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.
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