The process of apoptotic cell death is of fundamental importance for our understanding of the development, function, and regulation of the immune system. While the phenomenon of apoptosis is very well documented, virtually nothing was known regarding the biochemical events underlying the central mechanism of cell death until recently. In the past two years, a number of mammalian cysteine proteases have been identified that show homology to the C. elegans Ced-3 protein, which has been demonstrated to play a critical role in programmed cell death in the nematode. This set of proteases, the ICE/Ced-3 family, reside in most cells in an inactive form, and one or more of these becomes activated by proteolytic processing during apoptosis. The observation that inhibitors of these proteases can block several forms of apoptosis have led to the central hypothesis that the activation of one or more members of this family is responsible for mediating cell death in all forms of apoptosis. The investigator refers to this mechanism as the """"""""Executioner"""""""" and he will test the idea that members of this family are essential components. Based on preliminary studies, he proposes to address five specific aims in this project, all directed at delineating the activation, function, and regulation of these proteases during apoptotic cell death in lymphoid cells. Several of his approaches depend upon the use of cell-free systems he has developed in which normal nuclei are induced to undergo apoptotic changes when cultured with extracts from lymphoid cells. In his first aim, he will pursue his observation that cytochrome c induces apoptosis in his cell-free system by inducing the processing of the processing of the Ced-3-like proteases. In his second aim, he will examine how this cytochrome c is released from mitochondria, and whether there is a role for an upstream protease such as that induced by ligation of CD95. In his third aim, he will explore the role of the proteasome in activating and/or regulating critical Ced-3-subfamily proteases, or other members of the ICE/Ced-family. In the fourth aim he will examine the changes in subcellular localization, especially nuclear, upon activation of these proteases. Finally, in the fifth aim, the investigator will examine the function of a potent anti-apoptotic protein, Bcl-2, in terms of its effects on the activation, function, and localization of ICE/Ced-3 family proteases. Once characterized in detail, the activation of the critical proteases will eventually serve as a biochemical thread by which to trace the molecular events that begin at the surface and result in cell death in the immune system.
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