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（SOD 1）通过一百多个致病突变与肌萎缩侧索硬化症（ALS）相关。Rac 1 GT酶通过其在调节NADPH氧化酶（Nox）活性中的作用调节氧化剂水平。有趣的是，SOD 1最近显示出以核苷酸和氧化还原依赖性方式结合Rac 1以调节Rac 1活性。此外，SOD 1中的两个ALS相关突变显示出使SOD 1/Rac 1相互作用对氧化还原调节不敏感，假设通过Nox活性过高引起氧化剂水平增加。然而，这一前提是基于仅从两种细菌来源的突变体获得的有限定性结果。与天然SOD 1和人SOD 1在S. cerevisiae，E.大肠杆菌来源的人SOD 1缺乏N-末端乙酰化和足够的代谢。此外，我们最近发现，人红细胞中40-50%的SOD 1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SOD 1二聚体解离，这是SOD 1聚集的必要起始事件，也是母提案的焦点。在这里，我们的目的是提供一个定量的描述SOD 1/Rac 1的相互作用，其建议的关系，ALS，使用SOD 1分离的人红细胞和S。啤酒。我们将使用NMR和其他生物物理技术来表征和量化Rac 1/SOD 1相互作用的修饰和ALS相关突变的SOD 1的存在和不存在。最近发现SOD 1通过Rac 1与Nox复合物相互作用，这提供了SOD 1（dys）功能与导致细胞死亡的下游事件之间的潜在联系。因此，重要的是要阐明SOD 1修饰和家族性ALS突变如何影响与Rac 1的相互作用，这是本补充剂的目标。 公共卫生相关性：拟议工作的目标是确定一种机制，通过SOD 1对Rac 1进行监管。了解这一机制将有助于确定SOD 1的这种新的调节作用是否与ALS相关。此外，如果Rac 1/SOD 1复合物有助于ALS治疗。