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

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

La ceramica etrusca a vernice nera di Valle Trebba: dati archeologici e archeometrici a confronto

Il contributo si prefigge di affrontare da un punto di vista archeologico e archeometriche alcune delle problematiche relative allo studio delle ceramiche a vernice nera di produzione etrusca della necropoli di Valle Trebba della citt\ue0 etrusca di Spina. Nella prospettiva archeologica, viene presa in rassegna criticamente la bibliografia di riferimento e si approfondisce la problematica delle ciotole, una forma ceramica molto diffusa e dalla lunga vita produttiva dal tardo-arcaismo fino al III sec. a.C. Nella prospettiva archeometrica, si discutono i risultati delle analisi di un consistente gruppo di individui ceramici selezionati, arrivando alla identificazione di differenti tecniche di produzione nelle ceramiche locali e al riconoscimento di una produzione gi\ue0 di V sec. a.C

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

Ceramiche a vernice nera della necropoli etrusca di Valle Trebba, Spina (FE): dati archeologici e archeometrici

The project of study of the necropolis of Valle Trebba in Spina, coordinated by the Chair of Etruscology and Italic Archaeology of Bologna University in agreement with the Superintendence for Archaeological Heritage of Emilia Romagna, promotes a systematic analysis of black glazed pottery in a context of great proportions and relevance. Insights on local and imported productions of black glazed pottery in the Etruscan area of the Po Valley are few in order to date. Regarding Spina, the considerations on these productions in scientific literature are limited essentially to the autopsy of findings and the study of published funerary contexts. The sampling (64 samples) and the results of archaeometrical analysis presented here involved mainly Attic, Volterra and local black glazed pottery (end of the VIth-IIIrd century BC). The primary objective of this multidisciplinary approach in the study of black glazed pottery is to identify and single out specific techniques of each production, then to relate them to the autoptic criteria generally applied to the characterization of these ceramics in archaeological studies. The secondary purpose is to investigate the possible presence of vessels locally produced as an imitation of imports from Attica, that remained so far a hypothesis in scientific literature. The samples, previously cleaned of the glazed layer, pulverized and washed in deionized H2O, have been analyzed using mineral-geochemical methods. Therefore, the powders have been examined to determine the mineralogical composition by di raction analysis (Philips PW1710 Di ractometer) and thermal ones (TG, DTG, DTA) with heating from 20° C up to 1000° C, in a stream of CO2 and a heating rate of 20° C per minute (Apparatus Thermal Setaram LABSYS), and the chemical composition through X-Ray Fluorescence (XRF Philips PW1480). The thermal analysis allows us to quantify the loss in weight % (LOI) undergone by each sample at certain temperature ranges. In some samples, considered representative of homogeneous groups, thin section analysis to optical polarizing microscope has been performed to check more or less fine grain size and the presence of any included lithic and primary clinopyroxenes, further analysis to scanning electron microscope (SEM/EDS - Philips 515b/EDAX DX4) has been carried out to better detect phases not perceivable by the polarizing microscope (because of the fine particle size of the mixture). Subsequently, the chemical data have been processed statistically using bivariate plots and cluster analysis have been performed with the method of the average linkage to better highlight geochemical similarities of samples and to argue hypothesis of origin. During the processing of the cluster analysis, we also included chemical data of ndings of certain origin already studied (i.e. Marzabotto, Volterra, Adria, Suasa, Rimini, Faenza and Corinth), and data of three spacer rings found in the excavations of a productive area of the Etruscan town, as well as data of various clays samples carried out in the municipalities of the area (i.e. Ostellato, San Giovanni Masi and Argenta). This statistical analysis allows us to identify nine groups, mainly characterized by SiO2, Al2O3, Fe2O3, MgO, CaO among major elements and Cr, Ni, Zn, Rb, Zr among elements in trace. In all samples the predominant phase is quartz. K-feldspar is present in an amount from modest to signi cant and Plagioclase are recognized in an amount from modest to especially abundant in the samples of the Ist group. The clinopyroxenes, whose presence shows higher ring temperatures and a raw material rich in carbonates, are present in an amount from modest to abundant (Vth, VIth and IXth group). Calcite is usually scarcely present or absent in samples with high content of CaO: that confirms that the sampled findings have been baked at temperatures above 800° C, which allowed its almost complete dissociation. It was found the presence of hematite in almost all the samples in an amount from traces to modest, and also pyrite and maghemite are present in small amounts in some samples of color from light gray to dark gray: it is likely that the color is linked to the different content of these three stages, and it depends on the oxidant/reductant atmosphere to which the samples were subjected during cooking. Ist-IInd groups, identified as locally made and dated between IVth and IIInd century BC, evidently were created from raw materials poor in carbonates and elements derived from mafic rocks as ophiolite and rich in SiO2 (quartz-feldspathic skeleton). The Ist group is characterized by a homogeneous particle size and the presence of hematite which determines the characteristic red color. The IInd group demonstrates a different degree of clay purification and ring in a more reducing atmosphere. The comparison of these data with others from samples of local pottery belonging to the second half of Vth-mid IVth century BC confirms the continuity of production methods during time. The involvement in the analysis of samples of raw clay allows us to hypothesize that the deposits of raw material used for these vases were almost adjacent to the production area. The IVth group, which identified Attic pottery, comes from raw material poor in carbonates and poorer in quartz-feldspathic skeleton, but with a rich content in mafic elements. The group is rather compact in chemical and mineralogical characteristics. Analysis also confirms an Attic origin for outturned bowls and stemmed plates, vascular forms that were considered peculiar of the Adriatic and Po Valley market. Vth-IXth groups, characterized by a larger grain size than the previ- ous ones, are quite problematic. Vth-VIth groups consist of vases dating in the Vth century BC and characterized by forms predominantly of Attic influence, but their chemical-mineralogical characteristics distinguish them from Attic vases and from the remaining productions identified or used as comparison. The VIIth group is composed by a little stemmed plate, a typical local form in Spina, and by a bowl of Attic form. We could plausibly suppose that these two vases were locally made and chemically rather akin to the IInd group, thus confirming the hypothesis of a local imitation of Attic vases thanks to the bowl. We could suppose that the bowl, despite of being a close imitation of (or resembling) an Attic model, responds to a rather local request and perhaps also met an Etruscan taste in thin and sinuous vessel tectonics close to metal ware. At last, VIIIth-IXth groups are distinguished from all the others, in particular for a high content of CaO. The sample no. SVT 12, which belongs to an “Alto-Adriatico” figured oinochoe with trefoil mouth, is most likely a local product of Spina, even if the clay had a dfferent preparation from the one of Ist-IInd groups, and in particular by no. SVT 61, an “Alto-Adriatico” gured lekanis. We could divide “Alto- Adriatico” vessels at least into two productions, as noted in literature: the first one that does not differ from the black glazed pottery of the Ist group and the latter that instead has a clay rich in calcite, which may have been added to the starting raw material, likely for a whitening e ect. Finally, we would like to stress that the considerations we propose in this paper clearly show that only a systematic and multidisciplinary approach to the study of black glazed pottery supported by archaeometrical analysis can bring signi cant results not restricted only to a typological study

