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Programmed Cell Death In The Nervous System

神经系统中的程序性细胞死亡

基本信息

批准号：
7735271
负责人：
Richard James Youle
金额：
$ 159.85万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

We have studied programmed cell death in the nervous system and the biochemical mechanism of apoptosis. Recently we have focused on the Bcl-2 family of proteins that regulates the survival of neurons during development. One member of this family, Bax, is required for the normal death of neurons and elimination of the Bax gene results in excess neurons in adult animals. How Bax and other Bcl-2 family members regulate cell survival is unknown. We developed a series of monoclonal antibodies against Bcl-2, Bcl-xl, and Bax to probe their mechanism of action in vitro and in vivo. The antibodies revealed that Bax migrates from the cytosol to cell membranes during apoptosis. To explore Bax, Bcl-2 and Bcl-xl movement in neurons the green fluorescent protein has been used to tag and follow the proteins in living cells. We discovered that Bax and Bcl-xl move from the cytosol to the mitochondria as an essential step in their mechanism of apoptosis control. Point mutants of Bax that insert better into mitochondria are more toxic and mutants with less mitochondrial insertion are less toxic indicating that the C-terminus of Bax regulates the mitochondrial association. Therefore, we determined the three dimensional structure of Bax and found that the C-terminal tail fits into a hydrophobic pocket that has been thought to regulate hetero- and homo-dimer formation among Bcl-2 family members. This suggests a new model that dimer formation and mitochondrial docking are structurally linked to disengagement of the C-terminus. In contrast to Bax, Bcl-xl prevents neuron cell death due to many neurotoxic insults. Like Bax, Bcl-xl inserts into mitochondria during apoptosis. We have found one regulatory step that controls Bcl-xl subcellular location. Bad, a pro-apoptotic Bcl-2 family member, binds to Bcl-xl and triggers its docking to mitochondria. Bad binding to Bcl-xl is in turn regulated by phosphorylation and several neurotrophic factors that stimulate kinases prevent Bad from binding to Bcl-xl and thus prevent neuron death. The consequences of Bcl-2 family member binding to mitochondria have been also studied. Bax coalesces into large aggregates on the mitochondrial surface that co-localize with machinery for mitochondrial division and can activate mitochondrial division. Thus, the cell machinery that governs mitochondrial division participates in apoptosis.

我们已经研究了神经系统中的程序性细胞死亡和细胞凋亡的生化机制。最近，我们集中在Bcl-2家族的蛋白质，调节神经元的生存在发展过程中。该家族的一个成员Bax是神经元正常死亡所必需的，并且Bax基因的消除导致成年动物中过量的神经元。Bax和其他Bcl-2家族成员如何调节细胞存活尚不清楚。我们开发了一系列针对Bcl-2、Bcl-xl和Bax的单克隆抗体，以探测它们在体外和体内的作用机制。抗体显示Bax在凋亡过程中从细胞质迁移到细胞膜。为了探索Bax、Bcl-2和Bcl-xl在神经元中的运动，已经使用绿色荧光蛋白来标记和跟踪活细胞中的蛋白质。我们发现Bax和Bcl-xl从细胞质移动到线粒体作为其细胞凋亡控制机制中的重要步骤。插入线粒体更好的Bax点突变体毒性更强，线粒体插入较少的突变体毒性较低，表明Bax的C-末端调节线粒体缔合。因此，我们确定了Bax的三维结构，并发现C-末端尾部适合于一个疏水口袋，该口袋被认为调节Bcl-2家族成员之间的异源和同源二聚体形成。这表明二聚体形成和线粒体对接在结构上与C-末端的脱离有关的新模型。与Bax相反，Bcl-xl防止由于许多神经毒性损伤引起的神经元细胞死亡。与Bax一样，Bcl-xl在细胞凋亡期间插入线粒体。我们已经发现了一个控制Bcl-xl亚细胞定位的调节步骤。Bad是促凋亡Bcl-2家族成员，与Bcl-xl结合并触发其与线粒体的对接。Bad与Bcl-xl的结合又受到磷酸化的调节，并且刺激激酶的几种神经营养因子阻止Bad与Bcl-xl结合，从而防止神经元死亡。Bcl-2家族成员与线粒体结合的后果也已被研究。Bax在线粒体表面聚结成大的聚集体，与线粒体分裂的机制共定位，并可以激活线粒体分裂。因此，控制线粒体分裂的细胞机制参与了细胞凋亡。