Renato Acquaviva

ABIN-1 Binds to NEMO/IKKγ and Co-operates with A20 in Inhibiting NF-κB

Nuclear factor κB (NF-κB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-κB is dependent on the phosphorylation of the inhibitory subunit IκBα that is mediated by a multimeric, high molecular weight complex, called IκB kinase (IKK) complex. This is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO/IKKγ. The latter protein is essential for the activation of IKKs and NF-κB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-κB) as a NEMO/IKKγ-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-κB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-κB at the level of the IKK complex and that A20 inhibits activation of NF-κB by de-ubiquitination of NEMO/IKKγ. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKγ and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-κB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKγ and facilitates A20-mediated de-ubiquitination of NEMO/IKKγ, thus resulting in inhibition of NF-κB.

The neutrophil gelatinase-associated lipocalin (NGAL), a NF-B-regulated gene, is a survival factor for thyroid neoplastic cells

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.

Helicobacter pylori gamma-glutamyltranspeptidase upregulates COX-2 and EGF-related peptide expression in human gastric cells

Gastric mucosa responds to Helicobacter pylori‐induced 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)‐β‐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. pyloriγ‐glutamyltranspeptidase respectively. Acivicin, a selective γ‐glutamyltranspeptidase inhibitor, counteracted H. pylori‐induced upregulation of COX‐2 and EGF‐related peptide mRNA expression. An H. pylori isogenic mutant γ‐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. pyloriγ‐glutamyltranspeptidase‐induced upregulation of COX‐2 and EGF‐related peptide mRNA expression.

Thyroglobulin binding and TSH regulation of the RHL-1 subunit of the asialoglycoprotein receptor in rat thyroid

The ability of asialo-thyroglobulin to bind the thyroid RHL-1 subunit of the asialoglycoprotein receptor has been investigated. Ligand blot assays show that the recombinant carbohydrate recognition domain of the thyroid RHL-1 subunit specifically interacts with rat desialated thyroglobulin. Moreover, RT-PCR and Western blot assays show that TSH deprivation decreases RHL-1 expression in PC C13 thyroid differentiated cells whereas insulin deprivation does not have any effect. The simultaneous absence of both TSH and insulin dramatically decreases the level of RHL-1 expression.

Prediction of the secondary structure of the carboxy-terminal third of rat thyroglobulin

A secondary structure prediction has been made using the available primary sequence data of the proposed carboxy-terminal of rat thyroglobulin. The model predicts 22% alfa-helix, 28% beta-structure and 17% beta turns. Out of the 8 possible carbohydrate acceptor-sites (Asn-x-Ser/Thr), 3 (residues 136, 368, 782) are associated with peptide sequences which favour the formation of beta-turn or loop-structures and are located in high hydrophilic regions. The entire sequence is predicted to be made up of two domains: one of them is highly structured, contains the hormonogenic sites, a cluster of tyrosines and at least one carbohydrate acceptor site.

Conformational stability and DNA binding energetics of the rat thyroid transcription factor 1 homeodomain

The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF‐1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF‐1HD is a reversible two‐state transition. The protein is not stable against temperature, showing a denaturation temperature of 32°C in the absence of salt and 50°C at 75 mM KCl. The binding energetics of TTF‐1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25°C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 × 108M−1 and the binding enthalpy change amounts to −41 kJ mol−1. The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF‐1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF‐1HD with the target DNA sequence and TTF‐1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK‐2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF‐1HD. Proteins 2008.

Calcium interaction with bovine thyroglobulin: Stoichiometry and structural consequences of calcium binding☆

Gel filtration studies show that the thyroglobulin (Tg) molecule (dimer) binds from 18 to 50 Ca2+ ions. At pH 7.5 Tg binds 18 Ca2+ ions with a Kd of 1.3 x 10(-5) M, and 50 Ca2+ ions with a Kd of 5.5 x 10(-4) M. The binding of calcium to bovine thyroglobulin increases the absorption band of iodoamino acid residues at 315 nm. In the presence of Ca2+, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) is increased about 5-fold by Tg, with a shift in the fluorescence emission maximum from 505 to 490 nm. Thus, thyroglobulin possesses two classes of calcium binding sites with different affinities. The data reported indicate, also, that Ca2+ binding to Tg increases the hydrophobicity of the surface of the molecule.