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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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AIRE mediates E3 ligase activity through PHD1. (A) The PHD1 and PHD2 domains of AIRE showed homology with the PHDs from other proteins showing E3 ligase activity. *, the position of disease-causing mutations (C311Y and P326Q) used in the study. (B) The GST-PHD1, but not GST-PHD2, fusion protein exhibited polyubiquitylated proteins when rabbit reticulocyte lysate was used for in vitro ubiquitylation assays (top). SDS-PAGE of GST-fusion proteins used in the assay (10% input) is shown on the bottom. (C) Recombinant E1 and E2 (Ubc4) were used for in vitro ubiquitylation assays (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against GST (bottom). E3 ligase activity mediated through PHD1 is incubation dependent. *, nonspecific bands. (D) The full-sized recombinant AIRE protein was successfully and homogeneously expressed in Sf21 cells using a baculovirus expression system. The purified recombinant AIRE protein was subjected to immunoblot analysis with an antibody against His6-tag. (E) Ubc4 E2 preference by the full-sized recombinant AIRE in in vitro ubiquitylation assays (top). CHIP, a U-box–type E3 ligase, is included to verify the specificity and biological activities of E2s (middle; reference 22). Equal amount of E2s were used in the reaction (bottom). (F) Specificity of E3 ligase activity mediated by the full-sized recombinant AIRE in in vitro ubiquitylation assays.
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fig1: AIRE mediates E3 ligase activity through PHD1. (A) The PHD1 and PHD2 domains of AIRE showed homology with the PHDs from other proteins showing E3 ligase activity. *, the position of disease-causing mutations (C311Y and P326Q) used in the study. (B) The GST-PHD1, but not GST-PHD2, fusion protein exhibited polyubiquitylated proteins when rabbit reticulocyte lysate was used for in vitro ubiquitylation assays (top). SDS-PAGE of GST-fusion proteins used in the assay (10% input) is shown on the bottom. (C) Recombinant E1 and E2 (Ubc4) were used for in vitro ubiquitylation assays (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against GST (bottom). E3 ligase activity mediated through PHD1 is incubation dependent. *, nonspecific bands. (D) The full-sized recombinant AIRE protein was successfully and homogeneously expressed in Sf21 cells using a baculovirus expression system. The purified recombinant AIRE protein was subjected to immunoblot analysis with an antibody against His6-tag. (E) Ubc4 E2 preference by the full-sized recombinant AIRE in in vitro ubiquitylation assays (top). CHIP, a U-box–type E3 ligase, is included to verify the specificity and biological activities of E2s (middle; reference 22). Equal amount of E2s were used in the reaction (bottom). (F) Specificity of E3 ligase activity mediated by the full-sized recombinant AIRE in in vitro ubiquitylation assays.

Mentions: When aligned with other PHDs showing E3 ligase activity, both PHD1 and PHD2 of AIRE apparently demonstrated homology with the PHDs from those proteins (Fig. 1 A). To determine whether the PHD1 and/or PHD2 of AIRE have E3 ligase activity, we performed in vitro ubiquitylation assays. PHD1 and PHD2 were fused to the carboxyl terminus of GST and the purified fusion proteins were subjected to the assay using rabbit reticulocyte lysate as a source of E1 and E2. Polyubiquitylated proteins were observed from the GST-PHD1 fusion protein, whereas GST alone and the GST-PHD2 fusion protein did not have this form, suggesting that the PHD1 of AIRE mediates E3 ligase activity (Fig. 1 B). E3 ligase activity mediated through PHD1 was also demonstrated by the assay using recombinant E1 and E2 (Ubc4, see below), and this activity was incubation dependent (Fig. 1 C).


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)

AIRE mediates E3 ligase activity through PHD1. (A) The PHD1 and PHD2 domains of AIRE showed homology with the PHDs from other proteins showing E3 ligase activity. *, the position of disease-causing mutations (C311Y and P326Q) used in the study. (B) The GST-PHD1, but not GST-PHD2, fusion protein exhibited polyubiquitylated proteins when rabbit reticulocyte lysate was used for in vitro ubiquitylation assays (top). SDS-PAGE of GST-fusion proteins used in the assay (10% input) is shown on the bottom. (C) Recombinant E1 and E2 (Ubc4) were used for in vitro ubiquitylation assays (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against GST (bottom). E3 ligase activity mediated through PHD1 is incubation dependent. *, nonspecific bands. (D) The full-sized recombinant AIRE protein was successfully and homogeneously expressed in Sf21 cells using a baculovirus expression system. The purified recombinant AIRE protein was subjected to immunoblot analysis with an antibody against His6-tag. (E) Ubc4 E2 preference by the full-sized recombinant AIRE in in vitro ubiquitylation assays (top). CHIP, a U-box–type E3 ligase, is included to verify the specificity and biological activities of E2s (middle; reference 22). Equal amount of E2s were used in the reaction (bottom). (F) Specificity of E3 ligase activity mediated by the full-sized recombinant AIRE in in vitro ubiquitylation assays.
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Related In: Results  -  Collection

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fig1: AIRE mediates E3 ligase activity through PHD1. (A) The PHD1 and PHD2 domains of AIRE showed homology with the PHDs from other proteins showing E3 ligase activity. *, the position of disease-causing mutations (C311Y and P326Q) used in the study. (B) The GST-PHD1, but not GST-PHD2, fusion protein exhibited polyubiquitylated proteins when rabbit reticulocyte lysate was used for in vitro ubiquitylation assays (top). SDS-PAGE of GST-fusion proteins used in the assay (10% input) is shown on the bottom. (C) Recombinant E1 and E2 (Ubc4) were used for in vitro ubiquitylation assays (top). The ubiquitylation reactions were subjected to immunoblot analysis with antibodies against GST (bottom). E3 ligase activity mediated through PHD1 is incubation dependent. *, nonspecific bands. (D) The full-sized recombinant AIRE protein was successfully and homogeneously expressed in Sf21 cells using a baculovirus expression system. The purified recombinant AIRE protein was subjected to immunoblot analysis with an antibody against His6-tag. (E) Ubc4 E2 preference by the full-sized recombinant AIRE in in vitro ubiquitylation assays (top). CHIP, a U-box–type E3 ligase, is included to verify the specificity and biological activities of E2s (middle; reference 22). Equal amount of E2s were used in the reaction (bottom). (F) Specificity of E3 ligase activity mediated by the full-sized recombinant AIRE in in vitro ubiquitylation assays.
Mentions: When aligned with other PHDs showing E3 ligase activity, both PHD1 and PHD2 of AIRE apparently demonstrated homology with the PHDs from those proteins (Fig. 1 A). To determine whether the PHD1 and/or PHD2 of AIRE have E3 ligase activity, we performed in vitro ubiquitylation assays. PHD1 and PHD2 were fused to the carboxyl terminus of GST and the purified fusion proteins were subjected to the assay using rabbit reticulocyte lysate as a source of E1 and E2. Polyubiquitylated proteins were observed from the GST-PHD1 fusion protein, whereas GST alone and the GST-PHD2 fusion protein did not have this form, suggesting that the PHD1 of AIRE mediates E3 ligase activity (Fig. 1 B). E3 ligase activity mediated through PHD1 was also demonstrated by the assay using recombinant E1 and E2 (Ubc4, see below), and this activity was incubation dependent (Fig. 1 C).

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