Modification; Mutation; N-terminal; NADPH Oxidase; Neurodegenerative Disorders; Nucleotides; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxidopamine; Parents; Pathology; Phosphorylation; Physiological; Play; Process; Proteins; Reaction; Regulation; Relative (related person); Reporting; Role; Saccharomyces cerevisiae; Superoxides; Techniques; Work; analog; base; cell motility; cytotoxicity; design; dimer; mutant; oxidation; prevent; protein misfolding; public health relevance; research study

DESCRIPTION (provided by applicant): The anti-oxidant enzyme Cu,Zn superoxide dismutase (SOD1) is associated with amyotrophic lateral sclerosis (ALS) through more than one hundred causative mutations. The Rac1 GTPase regulates oxidant levels through its role in regulating NADPH oxidase (Nox) activity. Intriguingly, SOD1 was recently shown to bind Rac1 in a nucleotide and redox dependent manner to regulate Rac1 activity. Furthermore, two ALS-associated mutations in SOD1 were shown to render the SOD1/Rac1 interaction insensitive to redox regulation, hypothesized to cause increased oxidant levels via Nox hyperactivity. However, this premise is based on limited qualitative results obtained from only two bacterially-derived mutants. Unlike natural SOD1 and human SOD1 expressed in S. cerevisiae, E. coli-derived human SOD1 lacks both N-terminal acetylation and adequate metallation. In addition, we recently found that 40-50% of SOD1 in human erythrocytes is modified by glutathione - a key player in the cellular redox state. Glutathionylation promotes SOD1 dimer dissociation, which is a necessary initiating event for SOD1 aggregation and a focus of the parent proposal. Here, we aim to provide a quantitative description of the SOD1/Rac1 interaction, and its proposed relationship to ALS, using SOD1 isolated from human erythrocytes and S. cerevisiae. We will use NMR and other biophysical techniques to characterize and quantify the Rac1/SOD1 interaction in the presence and absence of modifications and ALS-associated mutations in SOD1. The recent discovery that SOD1 interacts with the Nox complex through Rac1 provides a potential link between SOD1 (dys)function and downstream events leading to cell death. It is therefore important to delineate how SOD1 modifications and familial ALS mutations affect the interactions with Rac1, which is the goal of this supplement. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to define a mechanism for the regulation of Rac1 by SOD1. Knowledge of this mechanism will lead to strategies for determining whether this new regulatory role of SOD1 is related to ALS. Furthermore if the Rac1/SOD1 the Rac1/SOD1 complex and contribute to ALS therapies.

描述（由申请人提供）：抗氧化酶铜、锌超氧化物歧化酶（SOD1）通过一百多个致病突变与肌萎缩侧索硬化症（ALS）相关。 Rac1 GTPase 通过调节 NADPH 氧化酶 (Nox) 活性来调节氧化剂水平。有趣的是，SOD1 最近被证明可以以核苷酸和氧化还原依赖性方式结合 Rac1，从而调节 Rac1 活性。此外，SOD1 中的两个 ALS 相关突变被证明使 SOD1/Rac1 相互作用对氧化还原调节不敏感，推测通过 Nox 过度活跃导致氧化剂水平增加。然而，这一前提是基于仅从两种细菌衍生的突变体获得的有限定性结果。与酿酒酵母中表达的天然 SOD1 和人类 SOD1 不同，大肠杆菌衍生的人类 SOD1 缺乏 N 末端乙酰化和足够的金属化。此外，我们最近发现人类红细胞中 40-50% 的 SOD1 被谷胱甘肽修饰，谷胱甘肽是细胞氧化还原状态的关键参与者。谷胱甘肽化促进 SOD1 二聚体解离，这是 SOD1 聚集的必要起始事件，也是母提案的重点。在这里，我们的目标是使用从人类红细胞和酿酒酵母中分离的 SOD1 来定量描述 SOD1/Rac1 相互作用及其与 ALS 的关系。我们将使用 NMR 和其他生物物理技术来表征和量化在 SOD1 存在或不存在修饰和 ALS 相关突变的情况下 Rac1/SOD1 相互作用。最近发现 SOD1 通过 Rac1 与 Nox 复合物相互作用，这提供了 SOD1（功能障碍）和导致细胞死亡的下游事件之间的潜在联系。因此，阐明 SOD1 修饰和家族性 ALS 突变如何影响与 Rac1 的相互作用非常重要，这也是本补充品的目标。 公共卫生相关性：拟议工作的目标是定义 SOD1 调节 Rac1 的机制。了解这一机制将有助于制定确定 SOD1 的新调节作用是否与 ALS 相关的策略。此外，如果 Rac1/SOD1 复合，则 Rac1/SOD1 有助于 ALS 治疗。