Inhibition of both the extrinsic and intrinsic death pathways through novel death -fold interactions
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Apoptosis is mediated by two central pathways, one involving cell surface death receptors (extrinsic pathway) and the other utilizing mitochondria (intrinsic pathway). These central death pathways transduce apoptotic signals through protein-protein interactions involving death-fold motifs. Death-fold domains constitute an evolutionarily conserved superfamily that includes CARD (Caspase Recruitment Domain), DD (Death Domain), and DED (Death Effector Domain), and which exert their effects through homotypic interactions.;ARC (A&barbelow;poptosis R&barbelow;epressor with a C&barbelow;aspase recruitment domain (CARD)) is a death fold protein which contains an N-terminal CARD and a C-terminal region rich in proline and glutamic acid residues. ARC is an anti-apoptotic regulator that is expressed in most cell types but is enriched in long-lived cells such as cardiac and skeletal myocytes, and neurons. While most apoptosis inhibitors oppose a single apoptosis pathway, ARC appears to inhibit both the extrinsic and intrinsic death pathways. However, the mechanisms by which ARC accomplishes this are not known.;In this thesis, we demonstrate that ARC, a single death-fold protein, inhibits both the extrinsic and intrinsic pathways through novel death-fold interactions. Antagonism of the extrinsic pathway is mediated by ARC's direct binding to Fas and FADD, which occurs through novel heterotypic interactions between the CARD of ARC and the DDs of Fas and FADD. These heterotypic interactions inhibit the traditional homotypic interactions between the DDs of Fas and FADD that are required for Fas-FADD binding and DISC (Death Inducing Signaling Complex) assembly. ARC inhibits the intrinsic pathway by direct binding to Bax, which requires ARC's CARD and the C-terminal regulatory domain of Bax, a novel interaction between a death-fold motif and a non-death-fold protein. The binding of ARC to Bax prevents Bax's conformational activation and translocation to the mitochondria in response to an apoptotic stimulus. The physiological relevance of these observations is demonstrated by the knockdown of endogenous ARC in muscle cells: DISC formation is potentiated and Bax activation and cell death occur spontaneously even in the absence of an apoptotic stimulus. Conversely, endogenous ARC is sufficient to prevent these events. Taken together, these studies establish a mechanism by which a single protein inhibits both extrinsic and intrinsic death pathways through novel death-fold interactions.;In this thesis, we also elucidate the mechanism by which the abundance of ARC protein rapidly decreases during apoptosis. While mRNA level remains constant, ARC's protein stability decreases in response to apoptotic stimuli. (Abstract shortened by UMI.).