Protein Misfolding and Aggregation

说明(申请人提供)：抗氧化酶铜锌超氧化物歧化酶(SOD1)通过100多种致病突变与肌萎缩侧索硬化症(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，SOD1最近被证明以核苷酸和氧化还原依赖的方式与rac1结合来调节rac1的活性。此外，SOD1中与ALS相关的两个突变被证明使SOD1/rac1相互作用对氧化还原调节不敏感，推测通过NOx过度活性导致氧化剂水平增加。然而，这一前提是基于仅从两个细菌来源的突变体获得的有限的定性结果。与在酿酒酵母中表达的天然SOD1和人SOD1不同，大肠杆菌来源的人SOD1既缺乏N端乙酰化，也缺乏足够的金属化。此外，我们最近发现，人红细胞中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。谷胱甘肽促进SOD1二聚体的解离，这是SOD1二聚体聚集的必要启动事件，也是亲代提议的焦点。在这里，我们的目标是使用从人红细胞和酿酒酵母中分离的SOD1来定量描述SOD1/rac1的相互作用，以及它与ALS的关系。我们将使用核磁共振和其他生物物理技术来表征和量化在SOD1存在和不存在修饰和ALS相关突变的情况下rac1/SOD1的相互作用。最近发现SOD1通过rac1与NOx复合体相互作用，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，这是本补充材料的目标。 公共卫生相关性：拟议工作的目标是定义一种机制，用于由SOD1调节rac1。对这一机制的了解将导致确定SOD1的这种新的调节作用是否与ALS有关的策略。此外，如果rac1/sod1激活了rac1/sod1复合体，则有助于ALS的治疗。

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.