A Novel Mechanism to Regulate Caspase-8 Activation on the Mitochondrial Membrane

描述(申请人提供)：环境危害和压力，实验和环境致癌物质，环境暴露于紫外线和电离辐射，以及抗癌治疗药物(包括化疗药物、辐射和辅助治疗药物)通过内在或线粒体死亡途径导致细胞死亡。异常的细胞凋亡反应有助于环境危害和致癌物诱导的肿瘤的发生。细胞凋亡缺陷也与肿瘤细胞对抗肿瘤药物的耐药性密切相关。阐明线粒体死亡途径的分子机制将有助于理解细胞如何响应环境应激、危险和治疗。不同信号通路中细胞凋亡的本质都集中在caspase的激活上。在Fas(CD95)、TNFR和TRAIL受体启动的细胞凋亡信号中，caspase-8是最上游的caspase。正常情况下，膜上死亡受体的结合将FADD和proaspase-8/10招募到死亡诱导信号复合体(DISC)，激活caspase-8，最终激活caspase-3执行细胞凋亡。然而，最近的证据表明，线粒体上存在显著水平的caspase-8。线粒体定位的caspase-8是如何在线粒体水平上被激活和调节的，目前尚不清楚。我们最近发表的和初步的观察结果表明：1)各种内在的凋亡刺激激活上皮性癌细胞和线粒体中的caspase-8似乎是FADD介导的caspase-8在这些凋亡系统中激活的关键平台，以及2)线粒体上的FADD在诱导凋亡时，寡聚为更高分子量的蛋白质复合体。因此，我们假设在内源性刺激诱导的细胞凋亡过程中，线粒体在启动FADD介导的caspase-8激活和盘组装过程中起着关键的信号中心作用。线粒体上caspase-8激活的启动可能是细胞凋亡诱导过程中最早的事件，也可能是与caspase-9激活平行的早期事件之一。阐明上述新的调控机制不仅有助于我们更好地了解caspase激活是如何启动和调控的，而且也为设计新的抗癌治疗药物奠定了基础。本文提出了两个具体目标来检验这一假设：1)。建立内在的细胞凋亡刺激诱导FADD介导的线粒体caspase-8激活；以及2)。目的：研究线粒体盘状复合体的形成和活性以及抗凋亡蛋白(即Bclxl、c-fllip)对其的调节作用。环境危害和压力、实验致癌物质和环境致癌物质、环境暴露于紫外线和电离辐射会导致细胞死亡或凋亡。阐明细胞凋亡的分子机制将有助于理解细胞如何应对环境压力和危害，对改善人类健康具有重要意义。

DESCRIPTION (provided by applicant): Environment hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation, and anti-cancer therapeutics (including chemotherapeutic drugs, radiation, and adjuvant therapeutics) cause cell death through intrinsic or mitochondrial death pathway. Abnormal apoptotic response contributes to tumor development induced by environmental hazards and carcinogens. Defects in apoptosis are also intimately associated with tumor cell resistance to anti-neoplastic agents. Elucidating the molecular mechanisms of the mitochondrial death pathway will facilitate understanding of how cells respond to environmental stresses, hazards and therapeutics. The essence of apoptosis in various signaling pathways converges on the activation of caspases. Caspase-8 acts as the most upstream caspase in apoptotic signaling initiated by Fas (CD95), TNFR, and TRAIL receptors. Normally, engagement of death receptors on the plasma membrane recruits FADD and procaspase-8/10 to the death-inducing signaling complex (DISC) to activate caspase-8, which ultimately activate caspase-3 to execute apoptosis. Recent evidence, however, indicates the presence of significant levels of caspase-8 on the mitochondria. How mitochondrially-localized caspase-8 is activated and regulated at the mitochondrial levels remains unknown. Our recent published and preliminary observations demonstrate that: 1) a variety of intrinsic apoptotic stimuli activate caspase-8 in epithelial cancer cells and mitochondria appear to function as a critical platform for FADD-mediated caspase-8 activation in these apoptotic systems, and 2) FADD on the mitochondria, upon apoptotic induction, oligomerizes to higher molecular weight protein complex. Accordingly, we hypothesize that mitochondria function as crucial signaling center to initiate FADD-mediated caspase-8 activation and DISC assembly during apoptosis induced by intrinsic stimuli. The initiation of caspase-8 activation on the mitochondria may represent the earliest event or one of the earlier events in parallel with caspase-9 activation during apoptosis induction. Elucidation of the afore-mentioned novel regulatory mechanisms should not only help us better understand how caspase activation is initiated and regulated but also lay a foundation for designing novel anti-cancer therapeutics. Two Specific Aims are proposed to test the hypothesis: 1). To establish that intrinsic apoptotic stimuli induce FADD-mediated caspase-8 activation on the mitochondria; and 2). To characterize the formation and activity of DISC-like complex on the mitochondria and its regulation by antiapoptotic proteins (i.e., Bcl-xL, c-FLIP). Environmental hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation cause cell death or apoptosis. Elucidating the molecular mechanisms of apoptotic cell death will facilitate the understanding of how cells respond to environmental stresses and hazards, and will be highly significant in improving human health.