1,720,957 research outputs found
Effects of polymorphic variation on the mechanism of Endoplasmic Reticulum Aminopeptidase 1
No full textEndoplasmic Reticulum Aminopeptidase 1 (ERAP1) generates antigenic peptides for loading onto Major Histocompatibility Class I molecules (MHCI) and can regulate adaptive immune responses. During the last few years, many genetic studies have revealed strong associations between coding Single Nucleotide Polymorphisms (SNPs) in ERAP1 and common human diseases ranging from viral infections to cancer and autoimmunity. Functional studies have established that these SNPs affect enzyme activity resulting to changes in antigenic peptide processing, presentation by MHCI and cellular cytotoxic responses. These disease-associated polymorphisms are, however, located away from the enzyme's active site and are interspersed to different structural domains. As a result, the mechanism by which these SNPs can affect function remains largely elusive. ERAP1 utilizes a complex catalytic mechanism that involves a large conformational change between inactive and active forms and has the unique property to trim larger peptides more efficiently than smaller ones. We analyzed two of the most consistently discovered disease-associated polymorphisms, namely K528R and Q730E, for their effect on the ability of the enzyme to select substrates based on length and to undergo conformational changes. By utilizing enzymatic and computational analysis we propose that disease-associated SNPs can affect ERAP1 function by influencing: (i) substrate length selection and (ii) the conformational distribution of the protein ensemble. Our results provide novel insight on the mechanisms by which polymorphic variation distal from the active site of ERAP1 can translate to changes in function and contribute to immune system variability in humans.</p
Screening identifies thimerosal as a selective inhibitor of endoplasmic reticulum aminopeptidase 1
No full textWe employed virtual screening followed by in vitro evaluation to discover novel inhibitors of ER aminopeptidase 1, an important enzyme for the human adaptive immune response that has emerged as an attractive target for cancer immunotherapy and the control of autoimmunity. Screening hits included three structurally related compounds carrying the (E)-N′-((1H-indol-3-yl)methylene)-1H-pyrazole-5-carbohydrazide scaffold and (2-carboxylatophenyl)sulfanyl-ethylmercury as novel ERAP1 inhibitors. The latter, also known as thimerosal, a common component in vaccines, was found to inhibit ERAP1 in the submicromolar range and to present strong selectivity versus the homologous aminopeptidases ERAP2 and IRAP. Cell-based analysis indicated that thimerosal can effectively reduce ERAP1-dependent cross-presentation by dendritic cells in a dose-dependent manner.</p
Study of the specificity and mechanism of function of aminopeptidases ERAP1, ERAP2 and IRAP
No full textERAP1, ERAP2 and IRAP aminopeptidases are important enzymes of the human adaptive immune system and play key roles in the antigen processing and presentation pathway. Their primary function is to produce mature antigenic epitopes which can bind onto MHC class I molecules. Therecognition of MHCI-epitope complexes by cytotoxic T lymphocytes results in the destruction of the infected or transformed cells. In the present thesis we focused on elucidating the mechanism of action and inhibition of ERAP1, the enzyme with the most profound effect in antigen processing. Specifically, by using recombinant DNA technology we expressed ERAP1 alleles with different polymorphic contents and studied their influence on the enzyme function. We demonstrated that ERAP1 polymorphisms can act by two distinct but synergetic mechanisms: influencing the enzyme activity or affecting the substrate length specificity. Furthermore, we analyzed how the conformational change of ERAP1 can affect its enzymatic activity. For that purpose we expressed recombinant ERAP1 variants, with perturbed inter-domain interactions responsible for stabilizing the closed conformation of the enzyme. Our results showed that these interactions are important for conformational plasticity, active site structural integrity and the specialization of the enzyme for antigenic peptide precursors. Finally, due to the emerging importance of pharmaceutical inhibition of these aminopeptidases, we screened, evaluated and characterized a set of non-peptidic compounds as potential inhibitors of these enzymes. Our results showed that the pharmaceutical compound Thimerosal is a potent and selective inhibitor of ERAP1.Οι αμινοπεπτιδάσες ERAP1, ERAP2 και IRAP αποτελούν σημαντικά συστατικά του επίκτητου ανοσοποιητικού συστήματος του ανθρώπου, συμμετέχοντας ενεργά στους μηχανισμούς αντιγονοπαρουσίασης. Λειτουργία τους είναι η παραγωγή ώριμων αντιγονικών επιτόπων, κατάλληλων για πρόσδεση σε μόρια τάξης I του MHC. Τα σύμπλοκα επιτόπων-MHCIαναγνωρίζονται στη συνέχεια από κυτταροτοξικά Τ λεμφοκύτταρα, με αποτέλεσμα την εξάλειψη μολυσμένων ή μετασχηματισμένων κυττάρων. Στην παρούσα διατριβή εστιάσαμε τη μελέτη μας στην ανάλυση του μηχανισμού δράσης και αναστολής της ERAP1, του ενζύμου με τη μεγαλύτερη συνεισφορά στην επεξεργασία αντιγόνων. Συγκεκριμένα, χρησιμοποιώντας τεχνολογία ανασυνδυασμένου DNA εκφράσαμε αλλήλια ERAP1 με διαφορετικούς συνδυασμούς πολυμορφισμών και μελετήσαμε την επίδρασή τους στη λειτουργία του ενζύμου. Διαπιστώσαμε ότι οι πολυμορφισμοί ασκούν τη δράση τους μέσω δύο ξεχωριστών αλλά συνεργιστικών μηχανισμών: επηρεάζοντας τη δραστικότητα του ενζύμου ή την προτίμηση των υποστρωμάτων του αναλόγως του μήκους τους. Στη συνέχεια, επικεντρωθήκαμε στη λειτουργική σημασία αλλαγής διαμορφώσεων της ERAP1. Για το σκοπό αυτό εκφράσαμε ανασυνδυασμένα αλλήλια ERAP1, στα οποία διαταράξαμε με τοποειδική μεταλλαξιγένεση ένα σύνολο αλληλεπιδράσεων που σταθεροποιούν την κλειστή διαμόρφωση του ενζύμου. Τα αποτελέσματα έδειξαν ότι οι αλληλεπιδράσεις αυτές είναι κρίσιμες για τη δομική πλαστικότητα του ενζύμου, τη δομική οργάνωση του ενεργού κέντρου καθώς και ότι παίζουν σημαντικό ρόλο στην εξειδίκευση του ενζύμου για πρόδρομα αντιγονικά πεπτίδια. Τέλος, λόγω της αναδυόμενης φαρμακευτικής σημασίας της αναστολής της ERAP1, έγινε διαλογή, αξιολόγηση καιχαρακτηρισμός του μηχανισμού ενός συνόλου μη πεπτιδικών ενώσεων ως πιθανών αναστολέων αυτών των ενζύμων. Τα αποτελέσματα μας αποκάλυψαν ότι η φαρμακευτική ουσία Thimerosal είναι ισχυρός και εκλεκτικός αναστολέας της ERAP1
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Critical role of interdomain interactions in the conformational change and catalytic mechanism of endoplasmic reticulum aminopeptidase 1
No full textEndoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.</p
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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