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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.Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), 1 also 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). This syndrome is characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiasis of mucous membranes, and ectodermal disorders. APECED is caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21q22.3 (2-4). The AIRE gene has recently been cloned by two independent groups of investigators (5, 6). The AIRE gene consists of 14 exons coding for a 2445-base pair mRNA transcript, and the translated product is expected to have 545 amino acids with a predicted molecular mass of 57.5 kDa. The predicted AIRE protein has several domains indicative of a transcriptional regulator protein (6). AIRE harbors two zinc fingers of plant homeodomain (PHD) type. A putative DNA binding domain named SAND as well as four nuclear receptor binding LXXLL motifs, an inverted LXXLL domain, and a variant of the latter (FXXLL) hint that this protein functions as a transcription coactivator (5-7). Furthermore, a highly conserved N-terminal 100-amino acid domain in AIRE has a significant homology to the homogenously staining (HSR) domain of Sp100 and Sp140 proteins (7). This domain has been shown to function as a dimerization domain in several Sp-100 related proteins (8). At the subcellular level, AIRE can be found in the cell nucleus in a speckled pattern in domains resembling promyelocytic leukemia nuclear bodies, also known as ND10, nuclear dots, or potential oncogenic domains, associated with the AIRE homologous nuclear proteins Sp100, Sp140, and Lysp100 (9).Interestingly, 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. A glutathione S-transferase pull-down assay showed that AIRE formed homodimers in vitro, probably through the N-terminal domain (...

Section: Discussionmentioning

confidence: 99%

The Autoimmune Regulator (AIRE) Is a DNA-binding Protein

Kumar

Laloraya

Wang

et al. 2001

148

123

“…The B834 cells were grown in M9 medium supplemented with all natural amino acids except methionine, plus 50 mg/liter selenomethionine. Proteins were first purified by Ni 2ϩ affinity chromatography, as described previously (33). The His 6 tag was removed by incubation at 23°C with tobacco etch virus protease.…”

Section: Methodsmentioning

confidence: 99%

Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer

Bottomley

Muraglia

Bazzo

et al. 2007

Self Cite

362

476

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C 12 -acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an ␣-␤-␣ fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-Å resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.Evolution has endowed bacteria with a wide variety of diffusible chemical signals that are used for communication both within and between species. The most common form of intercellular communication in Gram-negative bacteria is "quorum sensing," so named because the bacteria initiate coordinated activities only when a "quorum" population density is reached (1). This synchronization of individual behavior into cooperative group activity can have many different benefits, e.g. to produce virulence factors only when the bacterial population is sufficiently dense to survive host immune responses (2-4).The first quorum sensing system described was that of Vibrio fischeri, a symbiont of the Hawaiian bobtail squid (5, 6). The squid has a "light organ" wherein it provides nutrition for V. fischeri. Upon reaching high population density the bacteria produce bioluminescence enabling the squid to cancel its own shadow and thereby avoid predation (7). The quorum sensing mechanism triggering bioluminescence depends on the bacterial synthase LuxI, which constitutively produces a signal molecule, an acylhomoserine lactone (acyl-HSL or AHL), 2 the autoinducer. The AHL diffuses through the bacterial envelope, and, upon reaching a threshold concentration, the AHL binds and activates its receptor LuxR, a transcriptional activator of the luciferase operon (1). Many different Gram-negative bacteria have now been shown to use quorum sensing systems composed of LuxI/LuxR homologues and AHL autoinduc...

