NORMAL GENOMIC

• Lademann U, Kallunki T, Jaattela M
• A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins.
• Cell Death Differ. 2001; 8: 265-272
• Display abstract

• A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.

• Lee EG et al.
• Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice.
• Science. 2000; 289: 2350-4
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• A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappaB (NF-kappaB) activity and tumor necrosis factor (TNF)-mediated programmed cell death (PCD). TNF dramatically increases A20 messenger RNA expression in all tissues. Mice deficient for A20 develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely. A20-deficient cells fail to terminate TNF-induced NF-kappaB responses. These cells are also more susceptible than control cells to undergo TNF-mediated PCD. Thus, A20 is critical for limiting inflammation by terminating TNF-induced NF-kappaB responses in vivo.

• White AM, Yoshimura T, Smith AW, Westwick J, Watson ML
• Airway inflammation induced by recombinant guinea pig tumor necrosis factor-alpha.
• Am J Physiol. 1997; 273: 52430-52430
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• We have cloned and expressed recombinant guinea pig tumor necrosis factor-alpha (gpTNF-alpha) and examined its inflammatory activities after tracheal instillation in guinea pigs. A 1,071-bp cDNA, including the region encoding the full-length 234-amino acid gpTNF-alpha protein, was cloned from concanavalin A-stimulated guinea pig splenocytes. The 154-amino acid protein corresponding to secreted gpTNF-alpha was expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and cleaved to yield a 17-kDa protein. gpTNF-alpha had a cytotoxic effect on WEHI 164 cells and was detected by goat anti-murine tumor necrosis factor-alpha (TNF-alpha) antibody in Western blots. Intratracheal instillation of gpTNF-alpha (50-150 ng) caused pronounced and dose-dependent airway eosinophilia. Incubation of gpTNF-alpha with rabbit anti-murine TNF-alpha sera or heating the gpTNF-alpha before instillation reduced bronchoalveolar lavage (BAL) eosinophils to near control levels. Maximum BAL eosinophilia was observed at 24 h, but eosinophil numbers remained significantly above vehicle-treated animals for 72 h. Hence, gpTNF-alpha elicits a pronounced and protracted eosinophil accumulation in the guinea pig lung.

• De Valck D, Heyninck K, Van Criekinge W, Vandenabeele P, Fiers W, Beyaert R
• A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins.
• Biochem Biophys Res Commun. 1997; 238: 590-4
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• The A20 protein, which belongs to a class of Cys2/Cys2 zinc finger proteins, has been characterized as an inhibitor of NF-kappaB activation. In order to clarify its molecular mechanism of action, the yeast two-hybrid system was used to screen for interacting proteins. We report that different isoforms of 14-3-3 proteins, viz. eta and zeta, are able to bind A20, involving the 14-3-3-binding motif RSKSDP located between zinc fingers 3 and 4. However, A20 mutants that no longer associated with 14-3-3 proteins could still fully inhibit NF-kappaB activation induced by tumor necrosis factor, interleukin-1beta or phorbol 12-myristate 13-acetate, thus excluding a crucial role for 14-3-3 interaction in this A20 function.

• Zucker K, Lu P, Fuller L, Asthana D, Esquenazi V, Miller J
• Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E. coli.
• Lymphokine Cytokine Res. 1994; 13: 191-6
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• We have utilized the reverse transcription polymerase chain reaction (RT-PCR) to clone the protein coding region of canine tumor necrosis factor (TNF-alpha) cDNA. The gene displays 90% sequence homology to the corresponding human TNF-alpha cDNA. The predicted initial translation product is 233 amino acids and shows 88% homology to the human counterpart, and 92% homology with the human putative mature TNF-alpha protein. The canine TNF-alpha clone was used to engineer bacteria to express large amounts of the mature form of recombinant protein. A monoclonal antibody against human TNF-alpha cross-reacted with canine rTNF-alpha using Western blot and ELISA analysis. The purified canine rTNF-alpha had a cytotoxic effect on WEHI 164 clone 13 cells as well as increasing the cell surface expression of major histocompatibility class II antigens on canine kidney cortical cell line (MDCK) in vitro. The availability of canine rTNF-alpha will allow further studies on its role in immunoregulatory mechanisms in the canine transplantation model, both by itself and in conjunction with the already available canine specific recombinant interferon-gamma.