The protein ERp57 contributes to EGF receptor signaling and internalization in MDA-MB-468 breast cancer cells.

The disulfide isomerase ERp57 is a soluble protein mainly located in the endoplasmic reticulum, where it acts in the quality control of newly synthesized glycoproteins, in association with calreticulin and calnexin. It has been also detected in other cell compartments, such as the cytosol, the plasma membrane and the nucleus. In these locations it is implicated in various processes, participating in the rapid response to calcitriol, modulating the activity of STAT3 and being requested for the pre-apoptotic exposure of calreticulin on the plasma membrane. In the present work, the involvement of ERp57 in the activity of the EGF receptor was evaluated for the first time. EGFR is a tyrosine kinase receptor, which is able to activate numerous signaling cascades, leading to cell proliferation and inhibition of apoptosis. In the MDA-MB-468 breast adenocarcinoma cells, which overexpress EGFR, ERp57 expression has been knocked down by siRNA and the effects on EGFR have been studied. ERp57 silencing did not affect EGFR protein expression, cell membrane exposure or EGF binding, whereas the internalization and the phosphorylation of the receptor were impaired. The implication of ERp57 in the activity of EGFR, whose upregulation is known to be associated with tumors, could be relevant for cancer therapy.

ERp57/GRP58: a protein with multiple functions.

The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.

Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line.

Chromatin immunoprecipitation in M14 melanoma cells showed that the protein ERp57 (endoplasmic reticulum protein 57) binds to DNA in the proximity of STAT3 in a subset of STAT3-regulated genes. In the same cells, IL-6 induced a significant increase of the expression of one of these genes, i.e. CRP. Upon depletion of ERp57 by RNA interference, the phosphorylation of STAT3 on tyrosine 705 was decreased, and the IL-6-induced activation of CRP expression was completely suppressed. In vitro experiments showed that ERp57 is also required for the binding of STAT3 to its consensus sequence on DNA. Thus ERp57, previously shown to associate with STAT3 in the cytosol and in the nuclear STAT3-containing enhanceosome, is a necessary cofactor for the regulation of at least a subset of STAT3-dependent genes, probably intervening both at the site of STAT3 phosphorylation and at the nuclear level.

IFI16 and NM23 bind to a common DNA fragment both in the P53 and the cMYC gene promoters.

In the melanoma M14 cell line, we found that the antimetastatic protein NM23/nucleoside diphosphate kinase binds to the promoters of the oncogene cMYC and of P53, a gene often mutated in human cancer (Cervoni et al. [2006] J. Cell. Biochem. 98:421-428). In a further study, we find now that IFI16, a transcriptional repressor, in both promoters binds to the G-rich fragment that also binds NM23/NDPK. These fragments possess non-B DNA structures. Moreover, by sequential chromatin immunoprecipitation (re-ChIP) we show that the two proteins (IFI16 and NM23/NDPK) are simultaneously bound in vivo to the same DNA fragments. Since P53 stimulates apoptosis and inhibits cellular growth, and cMYC promotes cell growth and, in several instances, also apoptosis, the presence of NM23 and IFI16 on the same DNA fragments suggests their common involvement in the reduced development of some tumors.

The binding of antibiotics to ERp57/GRP58.

The effects of five antibiotics, previously described as ligands of protein disulfide isomerase PDI, have now been studied on the homologous protein ERp57. They bind to this protein with much higher affinity than to PDI, and some of them inhibit the reductase and the DNA-binding activities of ERp57. In view of the high affinity of vancomycin, erythromycin and streptomycin, some effects of their interaction with this protein might be expected in vivo.

Cooperative activity of Ref-1/APE and ERp57 in reductive activation of transcription factors.

ERp57, a protein disulfide isomerase localized mainly in the endoplasmic reticulum, has also been found in lesser amounts in the cytosol and nucleus, where its function is still not characterized. We report here that ERp57 displays affinity for Ref-1, a protein involved in DNA repair as well as in the reduction and activation of transcription factors. Immunoprecipitation experiments showed that Ref-1 and ERp57 also interact in vivo in at least three types of cultured human cells, namely HepG2, M14, and Raji. Oxidative stress increased the amount of nuclear Ref-1 associated with ERp57. Moreover, ERp57 reduced by the thioredoxin-reductase/thioredoxin system stimulated the binding of AP-1 to its consensus sequence on DNA, and HeLa cells stably transfected and overexpressing ERp57 were protected against hydrogen peroxide-induced cell killing. Accordingly, ERp57 appears to cooperate with Ref-1 in the regulation of gene expression mediated by redox-sensitive transcription factors and in the adaptive response of the cell to oxidative insult.

