Influence of ellagitannins extracted by pomegranate fruit on disulfide isomerase PDIA3 activity

Pomegranate fruit is a functional food of high interest for human health due to its wide range of phytochemicals with antioxidant properties are implicated in the prevention of inflammation and cancer. Ellagitannins, such as punicalagin and ellagic acid, play a role as anti-atherogenic and neuroprotective molecules in the complex fighting against the degenerative diseases. The aim of this work was to evaluate the composition in punicalagins and ellagic acid of differently obtained extracts from whole fruit, peels and juices, prepared by squeezing or by centrifugation, of pomegranate belonging to different cultivars. Moreover, a wider phenolic fingerprint was also determined. The bioactivity of the extracts was tested on the redox activity of PDIA3 disulfide isomerase, an enzyme involved in the regulation of several cellular functions and associated with different diseases such as cancer, prion disorders, Alzheimer’s and Parkinson’s diseases. The results demonstrate that the different ratios between punicalagin and ellagic acid modulate the enzyme activity and other ellagitannins could interfere with this activity

Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane- associated receptor, which has been discovered as the disul de isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We rst studied the interaction through bioinformatics and uorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’- ERp57, containing the rst three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10−9 M

Antisense to Epstein Barr virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBV-transformed B lymphocytes

It is generally accepted that Epstein-Barr virus (EBV) latent genes EBNA-2, EBNA-3A, -3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A, -3B and -3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors

Recoding of nonsense mutation as a pharmacological strategy

Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough

Shmt2: a stat3 signaling new player in prostate cancer energy metabolism

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotyp

ERp57/PDIA3 A PLEIOTROPIC MEMBER OF THE PDIs FAMILY

Protein disulfide isomerases (PDIs) are an important cellular oxidoreductase enzyme family including several structurally related components. PDIs are involved in protein folding, catalyzing the formation and remodeling of disulfide bonds, and are mainly located in the endoplasmic reticulum (ER). The ERp57/PDIA3 has a noncanonical ER retention signal (QEDL) on the C-terminal domain and a Lys-rich nuclear localization signal, which binds with high affinity to a specific site of importin, responsible for the nuclear import process [1]. ERp57/PDIA3, found in different extracellular and subcellular locations, is involved in multiple processes [2,3]. The nuclear localization of ERp57/PDIA3 was discovered many years ago, nevertheless, its role in the nucleus is not well understood. It has been shown that ERp57/PDIA3 altered in different cancer cell lines. This protein participates in the signal transduction processes of STAT3 pathways [4,5], and binds specific DNA fragments in a melanoma cell line [6]. In respect of its localization on the plasma membrane, the ERp57/PDIA3 seems involved in EGFR signaling and internalization, as evidenced by the silencing of ERp57/PDIA3 in MDA-MB-468 cells [7] and it was identified also as an alternative cell membrane receptor for active forms of vitamin D3 that regulates some phenotypic functions and nongenomic response [8,9]. More recently it has been hypothesized as a pharmacological target in glioblastoma because ERp57/PDIA3 inhibition induced cytotoxic effects in two different glioblastoma cell lines (T98G and U-87 MG cells)[10]. Its involvement in cancer progression and other diseases suggests a potential use of ERp57/PDIA3 both as a marker and a therapeutic target. ERp57/PDIA3 is an important research target considering its various subcellular locations and its involvement in the cellular response

Unexpected Plasma Membrane Location for a Disulfide Isomerase Protein

The plasma membrane is a fundamental cell compartment necessary to separate the cell interior from the external environment. The membrane regulates the exchange of ions and solutes, is involved in the cell-cell communication and adhesion, as well as in signaling mechanisms. Most of these processes require the participation of specialized proteins, which may be embedded in the phospholipid bilayer or recruited to the cell membrane in response to a particular stimulus. ERp57, a member of the disulfide isomerase family (PDIs), is a soluble protein that is mainly located in the lumen of the endoplasmic reticulum (ER), but that has also been found in the nucleus, cytosol, mitochondria and cell membrane. In the ER, it accomplishes the disulfide bonds reshuffling in newly synthesized glycoproteins, in complex with the lectins calreticulin and calnexin. At present, the functions in the other compartments are still unclear. Several studies have suggested a possible involvement of ERp57 in receptors signalling. On the plasma membrane it has been previously described as being associated with vasopressin and angiotensin II receptors, as well as with STAT3. Furthermore, ERp57 is considered a cell membrane receptor itself in intestinal epithelial cells, where it binds the biologically active form of vitamin D3 (calcitriol) and appears to be responsible for the rapid response to the steroid hormone. In order to investigate these processes, we first analyzed the presence of ERp57 on the plasma membrane in four different cell lines (cancer and immortalized cells) by means of a surface biotinylation method. Our results indicate that ERp57 is present on the plasma membrane of the three analyzed cancer cell lines (HeLa, Raji, M14), but absent in the immortalized one (HaCaT). Moreover, immunofluorescence assays and Western blotting analyses performed in our lab revealed that ERp57 is redistributed in the cell following treatment with calcitriol. Further studies to establish the role of ERp57 in signal transduction and in the protein trafficking from the membrane to the nucleus are presently ongoing

DNA damage and repair: from molecular mechanisms to health implications

DNA is subjected to several modifications, resulting from endogenous and exogenous sources. The cell has developed a network of complementary DNA-repair mechanisms, and in the human genome, >130 genes have been found to be involved. Knowledge about the basic mechanisms for DNA repair has revealed an unexpected complexity, with overlapping specificity within the same pathway, as well as extensive functional interactions between proteins involved in repair pathways. Unrepaired or improperly repaired DNA lesions have serious potential consequences for the cell, leading to genomic instability and deregulation of cellular functions. A number of disorders or syndromes, including several cancer predispositions and accelerated aging, are linked to an inherited defect in one of the DNA-repair pathways. Genomic instability, a characteristic of most human malignancies, can also arise from acquired defects in DNA repair, and the specific pathway affected is predictive of types of mutations, tumor drug sensitivity, and treatment outcome. Although DNA repair has received little attention as a determinant of drug sensitivity, emerging knowledge of mutations and polymorphisms in key human DNA-repair genes may provide a rational basis for improved strategies for therapeutic interventions on a number of tumors and degenerative disorders

Interactions of Epstein-Barr virus origins of replication with nuclear matrix in the latent and in the lytic phases of viral infection

Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein-Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection

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