The transcription factor NF-kappaB is critical in inflammatory gene expression, lymphocyte proliferation and cell death/survival decisions. Its activity is controlled by three inhibitory proteins, iKappaBalpha -beta -epsilon, whose synthesis and protein half-life is tightly regulated. Stimulus-induced degradation of IkappaB proteins associated with NFkappaB studied in great detail. The goals of the work proposed here are to characterize and contrast half-life regulation of NF-kappaB-bound and """"""""free"""""""" IkappaB proteins in resting cells and those responding to inflammatory stimuli and UV irradiation. The mechanisms and potential signaling pathways that determine these degradation rates will be characterized, and importantly, the role of the interaction with NF-kappaB in determining half-regulation will be examined. These results will serve to refine a computational model of NF-kappaB signaling significantly, that will allow us to examine the role of regulating these previously neglected degradation rates in NF-kapppaB signaling. First, we will focus on the equilibrium state in resting cells: Robustness of the signaling system vis-a-vis transient perturbations (""""""""noise"""""""") or long-term perturbations due to chronic changes in expression levels of the signaling components for example. Such perturbations may be caused by genetic changes that may be associated with inflammatory disease or tumorigenesis. Next, we will focus on the performance of the IkappaB/NF-kappaB signaling system during signal transduction, especially in response to weak or transient stimuli. Computational predictions will be examined experimentally using mutant cell lines generated from knockout mice and by lentivirus-mediated reconstitution of these with IkappaB mutant molecules. We will identify NF-kappaB target genes whose expression is sensitive to the regulation of IkappaB degradation rates and thereby demonstrate the specificity and physiological relevance of these mechanisms in human health and disease.
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