Nuclear factor B (NF-B) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-B is dependent on the phosphorylation of the inhibitory subunit IB␣ that is mediated by a multimeric, high molecular weight complex, called IB kinase (IKK) complex. This is composed of two catalytic subunits, IKK␣ and IKK␤, and a regulatory subunit, NEMO/IKK␥. The latter protein is essential for the activation of IKKs and NF-B, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-B) as a NEMO/IKK␥-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-B inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-B at the level of the IKK complex and that A20 inhibits activation of NF-B by de-ubiquitination of NEMO/IKK␥. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/ IKK␥ and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-B activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKK␥ and facilitates A20-mediated de-ubiquitination of NEMO/IKK␥, thus resulting in inhibition of NF-B.NF-B is a ubiquitously expressed family of transcription factors that controls the expression of numerous genes involved in immune and inflammatory responses (1). NF-B also plays an important role during cellular stress responses, due to its anti-apoptotic and proliferationpromoting functions (2). Aberrant activation of NF-B is a major hallmark of several inflammatory diseases such as arthritis (3, 4), and a variety of human cancers (5, 6). In resting cells, NF-B is sequestered in the cytoplasm in an inactive form by members of the inhibitory family of IB proteins (1). Various stimuli including pathogens, pathogen-related factors, and cytokines lead to phosphorylation of the inhibitory subunit IB␣ on specific serine residues (Ser 32 and Ser 36 ) (7) catalyzed by two IB kinases (IKKs), [3][4][5][6][7][8][9][10][11][12]. This step marks the IB protein for ubiquitination and subsequent degradation through a proteasome-dependent pathway (1). The active NF-B is then free for translocation to the nucleus, where it binds the B sequences present in the promoters of responsive genes. IKK␣ and IKK␤ reside in a larger kinase complex (700 -900 kDa), called the IB kinase complex (IKK complex), that also contains the essential regulatory subunit NEMO (also known as IKK␥) (13,14). Genetic studies suggest that NEMO/IKK␥ is absolutely required for the activation of IKKs and NF-B in response to different stimuli (13,15). NEMO/IKK␥ contains several coiled-coil domains, a leucine zipper, and a C-terminal zinc finger domain. These motifs are required for the correct assembly of the IKK complex (13) and recruitment of upstream signaling mediators (16). Numerous proteins have been demonstrated to interact with NEMO/IKK␥, as the kinase RIP and the inhibitor of NF-B A20 (17), the ...

NF-κB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-κB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-κB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-κB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-κB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-κB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

SummaryGastric mucosa responds to Helicobacter pyloriinduced cell damage by increasing the expression of COX-2 and EGF-related peptides. We sought to investigate the bacterial virulence factor/s and the host cellular pathways involved in the upregulation of COX-2, HB-EGF and amphiregulin in MKN 28 and AGS gastric mucosal cells. H. pylori strain CCUG 17874 was grown in Brucella broth supplemented with 0.2% (2,6-dimethyl)-b b b b -cyclodextrins. The soluble proteins released in the culture medium by the bacterium were fractionated by exclusion size and anion exchange chromatography. A single peak retaining the ability to upregulate COX-2 and HB-EGF mRNA and protein expression was obtained. SDS-PAGE analysis of the peak showed two peptides with an apparent molecular weight of 38 and 22 kDa, which were identified by automated Edman degradation analysis as the N-terminal and C-terminal peptides of H. pylori g g g g -glutamyltranspeptidase respectively. Acivicin, a selective g g g g -glutamyltranspeptidase inhibitor, counteracted H. pylori -induced upregulation of COX-2 and EGF-related peptide mRNA expression. An H. pylori isogenic mutant g g g g -glutamyltranspeptidase-deficient strain did not exert any effect on COX-2, HB-EGF and amphiregulin mRNA expression. Blockade of phosphatidylinositol-3 kinase and p38 kinase, but not MAP kinase kinase, inhibited H. pylori g g g g -glutamyltranspeptidase-induced upregulation of COX-2 and EGFrelated peptide mRNA expression.

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.