1,720,997 research outputs found
Secreted subtilisin gene family in Trichophyton rubrum
No full textpeer reviewedSecreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T rubrum and other fungi revealed a presumed ancestral lineage comprising T rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry. (C) 2004 Elsevier B.V. All rights reserved
Heat Shock Protein 9O Interaction Networks
No full textHsp90 est une chaperone majeure dans la cellule humaine où elle maintient l'homéostasie protéique, notamment en cas de stress cellulaire tel que l'hyperthermie. Même en conditions normales, des centaines de protéines, comprenant entre autres des kinases et des facteurs de transcription, dépendent d'Hsp90 pour être fonctionnelles. L'extraordinaire diversité de cette clientèle relie Hsp90 à la quasi-totalité des processus cellulaires ainsi qu'à des maladies. L'activité d'Hsp90 et ses multiples tâches sont régulées par une batterie de co-chaperones et par des modifications post-traductionnelles qui agissent sur sa dynamique structurelle. Le présent travail regroupe plusieurs projets qui ont pour but d'avancer les connaissances sur le mode de fonctionnement d'Hsp90, avec un accent mis sur les réseaux d'interaction d'Hsp90.
Tout d'abord, nous avons publié une revue des travaux sur les réseaux d'interaction d'Hsp90 dans le contexte de maladies en collaboration avec le groupe de Gabriela Chiosis (MSKCC, New York).
Nous avons ensuite mené un projet dont l'objectif était de décrire ces réseaux dans des cellules cancéreuses. L'existence d'un réseau spécifique dans les cancers, distinct d'un réseau normal dédié aux tâches de ménages, a en effet été postulée pour expliquer la plus grande sensibilité des cellules cancéreuses aux inhibiteurs d'Hsp90. Malheureusement, nous avons découvert certains défauts techniques de l'outil expérimental principal utilisé dans ce projet qui jettent de sérieuses résèrves sur les résultats obtenus avec cet outil, et nous avons été contraint d'abandonner le projet.
Le projet principal de ce travail était de comprendre la fonction de deux sites de phosphorylation d'Hsp90. Après avoir confirmé les études précédentes rapportant que ces sites sont abondamment phosphorylés dans une variété de cellules cancéreuses et normales et de conditions, nous avons découvert que la phosphorylation de ces sites modifie le réseau d'interaction d'Hsp90 et sa sécrétion. Une analyse approfondie nous a permis de démontrer que la structure globale d'Hsp90 est modifiée par ces phosphorylations, et que ce changement structurel pourrait être à l'origine des autres effets observés. Nous proposons ainsi une nouvelle hypothèse pour la fonction de ces sites, pouvant expliquer des effets apparemment sans liens à l'aide d'un seul mécanisme.
Les deux derniers projets ont été conduits en collaboration avec le groupe de Didier Picard de l'Université de Genève. L'un d'eux visait à élucider le rôle de STIPl, une co-chaperone centrale d'Hsp90. Notre contribution a été d'analyser les changements protéiques survenant quand STIPl est éliminé de cellules humaines, et son impact sur le réseau d'interaction d'Hsp90. Nous démontrons une élévation du niveau de plusieurs chaperones, et que l'interaction d'Hsp90 avec ces chaperones est réduite mais pas totalement abrogée. En particulier, le complexe binaire de type procaryotique Hsp70:Hsp90 est moins abondant mais toujours associé en l'absence de STIPl. La présence de STIPl dans le complexe ternaire de type eucaryotique Hsp70:STIP1:Hsp90 en revanche redirige le complexe vers une plus grande utilisation du système de dégradation des protéines. L'activité de STIPl pourrait ainsi moduler l'usage du système de dégradation des protéines par Hsp70 et Hsp90. La deuxième collaboration avec le groupe Picard a été de caractériser des cellules où soit Hsp90a soit Hsp90 ont été éliminés. A priori considéré létal, c'est la première lignée cellulaire dont Hsp90 est absente. Nous montrons que l'élimination de n'importe quel Hsp90 conduit à une augmentation du niveau de plusieurs chaperones, ainsi qu'à une réduction du niveau d'enzymes impliquées dans le métabolisme du cholestérol.
