Novel Insight on Nucleotide Regulation of Apoptosome Activation

描述（由申请人提供）：细胞凋亡缺陷与肿瘤发展密切相关，也是肿瘤细胞对抗肿瘤药物耐药性的原因。Caspase的激活是细胞凋亡的核心。细胞色素c（CC）启动的Apaf-1溶酶体形成代表了caspase-9（一种启动caspase）激活的关键起始事件，其最终激活效应caspase如caspase-3以执行细胞死亡。这种半胱天冬酶激活级联反应可以在体外通过向细胞裂解物中加入CC沿着低水平的ATP或dATP（~10-200 μ M）来重建，导致许多人得出的一般结论是核苷酸如dATP或ATP [（d）ATP]是促凋亡的。然而，大多数哺乳动物细胞具有4-10 mM的内源性核苷酸库。ATP和核苷酸的生理mM水平一般如何影响溶酶体形成和半胱天冬酶激活仍然是一个关键的未回答的问题。最近，我们提供了强有力的证据表明，生理相关水平的核苷酸有力地抑制CC诱导的，溶酶体介导的caspase-9的激活直接结合到CC和防止CC与Apaf-1相互作用。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。在这个K 01基金申请中，我提出了三个具体目标来验证这一假设：1）进一步研究核苷酸与CC的体外相互作用; 2）阐明核苷酸与CC的体内相互作用; 3）研究核苷酸与Apaf-1的相互作用及其对线粒体激活的影响。这些目标将使用细胞、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显着推进我们的理解，在分子水平上，核苷酸如何调节线粒体和半胱天冬酶的激活以及细胞死亡。新获得的知识也应该有重要的意义，在帮助设计新的抗癌治疗靶向凋亡机制。

DESCRIPTION (provided by applicant): Defects in apoptosis are intimately associated with tumor development and are also responsible for tumor cell resistance to anti-neoplastic agents. Caspase activation lies in the core of apoptotic cell death. 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. This cascade of caspase activation can be reconstituted in vitro with the addition of CC to cell lysates along with low levels of ATP or dATP (~10-200 ¿M), leading to the general conclusion by many that nucleotides such as dATP or ATP [(d)ATP] are proapoptotic. Most mammalian cells, however, possess an endogenous nucleotide pool of 4-10 mM. How the physiological, mM levels of ATP and nucleotides in general affect apoptosome formation and caspase activation remains a critical unanswered question. Recently, we have provided strong evidence that the physiologically relevant levels of nucleotides powerfully inhibit the CC-induced, apoptosome-mediated caspase-9 activation by binding directly to CC and preventing CC from interacting with Apaf-1. 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. In this K01 grant application, I propose three Specific Aims to test this hypothesis: 1) To further study nucleotide interaction with CC in vitro; 2) To elucidate nucleotide interaction with CC in vivo; and 3) To investigate nucleotide interaction with Apaf-1 and its impact on apoptosome activation. These aims will be accomplished using a combination of cellular, biochemical, and molecular approaches. The accomplishment of the proposed goals will significantly advance our understanding, at the molecular level, of how nucleotides regulate apoptosome and caspase activation as well as cell death. The newly obtained knowledge should also have important implications in helping to design novel anti-cancer therapeutics targeting the apoptotic machinery.