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AIRE functions as an E3 ubiquitin ligase.

Uchida D, Hatakeyama S, Matsushima A, Han H, Ishido S, Hotta H, Kudoh J, Shimizu N, Doucas V, Nakayama KI, Kuroda N, Matsumoto M - J. Exp. Med. (2004)

Bottom Line: Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity.The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity.These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.

ABSTRACT
Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

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Mutations in PHD1 affect E3 ligase activity. (A) Schematic representation of the PHD mutants used in the study. (B) Substitutions of the first cysteines into alanines in PHD1, or PHD1 and PHD2, but not in PHD2 alone, abolished E3 ligase activity (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against His6-tag (bottom). (C) Disease-causing mutations of PHD1 (C311Y and P326Q) resulted in loss of E3 ligase activity as assessed with in vitro ubiquitylation assays.
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fig2: Mutations in PHD1 affect E3 ligase activity. (A) Schematic representation of the PHD mutants used in the study. (B) Substitutions of the first cysteines into alanines in PHD1, or PHD1 and PHD2, but not in PHD2 alone, abolished E3 ligase activity (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against His6-tag (bottom). (C) Disease-causing mutations of PHD1 (C311Y and P326Q) resulted in loss of E3 ligase activity as assessed with in vitro ubiquitylation assays.

Mentions: The indispensability of PHD1 for E3 ligase activity was demonstrated with PHD mutants in which the first cysteines were mutated to alanines in either PHD1 (C299A), PHD2 (C434A), or both PHDs (C299A/C434A) of the full-sized recombinant AIRE (Fig. 2 A). Corresponding amino acid substitution in PHD of MEKK1 has demonstrated the significance of this cysteine for its E3 ligase activity (19). Although the wild-type and the PHD2 mutant (C434A) mediated polyubiquitylation, the PHD1 mutant (C299A) and the PHD1/PHD2 double mutant (C299A/C434A) showed no obvious polyubiquitylation (Fig. 2 B). Together with the data obtained from GST-PHD fusion proteins, these results indicate that AIRE mediates E3 ligase activity primarily through PHD1. Although we cannot completely exclude the possibility that PHD2 is involved in the full enzymatic activity, the absence of the tryptophan residue usually located between cysteines 6 and 7 of ligase-type PHDs might be consistent with the lack of E3 ligase activity from PHD2 (Fig. 1 A; reference 18).


AIRE functions as an E3 ubiquitin ligase.

Uchida D, Hatakeyama S, Matsushima A, Han H, Ishido S, Hotta H, Kudoh J, Shimizu N, Doucas V, Nakayama KI, Kuroda N, Matsumoto M - J. Exp. Med. (2004)

Mutations in PHD1 affect E3 ligase activity. (A) Schematic representation of the PHD mutants used in the study. (B) Substitutions of the first cysteines into alanines in PHD1, or PHD1 and PHD2, but not in PHD2 alone, abolished E3 ligase activity (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against His6-tag (bottom). (C) Disease-causing mutations of PHD1 (C311Y and P326Q) resulted in loss of E3 ligase activity as assessed with in vitro ubiquitylation assays.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211764&req=5

fig2: Mutations in PHD1 affect E3 ligase activity. (A) Schematic representation of the PHD mutants used in the study. (B) Substitutions of the first cysteines into alanines in PHD1, or PHD1 and PHD2, but not in PHD2 alone, abolished E3 ligase activity (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against His6-tag (bottom). (C) Disease-causing mutations of PHD1 (C311Y and P326Q) resulted in loss of E3 ligase activity as assessed with in vitro ubiquitylation assays.
Mentions: The indispensability of PHD1 for E3 ligase activity was demonstrated with PHD mutants in which the first cysteines were mutated to alanines in either PHD1 (C299A), PHD2 (C434A), or both PHDs (C299A/C434A) of the full-sized recombinant AIRE (Fig. 2 A). Corresponding amino acid substitution in PHD of MEKK1 has demonstrated the significance of this cysteine for its E3 ligase activity (19). Although the wild-type and the PHD2 mutant (C434A) mediated polyubiquitylation, the PHD1 mutant (C299A) and the PHD1/PHD2 double mutant (C299A/C434A) showed no obvious polyubiquitylation (Fig. 2 B). Together with the data obtained from GST-PHD fusion proteins, these results indicate that AIRE mediates E3 ligase activity primarily through PHD1. Although we cannot completely exclude the possibility that PHD2 is involved in the full enzymatic activity, the absence of the tryptophan residue usually located between cysteines 6 and 7 of ligase-type PHDs might be consistent with the lack of E3 ligase activity from PHD2 (Fig. 1 A; reference 18).

Bottom Line: Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity.The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity.These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.

ABSTRACT
Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

Show MeSH
Related in: MedlinePlus