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Hsp90 is a major chaperone in the human cell where it plays a key role in controlling protein homeostasis, notably in response to stresses such as heat shock. Even under normal conditions, hundreds of proteins depend on Hsp90 for proper folding and/or activity, including protein kinases, transcription factors and metabolic enzymes. The incredible diversity and vastness of Hsp90's clientele implicates Hsp90 in virtually all cellular processes as well as in some types of diseases.
The activity of Hsp90 in its thousands of tasks is regulated by an array of auxiliary co-chaperones and by post-translational modifications acting on Hsp90's structure and conformational dynamics. The present work gathers several projects aimed at understanding how Hsp90 fonctions in different contexts, with an emphasis on Hsp90's protein interaction networks.
We started by writing a review on the specific tapie of Hsp90 protein interaction networks in the context of diseases, in collaboration with the group of Gabriela Chiosis (MSKCC, New York). After this, our first research project aimed at characterizing Hsp90's interactions in the context of cancer. Indeed, the existence of a cancer specific Hsp90 "interactome", distinct from the normal Hsp90 network dedicated to housekeeping functions, was proposed to explain the observation that cancer cells are more sensitive to Hsp90 inhibition than normal cells. Unfortunately, we discovered technical flaws in the main affinity reagent that was to be used and we were not able to draw any solid conclusion from our experiments, forcing us to abandon the project.
The next main research project aimed at describing two phosphorylation sites in Hsp90, which are very abundant in vivo, but with no confirmed function. After corroborating early findings that phosphorylation at these sites is invariably high in cancer and normal cell lines under bath normal and stress conditions, we found out that phosphorylation modulates Hsp90's interactions and secretion. Further investigation revealed that these phospho-sites impact the global Hsp90 structural dynamics, which may be the cause for the aforementioned effects. We then developed a new hypothesis on the structural and functional effects of these phospho-sites in light of the existing knowledge. This hypothesis could help reconcile some apparently unrelated phenomena we observed and explain divergent results on this topic in the literature.
The third and fourth research projects were conducted in collaboration with the group of Didier Picard (University of Geneva). The first collaboration aimed at understanding the fundamental role of STIPl, a central Hsp90 co-chaperone. Our contribution was to analyze the proteomic changes occurring after STIPl knockout (KO) in human cell lines, as well as the Hsp90 network in absence of STIPl. The results show that STIPl KO upregulates proteins involved in the protein folding process as well as in the unfolded protein response, providing increased resistance to proteotoxic stresses. Accordingly, the prokaryotic-like Hsp70:Hsp90 complex remains associated and functional in the absence of STIPl. The results of this collaboration show that STIPl is able to drive the Hsp70:Hsp90 complex, directed at protein folding, towards the ternary Hsp70:STIP1:Hsp90 complex which promotes usage of the proteasome and protein degradation to maintain proteostasis.
The second collaboration aimed to characterize HEK293T cell lines where either Hsp90a or Hsp90 are knocked out. Initially thought to be essential, this is the first human cell line missing the cytosolic Hsp90 isoform. Proteomic analysis shows that Hsp90 KO of either isoform results in the upregulation of chaperones and the downregulation of the metabolism of steroids
Aspergillus Protein Degradation Pathways with Different Secreted Protease Sets at Neutral and Acidic pH
No full textAspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pool) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.Swiss National Foundation for Scientific Research [320030-1179641
Multi-panel, on-single-chip memristive biosensing
Get PDFMemristive biosensors have demonstrated excellent capabilities for ultrasensitive bio-detection. In the present work, memristive biosensing chips are designed, fabricated and implemented in a for the first time presented multi-panel on-chip detection for discrete sensing of a target molecule through separate functionalization of individual devices on the same chip. The biosensing scheme is validated by means of labeled (i.e. fluorescence) and label-free (i.e. electrical) characterization methods. This novel memristive multi-panel sensing paradigm paves the way for fast and ultrasensitive PoC (point-of-care) devices allowing the detection of specific targets in complex matrixes where non-specific molecules are present as well, and opening great potential for the application of memristive phenomena in multiplexed ultrasensitive bio-detection and theranostics
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe 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
Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium
No full textIn an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.</jats:p
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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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