Programmed Cell Death In The Nervous System

神经系统中的程序性细胞死亡

• 批准号: 6990654
• 负责人: Richard James Youle
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990654
• 关键词: BCL2 gene /protein; Bax gene /protein; apoptosis; cell growth regulation; cytotoxicity; developmental neurobiology; green fluorescent proteins; mitochondrial membrane; monoclonal antibody; neurons; neuroprotectants; neurotoxins; neurotrophic factors; phosphorylation

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 and can form pores in the mitochondrial membrane. These pores appear to release proteins that activate proteases to degrade cell contents during apoptosis.

我们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，我们重点关注在发育过程中调节神经元存活的 Bcl-2 蛋白家族。 Bax 是该家族的成员之一，它是神经元正常死亡所必需的，消除 Bax 基因会导致成年动物产生过多的神经元。 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，以探讨它们的体外和体内作用机制。抗体显示，在细胞凋亡过程中，Bax 从细胞质迁移到细胞膜。为了探索神经元中的 Bax、Bcl-2 和 Bcl-xl 运动，绿色荧光蛋白已被用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，这是其细胞凋亡控制机制中的一个重要步骤。更好地插入线粒体的 Bax 点突变体毒性更大，而线粒体插入较少的突变体毒性更小，表明 Bax 的 C 末端调节线粒体关联。因此，我们确定了 Bax 的三维结构，并发现 C 末端尾部适合一个疏水口袋，该口袋被认为可以调节 Bcl-2 家族成员之间异源和同源二聚体的形成。这提出了一种新模型，即二聚体形成和线粒体对接在结构上与 C 末端的脱离相关。与 Bax 相比，Bcl-xl 可以防止许多神经毒性损伤引起的神经元细胞死亡。与 Bax 一样，Bcl-xl 在细胞凋亡过程中插入线粒体。我们发现了一个控制 Bcl-xl 亚细胞位置的调控步骤。 Bad 是一种促凋亡 Bcl-2 家族成员，与 Bcl-xl 结合并触发其与线粒体对接。 Bad 与 Bcl-xl 的结合反过来又受到磷酸化和刺激激酶的几种神经营养因子的调节，可阻止 Bad 与 Bcl-xl 结合，从而防止神经元死亡。还研究了 Bcl-2 家族成员与线粒体结合的后果。 Bax 在线粒体表面聚结成大的聚集体，并可以在线粒体膜上形成孔。这些孔似乎会释放蛋白质，激活蛋白酶，在细胞凋亡过程中降解细胞内容物。