Abstract
Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alanine / metabolism
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Amino Acid Sequence
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Amino Acid Substitution
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Binding Sites
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Catalysis
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Cell Line
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Conserved Sequence
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Crystallography, X-Ray
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DNA-Binding Proteins
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Escherichia coli / genetics
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Gene Deletion
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Glutathione Transferase / metabolism
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Humans
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Hydrogen Bonding
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Intracellular Signaling Peptides and Proteins / chemistry
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / isolation & purification
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Intracellular Signaling Peptides and Proteins / metabolism*
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Kidney / cytology
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Models, Molecular
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Molecular Sequence Data
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NF-kappa B / antagonists & inhibitors*
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / isolation & purification
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Nuclear Proteins / metabolism*
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Polyubiquitin / chemistry
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Polyubiquitin / metabolism
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Precipitin Tests
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Protein Binding
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Sequence Homology, Amino Acid
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Static Electricity
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Substrate Specificity
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TNF Receptor-Associated Factor 6 / metabolism
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Tumor Necrosis Factor alpha-Induced Protein 3
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Ubiquitin / metabolism
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Ubiquitin-Protein Ligases / chemistry
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Ubiquitination*
Substances
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DNA-Binding Proteins
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Intracellular Signaling Peptides and Proteins
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NF-kappa B
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Nuclear Proteins
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TNF Receptor-Associated Factor 6
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Ubiquitin
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Polyubiquitin
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Ubiquitin-Protein Ligases
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Glutathione Transferase
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TNFAIP3 protein, human
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Tumor Necrosis Factor alpha-Induced Protein 3
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Alanine