Gamma interferon (IFN-gamma) is an important lymphokine and biological response modifier in host defense against cancer and various infectious diseases. It is well known as an enhancer or activator of macrophage, natural killer cell, and T-cell cytotoxicity against tumors. More recently, it has been shown that secretion of IFN-gamma is an essential part of the mechanism of action of tumor infiltrating lymphocytes. The objective of this proposal is to determine the structural basis for gamma interferon (IFN-gamma) activation of cells at the receptor level, which will ultimately facilitate the rational design of drugs with IFN-gamma agonist/antagonist properties. The approach is three-fold, involving IFN-gamma and receptor synthetic peptides, recombinant IFN-gamma mutants, and modeling based primarily on determination of the solution structure of IFN-gamma peptides and recombinant molecules using multidimensional nuclear magnetic resonance. The objectives will be achieved through the following specific aims: 1. Further characterization of the structural basis for the binding of N-terminal and C-terminal regions of IFN-gamma to receptor using residue substitutions, peptide truncations, and N-terminal and C-terminal peptides complexed together. 2. Identify sites on the IFN-gamma receptor that interact with receptor-binding domains of IFN-gamma. 3. Develop agonists/antagonists of IFN-gamma with high specific activity through peptide engineering and recombinant DNA technology. 4. Determine the solution structure of IFN-gamma-receptor binding domains as well as binding domains on receptor using state-of-the-art multidimensional nuclear magnetic resonance (NMR). 5. Determine the precise structural basis for binding of IFN-gamma to receptor by determining the solution structure of IFN-gamma receptor-binding domain alone and complexed to soluble receptor. 6. Use agonists/antagonists of IFN-gamma activity both in vivo and in tissue culture to assess their effects on IFN-gamma mediated functions. Finally, mutation and peptide and protein engineering that would increase IFN-gamma binding and agonist activity could lead to the creation of """"""""superferons"""""""".
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