“…A Second Self-interacting Region Occurs in the DEAF-1 SAND Domain-Gel chromatography had previously indicated that the DEAF-1 SAND domain may dimerize (24). To confirm this interaction and to further define the interacting motifs, GST fusion proteins with full-length DEAF-1 (GST-(1-565)) and a region containing the SAND domain (GST-(173-375)) were incubated with radiolabeled full-length DEAF-1 (Fig.…”

Section: Fig 5 the Deaf-1 Sand Zinc Binding Motif And Nls Region mentioning

confidence: 99%

“…A fusion protein between GST and amino acids 167-370 of DEAF-1 (peptide J, GST-167-370) was previously shown to bind a DNA ligand that contained two TTCG elements spaced apart by 9 base pairs (N52-69 probe), and mutation of the CG residue(s) in a single TTCG element (Mut 1) or in both TTCG elements (Mut 2) eliminated DNA binding (24). In addition, the shorter SAND domain (187-324) had previously bound the N52-69 probe as both a low and a high mobility species, and was also able to bind the Mut 1 probe but not the Mut 2 probe (24).…”

Section: Fig 5 the Deaf-1 Sand Zinc Binding Motif And Nls Region mentioning

confidence: 99%

See 1 more Smart Citation

Identification of a Nuclear Export Signal and Protein Interaction Domains in Deformed Epidermal Autoregulatory Factor-1 (DEAF-1)

Jensik

Huggenvik

Collard

2004

Self Cite

Deformed epidermal autoregulatory factor-1 (DEAF-1) is a DNA-binding protein required for embryonic development and linked to clinical depression and suicidal behavior in humans. Although primarily nuclear, cytoplasmic localization of DEAF-1 has been observed, and this suggests the presence of a nuclear export signal (NES). Using a series of fluorescent fusion proteins, an NES with a novel spacing of leucines (LXLX 6 LLX 5 LX 2 L) was identified near the COOH-terminal MYND domain at amino acids 454 -476. The NES was leptomycin B-sensitive and mutation of the leucine residues decreased or eliminated nuclear export activity. In vitro pull downs and an in vivo fluorescent protein interaction assay identified a DEAF-1/DEAF-1 protein interaction domain within the NES region. DNA binding had been previously mapped to a positively charged surface patch in the novel DNA binding fold called the "SAND" domain. A second protein-protein interaction domain was identified at amino acids 243-306 that contains the DNA-binding SAND domain and also an adjacent zinc binding motif and a monopartite nuclear localization signal (NLS). Deletion of these adjacent sequences or mutation of the conserved cysteines or histidine in the zinc binding motif not only inhibits protein interaction but also eliminates DNA binding, demonstrating that DEAF-1 protein-protein interaction is required for DNA recognition. The identification of an NES and NLS provides a basis for the control of DEAF-1 subcellular localization and function, whereas the requirement of protein-protein interaction by the SAND domain appears to be unique among this class of transcription factors.Deformed epidermal autoregulatory factor-1 (DEAF-1) 1 was first identified in Drosophila as a DNA-binding protein and potential regulator of the homeotic gene Deformed (1). The human, rat, and monkey homologs of Drosophila DEAF-1 (dDEAF-1) were previously called "nuclear DEAF-1 related" (NUDR) because of the limited protein similarity (46%) to dDEAF-1 (2). Genomic sequencing projects have confirmed a single gene in metazoan genomes with the overall structure of dDEAF-1, therefore we have adopted the DEAF-1 designation for all orthologs of the gene. DEAF-1 proteins are structurally defined as having both a DNA binding SAND domain (Sp100, AIRE-1, NucP41/75, DEAF-1) (3-5) and a carboxyl-terminal zinc finger motif called the MYND domain (myeloid translocation protein 8, Nervy, DEAF-1) (6, 7). Functional domains outside of these regions of DEAF-1 have yet to be extensively characterized.DEAF-1 appears to be an important factor in development and cancer. Loss of function mutations in DEAF-1 produce early embryonic arrest or segmentation defects in Drosophila, whereas overexpression of DEAF-1 can disrupt eye and wing development (8). DEAF-1 mRNA is widely expressed during mouse embryogenesis with elevated levels in several tissues, including the central nervous system and the dorsal root ganglia (9). DEAF-1 interacts with LMO4 and NLI (10), transcriptional regulators that mediate embryonic p...