Nucleotide Regulation of Apaf-1 Apoptosome and Apoptosis

描述(由申请人提供)：环境危害和压力、致癌物质和抗癌治疗通过内在或线粒体死亡途径导致细胞凋亡。异常的细胞凋亡反应常导致肿瘤的发生，而细胞凋亡缺陷与肿瘤细胞对抗肿瘤药物的耐药性密切相关。半胱氨酸天冬氨酸氨基转移酶激活是细胞凋亡的核心。在线粒体死亡途径中，细胞色素c(CC)启动的Apaf-1凋亡体的形成是caspase-9(一种启动子caspase)激活的关键启动事件，最终激活效应caspase如caspase-3来执行细胞死亡。APAF-1的凋亡体是如何在体内(即在刺激的细胞中)形成和调节的，目前还知之甚少。最近，我们提供了证据表明，生理水平的核苷酸通过直接与CC结合并阻止CC与APAF-1相互作用来抑制CC诱导的、凋亡体介导的caspase-9激活(Chandra等人，Cell 125,1333-1346,2006；附录I)。因此，CC介导的凋亡体组装和激活被阻断。在体内，联合微量注射核苷酸和CC使细胞对CC诱导的细胞凋亡产生抵抗，而实验中减少核苷酸会增强CC和凋亡刺激诱导的细胞死亡。这些观察结果使我们推测，生理水平的核苷酸，除了在核酸合成、中间代谢和生物能量学的维持中已被证实的作用外，还通过直接抑制CC介导的凋亡体形成和caspase激活而发挥关键的生存因素的作用。关于核苷酸对APAF-1凋亡体的调控，有几个关键问题尚未回答。我们提出以下三个具体目标来解决这些问题：1)在体外进一步研究核苷酸与CC的相互作用，以及在体内阐明核苷酸与CC的相互作用；2)研究核苷酸与APAF-1的相互作用及其对凋亡体激活的影响；以及3)研究APAF-1亚型表达和核苷酸相互作用对APAF-1凋亡体激活的影响。这些目标将通过细胞生物学、生化和分子方法的组合来实现。这些目标的实现将极大地促进我们对体内凋亡体调控的理解，并有助于我们了解细胞对环境应激的反应和癌细胞对抗肿瘤治疗的反应。

DESCRIPTION (provided by applicant): Environmental hazards and stress, carcinogens, and anti-cancer therapeutics cause apoptotic cell death through the intrinsic or mitochondrial death pathway. Abnormal apoptotic response often contributes to tumor development and defects in apoptosis are intimately associated with tumor cell resistance to anti- neoplastic agents. Caspase activation lies in the core of apoptotic cell death. In the mitochondrial death pathway, cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in caspase-9 (an initiator caspase) activation, which ultimately activates effector caspases such as caspase-3 to execute cell demise. How Apaf-1 apoptosome is formed and regulated in vivo (i.e., in the stimulated cells) remains poorly understood. Recently, we provided evidence that physiological levels of nucleotides inhibit the CC-induced, apoptosome-mediated caspase-9 activation by binding directly to CC and preventing CC from interacting with Apaf-1 (Chandra et al., Cell 125, 1333-1346, 2006; Appendix I). Consequently, the CC- mediated apoptosome assembly and activation are blocked. Co-microinjection of nucleotides and CC renders cells resistant to the CC-induced apoptosis in vivo whereas experimentally reducing nucleotides enhances both CC and apoptotic stimuli-induced cell death. These observations lead us to hypothesize that physiological levels of nucleotides, in addition to their well-established roles in nucleic acid synthesis, intermediate metabolism, and maintenance of bioenergetics, also function as critical prosurvival factors by directly inhibiting the CC-mediated apoptosome formation and caspase activation. There are several critical unanswered questions related to Apaf-1 apoptosome regulation by nucleotides. We propose the following three Specific Aims to address some of these questions: 1) To further study nucleotide interaction with CC in vitro and to elucidate nucleotide interaction with CC in vivo; 2) To investigate nucleotide interaction with Apaf-1 and its impact on apoptosome activation; and 3) To study the effects of Apaf-1 isoform expression and nucleotide interaction on Apaf-1 apoptosome activation. These goals will be achieved by a combination of cell biological, biochemical, and molecular approaches. The accomplishment of the proposed goals will greatly advance our understanding of apoptosome regulation in vivo and help us understand cellular response to environmental stress and cancer cell response to anti- tumor therapeutics.