Establishment of Cloning Genes for Protein-Protein Interaction Using Yeast System

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(1) Redox regulation of NF-kB has been investigated using the following systems.1) Pathogenetic role of NF-kB in rheumatoid arthritis (RA) has been identified using the synovial fibroblast culture fromthe patients. Co-localization of thioredoxin (TRX) and NF-kB was successfully demonstrated thus confirmed the redox regulation of NF-kB by TRX.2) Involvement of NF-kB in the cancer cell-endothelial cell adhesion was further investigated. Anti-oxidants were demonstrated to be effective in blocking NF-kB activation and cancer cell adhesion.3) We have established a recombinant cell line survival of which is under the control of NF-kB.This cell line has been shown to be applicable for the screening of novel anti-NF-kB compounds.(2) Activity of S.cerevisiae yAP-1, an AP-1-like transcription factor, is elevated by oxidation stress, and yAP-1 activates transcription from an AP-1 dependent reporter gene and TRX2 that is required for resistance to peroxidation. Furthemore yAP-1 migrates into nucleus upon oxidation stress and this change in localization is important for transcriptional regulation. Cystein residues in the essential C-terminal region are involved in this migration.(3) A bZip protein Fral has been isolated by the two-hybrid screening for protein specifically interacting with bHLHZip protein USF.Both of bZip and N terminal regions are required for this interaction. Expression of exogenous USF caused down-regulation of AP1 activity, which suggests that USF is a negative regulator of AP1. We have also isolated cDNA for a novel protein TAM2 that interacts with the activation region of HIV-1 Tat by the two-hybrid system. Since TAM2 pulled down both cdk7 and cyclinH,we suggest that Tat recruits TFIIH via TAM2 and activates initiation and elongation of transcription.