The binding of silibinin to ERp57.

The flavonoid silibinin is known to intervene in many cellular processes involved in a variety of pathologies, thus appearing a promising therapeutic tool. The molecular mechanisms responsible for these activities, however, have not been clearly defined, and although some of its interactions with proteins have been identified, the relative affinities are often too low to appear relevant in vivo. Here we describe the interaction of silibinin with the protein disulfide isomerase ERp57, characterized by a submicromolar dissociation constant. This interaction enhances the formation of a ERp57/REF-1 complex, and furthermore appears to affect the intracellular distribution of ERp57. This protein is involved in signaling pathways which are also affected by silibinin. This suggests that the ERp57-silibinin interaction might explain at least some of the biological effects caused by the flavonoid.

Upregulation of TPX2 by STAT3: identification of a novel STAT3 binding site.

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5'-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells.

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose-regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress-response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non-coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non-coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress-response genes.

DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells.

We isolated and analyzed by chromatin immunoprecipitation (ChIP) in viable M14 cells DNA sequences bound to the antimetastatic protein nucleoside diphosphate kinase (NM23/NDPK) to shed some light on the nuclear functions of this protein and on the mechanism by which it acts in development and cancer. We assessed the presence of selected sequences from promoters of platelet-derived growth factor A (PDGF-A), c-myc, myeloperoxidase (MPO), CD11b, p53, WT1, CCR5, ING1, and NM23-H1 genes in the cross-linked complexes. Quantitative PCR (Q-PCR) showed a substantial enrichment of the correlated oncosuppressor genes p53, WT1, ING1, and NM23-H1 in the immunoprecipitated (IP) DNA. This suggests that NM23/NDPK binding is involved in the transcription regulation of these genes. These results reveal new interactions that should help us to disclose the antimetastatic mechanism of NM23.

Proteins of the PDI family: unpredicted non-ER locations and functions.

Protein disulfide isomerases (PDIs) constitute a family of structurally related enzymes which catalyze disulfide bonds formation, reduction, or isomerization of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER). They act also as chaperones, and are, therefore, part of a quality-control system for the correct folding of the proteins in the same subcellular compartment. While their functions in the ER have been thoroughly studied, much less is known about their roles in non-ER locations, where, however, they have been shown to be involved in important biological processes. At least three proteins of this family from higher vertebrates have been found in unusual locations (i.e., the cell surface, the extracellular space, the cytosol, and the nucleus), reached through an export mechanism which has not yet been understood. In some cases their function in the non-ER location is clearly related to their redox properties, but in most cases their mechanism of action has still to be disclosed, although their propensity to associate with other proteins or even with DNA might be the main factor responsible for their activities.

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain.

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.

ERp57 is present in STAT3-DNA complexes.

STAT3 has been found constitutively activated in M14 melanoma cell line, as previously found in other melanoma cells. Using EMSA, DNA affinity experiments, and chromatin immunoprecipitation, STAT3 was found in M14 to bind the alpha2-macroglobulin gene enhancer in association with the protein disulfide isomerase isoform ERp57. The two proteins have also been found to be associated when bound to the SIE sequence in HepG2 cells stimulated by IL-6. In both cases an anti-ERp57 antibody hinders the binding of STAT3 to its consensus sequence on DNA, indicating that ERp57 is a necessary component of the DNA-bound STAT3 complex. Considering the functional association of the two proteins, the overexpression of ERp57 observed in a variety of transformed cells might be relevant to the oncogenic properties of STAT3.

Immunoprecipitation of DNA-protein complexes cross-linked by cis-diamminedichloroplatinum.

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described.

Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells.

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA-protein cross-linking experiments performed with two different cross-linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR-like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus.

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter.

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.

HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila.

HP1 is a conserved chromosomal protein, first discovered in Drosophila, which is predominantly associated with the heterochromatin of many organisms. Recently, it has been shown that HP1 is required for telomere capping, telomere elongation, and transcriptional repression of telomeric sequences. Several studies have suggested a model for heterochromatin formation and epigenetic gene silencing in different species that is based on interactions among histone methyltransferases (HMTases), histone H3 methylated at lysine 9 (H3-MeK9), and the HP1 chromodomain. This model has been extended to HP1 telomeric localization by data showing that H3-MeK9 is present at all of the telomeres. Here, we tested this model, and we found that the capping function of HP1 is due to its direct binding to telomeric DNA, while the silencing of telomeric sequences and telomere elongation is due to its interaction with H3-MeK9.