The purpose of this research proposal is to gain further understanding of the role of ubiquitinating enzymes in the activation of NF-kB. The transcription factor NF-kB is a key regulator of a number of different genes particularly those of the immune system. NF-kB is activated through the degradation of its inhibitor IkB by the ubiquitinproteasome pathway. This research proposal will evaluate the potential of the ubiquitination system for antiinflammatory intervention and can be summarized as follows: 1. Purification of the ubiquitin-protein ligase which is involved in the ubiquitination of IkBa (Ubiquitin-IkBa-ligase).
2. Characterization of the specificity of the Ubiquitin-IkB-ligase for IkBa versus other known substrates of the ubiquitination pathway. 3. Investigation of amino acid sequence requirements for ubiquitination in IkBa by characterizing the efficiency of ubiquitination of synthetic peptides.
4. Cloning of the cDNA of the ubiquitin-IkBa-ligase.
5. Comparison of activity of recombinant ubiquitin-IkBa-ligase with the purified enzyme.