The object of this proposal is to understand the details of the molecular processes involved in interleukin 1-induction of metalloproteinase- mediated inflammation. Human interleukin 1 (IL-1) has been shown to play important roles in mediating immune responses and inflammatory diseases such as in the joint destruction of rheumatoid arthritis. A model for rheumatoid involving IL-1-induced expression of target genes such as procellagenase and prostromelysin forms the basis for using human fibroblasts in many of the studies in this proposal. This model will be studied in concert with a program of molecular structural analysis, recombinant protein modification, and investigation of cellular transduction pathways in order to determine the details of the mechanism leading to IL-1 target cell activation. We previously generated a mutant human IL-1Beta protein that can now be characterized as ligand defective in only a subset of IL-1 signaling activates. Analysis of the effects of this mutant as well as native wild-type IL-1Beta and the naturally occurring IL-1 receptor antagonist proteins defines three distinct IL-1 partial functions. Definition of the three partial functions manifested differentially by these three molecules, namely: receptor binding; early signal transduction; and late signal transduction, provide a powerful tool for elucidating the mechanism of target cell activation by IL-1. Additionally, the construction and expression of mutant IL-1, IL-1 receptor antagonist, and IL-1 receptor binding and cytoplasmic domains is proposed in order to map essential regions for binding and signal transduction. Transfection of modified receptors into various cell lines is also proposed. Finally, an in vitro approach using affinity chromatography employing various glutathione S-transferase fusion proteins will be used to identify possible intracellular associations involving IL-1 receptors and other proteins.
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