Mechanism by which Human ES Cells Prime Bax at the Golgi for Rapid Apoptosis

描述（由申请人提供）：人类胚胎干细胞（hES）因其再生能力而受到相当多的关注。然而，包括he细胞凋亡在内的基本生物学途径在很大程度上仍未被探索。我们已经开始研究he细胞的凋亡途径，并发现了he细胞调节细胞死亡的新颖而有趣的机制。我们发现he细胞对DNA损伤高度敏感，所有细胞在6小时内死亡。哺乳动物细胞凋亡的关键介质是Bax。在大多数细胞中，Bax维持在细胞质中处于非活性构象，仅在凋亡刺激下才被激活。活化的Bax随后转运到线粒体，诱导细胞色素c的释放和半胱天冬酶的激活。值得注意的是，我们发现he细胞将Bax维持在其已经活跃的构象中。令人惊讶的是，活跃的Bax维持在高尔基体中，而不是在线粒体中，从而使hES细胞有效地减少了与预先激活的Bax相关的风险。我们的研究结果表明，DNA损伤后，活性Bax通过p53依赖机制迅速从高尔基体转移到线粒体。因此，维持Bax的活性形式是一种独特的机制，可以使hES细胞快速死亡，可能在胚胎发育的早期关键阶段阻止突变的传播。在本研究中，我们将研究这种新的、意想不到的机制，通过这种机制，细胞凋亡在hES细胞中受到调控。我们将特别关注Bax如何维持活性状态（目的1），确定它如何定位到高尔基体（目的2），并确定DNA损伤引发的分子事件，诱导活性Bax在hES细胞中从高尔基体快速易位到线粒体（目的3）。这些研究无疑将揭示细胞凋亡调控的关键方面，揭示干细胞生物学的关键特征，对再生医学产生重大影响。

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells have received considerable attention with regards to their regenerative capacity. However, basic biological pathways including apoptosis in hES cells remain largely unexplored. We have started to investigate the apoptotic pathway in hES cells and have uncovered novel and fascinating mechanisms by which hES cells regulate cell death. We found that hES cells are highly sensitive to DNA damage, with all cells dying by 6 hours. A critical mediator of apoptosis in mammalian cells is Bax. In most cells, Bax is maintained in the cytosol in an inactive conformation and becomes activated only in response to apoptotic stimuli. Activated Bax then translocates to the mitochondria to induce cytochrome c release and caspase activation. Remarkably, we found that hES cells maintain Bax in its already active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. Our results show that after DNA damage, active Bax rapidly translocated from the Golgi to mitochondria by a p53-dependent mechanism. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development. In this proposal, we will investigate this novel and unexpected mechanism by which apoptosis is regulated in hES cells. We will focus specifically on examining how Bax is maintained in an active state (Aim 1), determine how it localizes to the Golgi (Aim 2) and identify the molecular events triggered by DNA damage to induce the rapid translocation of active Bax from the Golgi to the mitochondria (Aim 3) in hES cells. These studies will undoubtedly uncover critical aspects of apoptosis regulation in cells and reveal key features of stem cell biology that can have significant impact for regenerative medicine.