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

描述（由申请人提供）：在本项目中，我们将确定铜锌超氧化物歧化酶（SOD1）在细胞存活和死亡调控中的作用。野生型（WT） SOD1是一种促生存蛋白，而与肌萎缩性侧索硬化症（ALS）相关的SOD1突变体在体内和体外都是有毒的。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，突变体SOD1特异性定位于脊髓线粒体，在那里形成抗sds的高分子量聚集体，结合并诱捕Bcl-2。（Pasinelli et al ., 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，并且突变SOD1具有新的毒性功能获得。因此，虽然WT SOD1可能通过与Bcl-2的相互作用来保护细胞免于死亡，但突变的SOD1可能通过与Bcl-2的异常结合并将Bcl-2转化为有毒或无功能的蛋白而变得有毒。为了支持这一假设，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来发挥其抗和促凋亡功能。本研究旨在研究WT和突变体SOD1的抗和促死亡功能，以及它们与Bcl-2的相互作用。最终目的是了解突变SOD1介导的毒性机制，并确定线粒体突变SOD1/Bcl-2复合物在ALS发病机制中的潜在作用。具体目的是：1)(A)确定WT SOD1的促存活活性是否取决于其与Bcl-2的结合，以及(B)确定突变体SOD1介导的毒性是否取决于其与Bcl-2的异常相互作用。2) (A)确定SOD1中与Bcl-2结合所必需的区域；(B)研究WT SOD1与Bcl-2和突变体SOD1与Bcl-2结合强度的差异。3) (A)确定Bcl-2与突变体SOD1和结合后是否发生构象修饰。(B)在突变型SOD1连锁ALS的细胞培养模型上，测试Bcl-2和SOD1样肽消除Bcl-2与突变型SOD1结合的潜在益处。4)确定Bcl-2是否介导突变体SOD1线粒体易位。5)利用转基因ALS小鼠和患者研究SOD1/ bcl -2突变体与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.