The investigators long-term objective is to understand the role of interleukin 1-b converting enzyme (ICE) family in normal and pathological conditions. The goal of this application is to characterize the mechanism and function of ICH-3, a member of the ICE family, in mediating apoptosis and inflammatory responses. Members of the mammalian ICE family are homologs of C. elegans cell death gene product ced-3. Increasing evidence suggests that the ICE protease family play important roles in controlling apoptosis. Ich-3-/- thymocytes are partially resistant to Fas induced apoptosis. Ich-3-/-embryonic fibroblast cells are resistant to granzyme B induced apoptosis. These results showed that ICH-3 may be a downstream component of Fas and GraB induced apoptotic pathway in certain cells. The first Specific aim is to continue characterization of the Ich-3 gene products and analyze the interaction of two products of Ich-3 locus. The second specific aim is to determine the mechanism of pro-ICH-3 activation. The members of the ICE family are synthesized as precursors and proteolytic activation is a critical regulatory step. Activation of ICH-3 in apoptosis will be determined by western blot analysis. The third specific Aim is to determine the mechanism of Ich-3 induction. Expression of Ich-3 is very low in normal condition and is highly induced upon stimulation by cellular stresses such lipoplysacharide (LPS) treatment and heat shock. The hypothesis that induction of Ich-3 is mediated by stress-activated MAP kinases JNK and p38 will be directly examined using dominant negative and constitutive active mutants of the JNK and p38 pathway. The fourth specific aim is to determine the substrates of ICH-3. Since expression of Ich-3 potentiates the ability of ICE in processing pro-IL-1b, the hypothesis is that one of the substrates of ICH-3 is an inhibitor of ICE. The fifth specific aim is to determine the role of ICH-3 in inflammatory responses. Ich-3-/- mice are resistant to lethality induced by LPS. The hypothesis is that the resistance of Ich-3 mutant mice to LIP lethality is caused at least in part by the resistance of the Ich-3 mutant cells to apoptosis. Vital organs of Ich-3-/- and wild type mice injected with LPS will be examined for apoptosis. These experiments will elucidate the ICH-3 pathway from signal tranduction, regulation of expression, mechanism of activation to the substrates of ICH-3. These works will have direct implication in control of apoptosis in normal and inflammatory conditions.
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