Abstract
The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.
Highlights
Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED),1 known as autoimmune polyglandular syndrome type 1 (APS1), is a rare autosomal recessive disorder common in isolated populations such as Finns, Sardinians, and Iranian Jews [1]
The processing of the recombinant human AIRE (rhAIRE) under oxidation and refolding conditions brought about a major shift in the pattern of migration of this molecule (Fig. 1A, lane 5)
A minor fraction of autoimmune regulator (AIRE) still appears at the 56-kDa position, the major bands appear at estimated molecular sizes of 110 and 220 kDa when the refolded rhAIRE was analyzed on native PAGE
Summary
Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), known as autoimmune polyglandular syndrome type 1 (APS1), is a rare autosomal recessive disorder common in isolated populations such as Finns, Sardinians, and Iranian Jews [1]. It has recently been shown that AIRE can activate transcription from a reporter gene when fused to a heterologous DNA binding domain. This activation required the full-length protein or the presence of more than one activation domain. It has been shown that AIRE interacts in vitro with CREBbinding protein (CBP) through the CH1 and CH3 conserved domains, which has led the investigators to suggest that the transcriptional activities of AIRE might be mediated through its physical interaction with the common coactivator CBP [10]. The signal processing is mediated by a family of cyclic AMP-responsive nuclear factors, including CREB, cAMP response element modulator (CREM), and activating transcription factor 1 (ATF-1) These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-responsive elements (CREs). We further demonstrate that AIRE exists in oligomerized forms in vivo and that phosphorylation of AIRE by cAMP-dependent PKA and/or PKC could trigger its dimerization
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