Functional interaction of AIRE with PIAS1 in transcriptional regulation.

AIRE (autoimmune regulator) promotes the establishment of self-tolerance by regulating gene expression in the thymus. Mutations in AIRE lead to an autoimmune disease, APECED. Here we have identified PIAS proteins as novel AIRE interaction partners. Although PIAS proteins function as E3 SUMO ligases, AIRE is not sumoylated. We expressed AIRE, wt PIAS1, and PIAS1 mutants with deleted SP-RING domain or SUMO interaction motif (SIM) in different cell lines and demonstrate that AIRE and PIAS1 localize to adjacent nuclear bodies (NBs). The expression of AIRE enhances the formation of PIAS1 NBs. The ability of PIAS1 to localize into NBs and interconnect with AIRE is neither dependent on the SP-RING domain nor the SIM. Further, we show that PIAS1 is able to attract AIRE into SUMO1-containing complexes and that the process is dependent on the SIM of PIAS1. PIAS1 and AIRE concurrently activate the human insulin promoter, a known target gene of AIRE, and the SP-RING is required for this activation. Moreover, AIRE represses and PIAS1 activates the CSTB promoter, used as a model for a housekeeping promoter, and both the SP-RING and SIM are needed for its activation by PIAS1. Collectively, our data suggest that AIRE and PIAS1 interact functionally to regulate the activities of the target genes of AIRE.

The monopartite nuclear localization signal of autoimmune regulator mediates its nuclear import and interaction with multiple importin alpha molecules.

Autoimmune regulator (AIRE) is a transcriptional regulator involved in establishing immunological self-tolerance. Mutations in the AIRE gene lead to the development of the autosomal, recessively inherited, organ-specific autoimmune disease, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). The AIRE protein is mainly localized in the cell nucleus where it is associated with nuclear bodies. The N-terminal part of AIRE has been previously shown to mediate nuclear localization of the protein. However, the functional nuclear localization signal (NLS) and nuclear import mechanisms of AIRE have not been identified. We show that, although the amino-acid sequence of AIRE contains a potential bipartite NLS consisting of amino acids 110-114 and 131-133, only the latter part constitutes a functional NLS. Furthermore, we show by in vitro binding assays that AIRE interacts with multiple members of the nuclear transport receptor importin alpha family, mainly alpha1, alpha3, and alpha5, and that these interactions depend on the intactness of the Arg-Lys-rich NLS of AIRE. In addition, we found that AIRE binds to the 'minor' NLS-binding site of importin alpha3 and alpha5 proteins consisting of the C-terminal armadillo repeats 7-9. Our findings strongly suggest that the nuclear import of AIRE is mediated by the classical importin alpha/beta pathway through binding to several importin alpha family members.

Functional analysis of SAND mutations in AIRE supports dominant inheritance of the G228W mutation.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare disorder caused by mutations in the autoimmune regulator gene (AIRE) and characterized by a variable combination of organ-specific autoimmune diseases. Studies on AIRE-deficient mice suggest that AIRE is an important factor in the establishment and maintenance of self-tolerance. The AIRE protein contains several structural domains often found in transcriptional regulators and functions as a transcriptional transactivator in vitro. To date, more than 50 patient mutations have been identified in the coding region of the AIRE gene. So far, APECED has been reported to be inherited in an autosomal recessive manner. However, in contrast to all other AIRE mutations, a novel mutation c.682T>G (p.G228W) in the DNA-binding and/or multimerization domain SAND was recently described to be inherited in a dominant fashion. We analyzed the effects of mutant AIRE proteins containing the patient mutations c.682T>G (p.G228W) and c.755C>T (p.P252L) located in the SAND domain on the properties of the wild-type AIRE in a heterozygous situation in vitro. In addition to the patient mutations, we analyzed the effects of a double mutation [c.727A>G;c.728A>C;c.739C>G;c740G>C] (p.K243A;R247A) of positively charged amino acids in the SAND domain. Of the mutants studied, only c.682T>G (p.G228W) mutant changed the subcellular localization and in addition severely disrupted the transactivating capacity of the wild-type AIRE. Our results indicate that the c.682T>G (p.G228W) mutant AIRE protein acts with a dominant negative effect by binding to the wild-type AIRE, thus preventing the protein from forming the complexes needed for transactivation.

APECED-causing mutations in AIRE reveal the functional domains of the protein.

A defective form of the AIRE protein causes autoimmune destruction of target organs by disturbing the immunological tolerance of patients with a rare monogenic disease, autoimmune polyendocrinopathy (APE)-candidiasis (C)-ectodermal dystrophy (ED), APECED. Recently, experiments on knockout mice revealed that AIRE controls autoimmunity by regulating the transcription of peripheral tissue-restricted antigens in thymic medullary epithelial cells. Thus, AIRE provides a unique model for molecular studies of organ-specific autoimmunity. In order to analyze the molecular and cellular consequences of 16 disease-causing mutations in vitro, we studied the subcellular localization, transactivation capacity, homomultimerization, and complex formation of several mutant AIRE polypeptides. Most of the mutations altered the nucleus-cytoplasm distribution of AIRE and disturbed its association with nuclear dots and cytoplasmic filaments. While the PHD zinc fingers were necessary for the transactivation capacity of AIRE, other regions of AIRE also modulated this function. Consequently, most of the mutations decreased transactivation. The HSR domain was responsible for the homomultimerization activity of AIRE; all the missense mutations of the HSR and the SAND domains decreased this activity, but those in other domains did not. The AIRE protein was present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturbed the formation of these complexes. In conclusion, we propose an in vitro model in which AIRE transactivates transcription through heteromeric molecular interactions that are regulated by homomultimerization and conditional localization of AIRE in the nucleus or in the cytoplasm.

Gelsolin-related familial amyloidosis, Finnish type, in a Portuguese family: clinical and neurophysiological studies.

We report a Portuguese family with familial amyloid polyneuropathy related to gelsolin. There were no known Finnish ancestors, but the same mutation as described in Finnish patients (G654A) was carried. Clinical and neurophysiological investigations were performed in four patients. Corneal lattice dystrophy affected all four patients; an axonal lesion of the facial nerve occurred in three patients; visual tract involvement was documented in one case; and corticospinal and posterior column dysfunction was present in one patient. Polarizing microscopy of skin and muscle samples demonstrated amyloid deposits in two patients; anti-gelsolin immunohistochemistry was positive for amyloidogenic gelsolin. The Finnish mutation of gelsolin protein (G654A) was detected in five family members. The utility of neurophysiological testing in the evaluation and follow-up of this type of amyloidosis is discussed.

Role of proprotein convertases in the pathogenic processing of the amyloidosis-associated form of secretory gelsolin.

Familial amyloidosis of the Finnish type (FAF) is caused by two proteolytic cleavages of mutant gelsolin leading to the accumulation of FAF amyloid in the patients' tissues. Here, we demonstrate that, in mouse pituitary corticotropic AtT20 cells, the enzyme responsible for the first cleavage of mutant secretory FAF gelsolin to FAF amyloid precursor is present in reasonable amounts. Furthermore, in At T20 cells stably expressing alpha1-PDX a potent inhibitor of most proprotein convertases, this cleavage was inhibited The present data provide strong evidence that proprotein convertases, possibly furin or PC5, are involved in the initialpathological cleavage of mutant secretory FAF gelsolin leading ultimately to the amyloid disease.