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的发展密切相关，也是肿瘤细胞对抗肿瘤药物产生耐药性的原因。Caspase的激活是凋亡细胞死亡的核心。细胞色素c （CC）启动的Apaf-1凋亡小体的形成代表了caspase-9（一种启动caspase）激活的关键启动事件，caspase-9最终激活caspase-3等效应caspase来执行细胞死亡。这种caspase激活的级联反应可以在体外通过在细胞裂解物中加入CC以及低水平的ATP或dATP （~10-200 μ M）来重建，从而导致许多人得出一般结论，如dATP或ATP [(d)ATP]等核苷酸是促凋亡的。然而，大多数哺乳动物细胞具有4- 10mm的内源性核苷酸库。ATP和核苷酸的生理、mM水平如何影响凋亡的形成和caspase的激活仍然是一个关键的未解之谜。最近，我们提供了强有力的证据，证明生理相关水平的核苷酸通过直接结合CC并阻止CC与Apaf-1相互作用，有效抑制CC诱导的凋亡体介导的caspase-9激活。因此，cc介导的凋亡细胞组装和激活被阻断。在体内共微量注射核苷酸和CC可使细胞抵抗CC诱导的细胞凋亡，而在实验中减少核苷酸可增强CC和凋亡刺激诱导的细胞死亡。

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.