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Cu，Zn超氧化物歧化酶（SOD1）通过一百多个致病突变与肌萎缩性侧索硬化症（ALS）有关。 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，最近显示SOD1以核苷酸和氧化还原依赖的方式结合Rac1来调节Rac1活性。此外，SOD1中的两个与ALS相关的突变显示出对氧化还原调节不敏感的SOD1/RAC1相互作用，据说通过NOX过度活跃会导致氧化剂水平升高。但是，此前提是基于仅从两个细菌衍生的突变体获得的有限定性结果。与在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末端乙酰化和适当的金属化。此外，我们最近发现，人类红细胞中的40-50％的SOD1被谷胱甘肽（谷胱甘肽）修改 - 谷胱甘肽是细胞氧化还原状态的关键参与者。谷胱甘肽促进了SOD1二聚体解离，这是SOD1聚集的必要启动事件，也是父求的重点。在这里，我们旨在使用从人类的红细胞和酿酒酵母中分离出的SOD1，对SOD1/RAC1相互作用及其与ALS的拟议关系进行定量描述。我们将使用NMR和其他生物物理技术来表征和量化RAC1/SOD1在存在和不存在SOD1中与ALS相关的突变的情况下进行的Rac1/SOD1相互作用。最近发现SOD1通过RAC1与NOX复合物相互作用的发现提供了SOD1（DYS）功能与导致细胞死亡的下游事件之间的潜在联系。因此，重要的是要描述SOD1修饰和家族性ALS突变如何影响与Rac1的相互作用，这是该补充的目的。 公共卫生相关性：拟议工作的目的是定义SOD1对RAC1进行调控的机制。对这种机制的了解将导致确定SOD1的新调节作用是否与ALS有关的策略。此外，如果Rac1/SOD1 Rac1/SOD1复合物并有助于ALS疗法。