A20: A novel zinc finger protein induced by tumor necrosis factor alpha.

Abstract

Tumor Necrosis Factor $\alpha$ (TNF) is a proinflammatory cytokine that is capable of eliciting a number of cellular responses including, cell death. An approach to identify the genetic changes induced by TNF uncovered a number of unique cDNA molecules. These cDNAs represented the immediate early genetic response of cells to TNF and as such, are likely to encode the mediators of TNF's biological effects. One of the cDNAs, termed A20, has a sequence that is not identical to any other known gene. The nucleotide sequence of the full-length cDNA revealed that A20 encodes a novel protein of 790 amino acids. The predicted protein sequence contained seven repeats of an element defined by the amino acid consensus pattern: C-X$\sb4$-C-X$\sb{11}$-C-X$\sb2$-C. This element is similar to the finger motifs found in Cys(x) type zinc finger proteins. Many Cys(x) finger proteins are known to bind zinc and DNA and to function as transcriptional factors. Neutron activation analysis was performed to determine if the finger-containing domain of A20 was also capable of binding zinc. The results of these experiments suggested that one zinc ion bound to each of the seven finger motifs in A20. Column chromatography revealed that in vitro translated A20 protein could bind double stranded genomic DNA. A20 specific antibodies could immunoprecipitate A20 protein from a variety of cells following treatment with TNF. Studies to determine the subcellular localization of A20 demonstrated that the protein was found predominantly in the nuclei of expressing cells. In a panel of isogenic breast carcinoma cell lines A20 expression was observed to correlate with cellular resistance to TNF cytotoxicity. Constitutive expression of A20 in NIH 3T3 and WEHI 164 cells following single gene transfection rendered these cells more resistant to TNF killing than control cells. Conversely, antisense RNA mediated inhibition of A20 expression caused NIH 3T3 cells to become more sensitive to TNF's cytotoxic effects. Thus, this work has identified a novel TNF-responsive zinc finger protein that mediates cellular resistance to TNF and has properties consistent with a function involving transcriptional regulation.