SOD1/Bc1-2 Complex: A Role in Regulating Motor Neuron Cell Death

描述(申请人提供)：在这个项目中，我们将确定铜锌超氧化物歧化酶(SOD1)在调节细胞存活和死亡中的作用。野生型(WT)SOD1是一种促进生存的蛋白，而肌萎缩侧索硬化症(ALS)相关的SOD1突变体在体外和体内都是有毒的。我们最近发现WT和突变体SOD1都与抗凋亡蛋白Bcl-2相互作用。然而，突变体SOD1与Bcl-2结合的性质与WT SOD1不同。与WT SOD1相反，突变型SOD1特异性地定位于脊髓线粒体，在那里它形成结合和捕获Bcl-2的耐十二烷基硫酸钠的高分子量聚集体。(Pasinelli等，2004，Neuron 43：19-30)。这些研究表明，WT SOD1在调节细胞存活和死亡方面具有潜在的新功能，而突变体SOD1具有新的毒性功能。因此，虽然WT SOD1可能通过与Bcl-2的相互作用来保护细胞免受死亡，但突变的SOD1可能通过异常地与Bcl2结合并将Bcl2转化为有毒或无功能的蛋白质而变得有毒。为了支持这一假说，我们现在有初步的数据表明，WT和突变体SOD1可能分别需要Bcl-2发挥其抗和促凋亡功能。根据目前的建议，我们打算表征WT和突变体SOD1的抗死亡和促死亡功能，以及它们各自与Bcl-2的相互作用。最终目的是了解突变的Sod1介导的毒性的机制(S)，并确定线粒体突变的Sod1/Bcl2复合体在ALS发病机制中的潜在作用。其具体目的是：(1)(A)确定WT SOD1的促生存活性是否取决于其与Bcl-2的结合；(B)确定突变型SOD1介导的毒性是否取决于其与Bcl-2的异常相互作用。2)(A)鉴定SOD1中与Bcl2结合所必需的区域(S)；(B)研究WTSOD1Bcl一2与突变型SOD1Bcl一2结合强度的差异。3)(A)确定Bcl-2与突变型SOD1结合后是否发生构象改变，以及(B)在我们建立的突变型SOD1连锁的ALS细胞培养模型上，测试取消Bcl2和突变型SOD1结合的类似肽对细胞的潜在益处。4)确定Bcl2是否介导突变型SOD1线粒体易位。5)利用转基因ALS小鼠和患者研究突变的SOD1/Bcl2聚集体与ALS的相关性。

DESCRIPTION (provided by applicant): In this project we will define a role for copper-zinc superoxide dismutase (SOD1) in the regulation of cell survival and death. While the wild-type (WT) SOD1 is a pro-survival protein, amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants are toxic both in vitro and in vivo. We recently found that both WT and mutant SOD1 interact with the anti-apoptotic protein Bcl-2. However, the nature of the mutant SOD1 binding with Bcl-2 differs from WT SOD1. Contrary to WT SOD1, mutant SOD1 specifically localizes to spinal cord mitochondria where it forms SDS-resistant high molecular weight aggregates that bind and entrap Bcl-2. (Pasinelli et al, 2004, Neuron 43: 19-30). These studies suggest a potentially novel function for WT SOD1 in regulating cell survival and death, and a novel, toxic gain-of-function for mutant SOD1. Thus, while WT SOD1 may protect against cell death through its interaction with Bcl-2, mutant SOD1 may become toxic by aberrantly binding to Bcl-2 and converting Bcl-2 into a toxic or non-functional protein. In support of this hypothesis, we now have preliminary data indicating that both WT and mutant SOD1 might require Bcl-2 to exert their anti-and-pro apoptotic function respectively. With the present proposal we intend to characterize the anti and pro-death function of WT and mutant SOD1 and their respective interactions with Bcl-2. The ultimate goal is to understand the mechanism(s) of mutant SOD1-mediated toxicity and to define a potential role for the mitochondrial mutant SOD1/Bcl-2 complex in ALS pathogenesis. The specific aims are: 1) (A) To determine whether WT SOD1 pro-survival activity depends on its binding to Bcl-2, and (B) to determine whether mutant SOD 1-mediated toxicity depends on the aberrant interaction with Bcl-2. 2) (A) To identify the region(s) in SOD1 essential for the binding with Bcl-2, and (B) to study the difference in binding strength between WT SOD1 and Bcl-2 and mutant SOD1 and Bcl-2. 3) (A) To determine whether Bcl-2 undergoes conformational modifications upon binding with mutant SOD1 and.(B) to test the potential benefit of Bcl-2 and SOD1 like-peptides that abolish binding between Bcl-2 and mutant SOD1 on our cell culture model of mutant SOD1-linked ALS. 4) To determine whether Bcl-2 mediates mutant SOD1 mitochondrial translocation. 5) To study the correlation between mutant SOD1/Bcl-2-containing aggregates and ALS using transgenic ALS mice and patients.