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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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Effect of PHD mutations on the transcriptional transactivating properties of AIRE. (A) Various forms of full-length AIRE including disease-causing mutations were fused with Gal4-binding domain and transfected into mTEC cells together with a reporter plasmid. Firefly luciferase activities were normalized with respect to the Renilla luciferase activities used for assessment of the transfection efficiency. No significant reduction of Gal4 activation was observed from the disease-causing mutations of PHD1 (C311Y and P326Q), whereas the PHD2 mutant (C434A) showed loss of transcriptional activation. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of five repeat experiments. (B) Isolated PHDs were tested for the transcriptional activation with the same system described in A. The fragments used are as follows (see Figs. 1 A and 2 A): PHD1, amino acids 292–341; PHD1 plus PRR, 292–432; PHD2, 433–545; PRR plus PHD2, 342–545; PHD1 plus PRR plus PHD2, 292–545; and PRR, 342–432. PHD2 suffices for the transcriptional activation in this assay, whereas PHD1 has no such activities. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of three repeat experiments.
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fig3: Effect of PHD mutations on the transcriptional transactivating properties of AIRE. (A) Various forms of full-length AIRE including disease-causing mutations were fused with Gal4-binding domain and transfected into mTEC cells together with a reporter plasmid. Firefly luciferase activities were normalized with respect to the Renilla luciferase activities used for assessment of the transfection efficiency. No significant reduction of Gal4 activation was observed from the disease-causing mutations of PHD1 (C311Y and P326Q), whereas the PHD2 mutant (C434A) showed loss of transcriptional activation. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of five repeat experiments. (B) Isolated PHDs were tested for the transcriptional activation with the same system described in A. The fragments used are as follows (see Figs. 1 A and 2 A): PHD1, amino acids 292–341; PHD1 plus PRR, 292–432; PHD2, 433–545; PRR plus PHD2, 342–545; PHD1 plus PRR plus PHD2, 292–545; and PRR, 342–432. PHD2 suffices for the transcriptional activation in this assay, whereas PHD1 has no such activities. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of three repeat experiments.

Mentions: It has been previously demonstrated that AIRE has transcription-activating properties when fused to a heterologous DNA-binding domain (20, 24), and many PHD-disrupting mutations result in decreased transcriptional activity. We investigated whether the loss of E3 ligase activity resulting from the disease-causing mutations C311Y and P326Q is associated with alterations in the transcription-activating properties assessed with a series of Gal4-binding assays. In light of the fact that AIRE expression is most prominent in the thymic medulla (7, 8), we used mouse mTEC (1C6; reference 23), which does not express endogenous AIRE as assessed by standard RT-PCR (unpublished data), for the transfection. As expected, the full-length wild-type AIRE induced activation of the reporter genes in mTEC (Fig. 3 A). The disease-causing mutant forms of PHD1 (C311Y and P326Q), which lack E3 ligase activity, exhibited normal transcriptional activation. In contrast, a PHD2 mutant (C434A) with normal E3 ligase activity showed dramatically reduced transcriptional activation.


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)

Effect of PHD mutations on the transcriptional transactivating properties of AIRE. (A) Various forms of full-length AIRE including disease-causing mutations were fused with Gal4-binding domain and transfected into mTEC cells together with a reporter plasmid. Firefly luciferase activities were normalized with respect to the Renilla luciferase activities used for assessment of the transfection efficiency. No significant reduction of Gal4 activation was observed from the disease-causing mutations of PHD1 (C311Y and P326Q), whereas the PHD2 mutant (C434A) showed loss of transcriptional activation. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of five repeat experiments. (B) Isolated PHDs were tested for the transcriptional activation with the same system described in A. The fragments used are as follows (see Figs. 1 A and 2 A): PHD1, amino acids 292–341; PHD1 plus PRR, 292–432; PHD2, 433–545; PRR plus PHD2, 342–545; PHD1 plus PRR plus PHD2, 292–545; and PRR, 342–432. PHD2 suffices for the transcriptional activation in this assay, whereas PHD1 has no such activities. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of three repeat experiments.
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Related In: Results  -  Collection

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fig3: Effect of PHD mutations on the transcriptional transactivating properties of AIRE. (A) Various forms of full-length AIRE including disease-causing mutations were fused with Gal4-binding domain and transfected into mTEC cells together with a reporter plasmid. Firefly luciferase activities were normalized with respect to the Renilla luciferase activities used for assessment of the transfection efficiency. No significant reduction of Gal4 activation was observed from the disease-causing mutations of PHD1 (C311Y and P326Q), whereas the PHD2 mutant (C434A) showed loss of transcriptional activation. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of five repeat experiments. (B) Isolated PHDs were tested for the transcriptional activation with the same system described in A. The fragments used are as follows (see Figs. 1 A and 2 A): PHD1, amino acids 292–341; PHD1 plus PRR, 292–432; PHD2, 433–545; PRR plus PHD2, 342–545; PHD1 plus PRR plus PHD2, 292–545; and PRR, 342–432. PHD2 suffices for the transcriptional activation in this assay, whereas PHD1 has no such activities. The results are expressed as the mean ± SEM for triplicate wells during one representative experiment from a total of three repeat experiments.
Mentions: It has been previously demonstrated that AIRE has transcription-activating properties when fused to a heterologous DNA-binding domain (20, 24), and many PHD-disrupting mutations result in decreased transcriptional activity. We investigated whether the loss of E3 ligase activity resulting from the disease-causing mutations C311Y and P326Q is associated with alterations in the transcription-activating properties assessed with a series of Gal4-binding assays. In light of the fact that AIRE expression is most prominent in the thymic medulla (7, 8), we used mouse mTEC (1C6; reference 23), which does not express endogenous AIRE as assessed by standard RT-PCR (unpublished data), for the transfection. As expected, the full-length wild-type AIRE induced activation of the reporter genes in mTEC (Fig. 3 A). The disease-causing mutant forms of PHD1 (C311Y and P326Q), which lack E3 ligase activity, exhibited normal transcriptional activation. In contrast, a PHD2 mutant (C434A) with normal E3 ligase activity showed dramatically reduced transcriptional activation.

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