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

描述（由申请人提供）：环境危害和应激、实验和环境致癌物、环境暴露于紫外线和电离辐射以及抗癌治疗（包括化疗药物、辐射和辅助治疗）通过内源性或线粒体死亡途径引起细胞死亡。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发展。凋亡缺陷也与肿瘤细胞对抗肿瘤剂的抗性密切相关。阐明线粒体死亡途径的分子机制将有助于理解细胞如何应对环境压力，危害和治疗。细胞凋亡的本质在各种信号通路中汇聚于半胱天冬酶的激活。胱天蛋白酶-8在由Fas（CD 95）、TNFR和TRAIL受体启动的凋亡信号传导中充当最上游的胱天蛋白酶。正常情况下，质膜上死亡受体的参与将FADD和半胱天冬酶原-8/10募集到死亡诱导信号复合物（DISC）以激活半胱天冬酶-8，其最终激活半胱天冬酶-3以执行凋亡。然而，最近的证据表明线粒体上存在显著水平的半胱天冬酶-8。如何在线粒体水平上激活和调节脑内定位的caspase-8仍然是未知的。我们最近发表的和初步的观察结果表明：1）多种内在的凋亡刺激激活上皮癌细胞中的半胱天冬酶-8，并且线粒体似乎在这些凋亡系统中充当FADD介导的半胱天冬酶-8激活的关键平台，和2）线粒体上的FADD在凋亡诱导后寡聚化为更高分子量的蛋白质复合物。因此，我们假设线粒体作为重要的信号中心，启动FADD介导的caspase-8激活和DISC组装在细胞凋亡诱导的内在刺激。线粒体上半胱天冬酶-8激活的起始可能代表细胞凋亡诱导期间与半胱天冬酶-9激活平行的最早事件或较早事件之一。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节的，而且还为设计新的抗癌治疗方法奠定了基础。提出了两个具体的目的来检验假设：1）。确定内在凋亡刺激诱导线粒体上FADD介导的半胱天冬酶-8活化;和2）。为了表征线粒体上DISC样复合物的形成和活性以及抗凋亡蛋白（即，Bcl-xL，c-FLIP）。环境危害和压力，实验和环境致癌物，环境暴露于紫外线和电离辐射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，并将在改善人类健康方面具有重要意义。

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.