Quality Assessment of Investigational Medicinal Products in COVID-19 Clinical Trials: One Year of Activity at the Clinical Trials Office

One year after the spread of the pandemic, we analyzed the assessment results of the quality documentation submitted to the Clinical Trials Office of the Italian Medicines Agency as part of the request for authorization of clinical trials with a COVID-19 indication. In this article, we report the classification of the documentation type, an overview of the assessment results, and the related issues focusing on the most frequently detected ones. Relevant data regarding the Investigational Medicinal Products (IMPs) tested in COVID-19 clinical trials and their quality profiles are provided in the perspective of increasing transparency and availability of information. Some criticalities that have been exacerbated by the management of clinical trials during the emergency period are highlighted. Results confirm that IMPs tested in authorized COVID-19 clinical trials are developed in agreement with the same legal requirements for quality, safety, and efficacy as for any other medicinal product in the European Union (EU). The same strong regulatory framework applies, and there is no lowering in the safety profile due to the pandemic; authorized IMPs meet the highest standards of quality. The regulatory network should capitalize on lessons learned from the emergency setting. Some take-home messages are provided that could support the regulatory framework to expand its boundaries by innovating and evolving even though remaining strong and effective

The binding of silibinin to ERp57

International audienceThe flavonoid silibinin is known to intervene in many cellular processes involved in a variety of pathol-ogies, 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

ERp57/PDIA3 binds specific DNA fragments in a melanoma cell line

ERp57/PDIA3 is a ubiquitously expressed disulfide isomerase protein, which acts in concert with calreticulin and calnexin in the folding of glycoproteins destined to the plasma membrane or to be secreted. Its canonical compartment is the endoplasmic reticulum, where it acts as a chaperone and redox catalyst, but non canonical locations have been described as well, and ERp57 has been found associated with DNA and nuclear proteins. In previous work performed in HeLa cells, ERp57 has been demonstrated to bind specific DNA sequences involved in the stress response. The direct interaction with the DNA sequences identified as ERp57-targeted regions in HeLa cells has now been confirmed in a melanoma cell line. Furthermore, the ERp57 silencing, achieved by RNA interference, has produced a significant down-regulation of the expression of target genes. The possible involvement of other proteins in complex with ERp57 has been studied by an in vitro biotin-streptavidin based binding assay and the interacting protein APE/Ref-1 has been also assessed for its direct association with the ERp57 target regions. In conclusion, nuclear ERp57 interacts in vivo with DNA fragments in melanoma cells and is potentially involved in the transcriptional regulation of its target genes. (C) 2013 Elsevier B.V. All rights reserved