Nucleotide Regulation of Apaf-1 Apoptosome and Apoptosis

描述（由申请人提供）：环境危害和压力、致癌物和抗癌治疗剂通过内在或线粒体死亡途径引起细胞凋亡。异常的细胞凋亡反应通常促进肿瘤的发展，并且细胞凋亡中的缺陷与肿瘤细胞对抗肿瘤剂的抗性密切相关。Caspase的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溶酶体形成代表了半胱天冬酶-9（引发剂半胱天冬酶）活化中的关键起始事件，其最终激活效应半胱天冬酶如半胱天冬酶-3以执行细胞死亡。Apaf-1核糖体如何在体内形成和调节（即，在受刺激的细胞中）仍然知之甚少。最近，我们提供的证据表明，生理水平的核苷酸通过直接结合CC并阻止CC与Apaf-1相互作用来抑制CC诱导的、溶酶体介导的胱天蛋白酶-9活化（Chandra等人，Cell 125，1333-1346，2006;附录I）。因此，CC介导的溶酶体组装和活化被阻断。共显微注射的核苷酸和CC使细胞抵抗CC诱导的细胞凋亡在体内，而实验减少核苷酸增强CC和凋亡刺激诱导的细胞死亡。这些观察结果使我们假设，生理水平的核苷酸，除了它们在核酸合成，中间代谢和维持生物能量学中的既定作用外，还通过直接抑制CC介导的溶酶体形成和半胱天冬酶激活来作为关键的促生存因子。有几个关键的未回答的问题有关Apaf-1核糖体的调节核苷酸。我们提出了以下三个具体目标来解决这些问题：1）进一步研究核苷酸与CC在体外的相互作用，并阐明核苷酸与CC在体内的相互作用; 2）研究核苷酸与Apaf-1的相互作用及其对Apaf-1的激活的影响; 3）研究Apaf-1亚型表达和核苷酸相互作用对Apaf-1激活的影响。这些目标将通过细胞生物学、生物化学和分子方法的组合来实现。所提出的目标的实现将极大地推进我们对体内溶酶体调控的理解，并帮助我们理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.