Liberato Marzullo

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-κB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKγ, increasing availability of IKKγ and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-κB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: new hints for actin folding.

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.

BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells.

BAG3, a member of the BAG co‐chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV‐1‐associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase‐3 activation by HIV‐1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection. J. Cell. Physiol. 218: 264–267, 2009.

BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages

The incidence and death rate of pancreatic ductal adenocarcinoma (PDAC) have increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumour formation and development of metastasis. Here we report that PDAC cells secrete BAG3, which binds and activates macrophages, inducing their activation and the secretion of PDAC supporting factors. We also identify IFITM-2 as a BAG3 receptor and show that it signals through PI3K and the p38 MAPK pathways. Finally, we show that the use of an anti-BAG3 antibody results in reduced tumour growth and prevents metastasis formation in three different mouse models. In conclusion, we identify a paracrine loop involved in PDAC growth and metastatic spreading, and show that an anti-BAG3 antibody has therapeutic potential.

Molecular bases of copper and iron deficiency-associated dyslipidemia: a microarray analysis of the rat intestinal transcriptome

As essential cofactor in many proteins and redox enzymes, copper and iron are involved in a wide range of biological processes. Mild dietary deficiency of metals represents an underestimated problem for human health, because it does not cause clear signs and clinical symptoms, but it is associated to long-term deleterious effects in cardiovascular system and alterations in lipid metabolism. The aim of this work was to study the biological processes significantly affected by mild dietary deficiency of both metals in rat intestine, in order to better understand the molecular bases of the systemic metabolic alterations, as hypercholesterolemia and hypertriglyceridemia observed in copper-deficient rats. A gene-microarray differential analysis was carried out on the intestinal transcriptome of copper- and iron-deficient rats, thus highlighting the biological processes significantly modulated by the dietary restrictions. The gene array analysis showed a down-regulation of genes involved in mitochondrial and peroxisomal fatty acids beta-oxidation and an up-regulation of genes involved in plasmatic cholesterol transport (apoprotein E and lecithin:cholesterol acyltransferase) in copper deficiency. Furthermore, a severe down-regulation of ApoH was pointed out in iron-deficient animals.

Identification of a synaptosome-associated form of BAG3 protein.

3104 Cell Cycle 2008; Vol. 7 Issue 19 BAG3 protein belongs to the family of co-chaperones sharing BAG domain, through which they bind to heat shock protein (Hsp) 70; furthermore, through BAG or different domains, members of the family can interact with steroid hormone receptors, Raf-1 kinase and other proteins.1-4 Basal levels of BAG3 have been detected in a restricted panel of normal tissues, including muscle, heart, kidney, lung, liver and placenta,2 and in some tumours, such as lymphoid leukaemias,5,6 pancreatic cancers,7 thyroid carcinomas8 and others;2 on the other hand, BAG3 expression can be induced in normal leukocytes and other cell types by exposure to stressful stimuli, that activate heat shock factor (HSF)-1; this, in turn, acts on bag3 promoter.2,9 BAG3 protein sustains cell survival in a number of cell systems, mostly by influencing, via Hsp70, levels and/or activity of some anti-apoptotic proteins.2,5,6,8-10 Furthermore, we recently showed that BAG3 regulates cyclin B1 expression and cell cycle in neuroblastoma cells.11 The glia-neuron unit represents a specialized supracellular structure that allows peripheral regions of the neuron to promptly respond to challenges, including those affecting cell cycle and survival (ischemic or hemorrhagic insults, exposure to toxins or drugs, etc.,) or learning events.12 We therefore investigated whether BAG3 protein was present in synaptosomes, where it might contribute in tuning cell state and modulating synaptic response. Synaptosomal structures were prepared by rat brain cell fractionation, as described,13 and analyzed by Western blot with two different anti-BAG3 antibodies. First we used a polyclonal antibody (TOS-2), detecting BAG3 protein at its known molecular weight of 75 kD:2 the signal was clear in total (cortex + hippocampus) homogenate and in several subcellular fractions, while much less evident in the synaptosomal fraction. However, when we used a monoclonal antibody (AC-1) raised against a BAG3 N-terminal peptide, we observed the presence of a lower (about 40 Kd) band in synaptosomes, with an intensity even higher than in the homogenate (Fig. 1A). Those results indicated that: (i) BAG3 protein was expressed in rat brain, and (ii) another protein, possibly corresponding to a smaller form of BAG3, is highly present in synaptosomes. To verify the identity of such protein, we immunoprecipitated the synaptosomal preparation with AC-1 and analysed the immunoprecipitated proteins by Western blot; the resulting band at 40 kD was subjected to an in gel digestion procedure using trypsin and resulting peptide mixtures were analysed by mass spectrometry. MALDI/MS and LC/MSMS analyses14,15 allowed identifying 13 peptides, whose molecular weights were in good agreement with those expected for BAG3 (mass accuracy 5–15 ppm). The identities of four of those peptides were also confirmed by tandem mass spectra. The observed peptides allowed a 35% sequence coverage: the N-terminal tryptic peptide (1–21) was detected, whereas the last fragment identified in the C-terminal portion of the protein was peptide 453–462. On the basis of these data, it could be deduced that this protein consists of a shorter form of BAG3, still possessing its N-terminal end, but lacking its C-terminal (Fig. 1B). *Correspondence to: Antonio Giuditta; Dipartimento delle Scienze Biologiche; Via Mezzocannone 8; Napoli 80134, Italia; Tel.: +39.081.2535089; Fax: +39.081.2535090; Email: giuditta@unina.it

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Copper promotes TFF1-mediated Helicobacter pylori colonization.

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.

Copper-binding activity of Trefoil factor 1 (TFF1): a new perspective in the study of the multifunctional roles of TFFs.

Trefoil factors (TFFs) are gastrointestinal peptides playing an essential role in the epithelial restitution. Among the three known TFF peptides, TFF1 is characterized by three disulfide bonds producing a compact globular structure and an extended and disordered tail formed by amino- and carboxy-termini. The presence of a cysteine surrounded by several negatively charged residues in this region of the protein, highly conserved in different species, suggests the possible formation of a metal-binding site. Affinity chromatography and mass spectrometric analyses allowed us to demonstrate a selective binding affinity of TFF1 for copper. The binding induces conformational changes in the tertiary structure as demonstrated by circular dichroism experiments, while limited proteolysis revealed an altered access to the cleavage sites in the amino- and carboxy-termini. The results of this study reveal a new property of TFF1 and suggest that copper could influence its biological activities by interfering with the dimerization of the peptide and/or the interaction with mucins or putative TFF receptors.

Role of BAG3 in cancer progression: A therapeutic opportunity

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours.