1,720,992 research outputs found
Structural and Functional characterisation of SteE, a Salmonella type III secretion system effector
No full textUpon Salmonella infection, bacterial effectors proteins are delivered into host cells by specialised multiprotein secretion systems. They modulate a range of host cellular processes, helping prevent bacterial clearance and promoting bacterial replication and survival within the host cell. Although many Salmonella effectors have been characterised, the type III secretion system effector SteE remains largely uncharacterised in terms of structure and function. This study revealed that SteE is predominately an intrinsically disordered protein, which may mimic the disordered regions commonly found in eukaryotic signalling proteins. Interaction studies with recombinant proteins demonstrated that SteE forms a stable and direct interaction with GSK3β, one of its identified host interaction partners, and addition of ATP increased SteE/GSK3 complex formation. Complex stabilisation is highly dependent on the conserved T91 residue of SteE, which GSK3β directly phosphorylates. SteE interaction with GSK3β is also dependent on conserved sequence motifs in SteE, one of which mimics a canonical GSK3β phosphorylation site. While SteE has multiple GSK3 binding sites, SteE interacts with the canonical substrate binding site in GSK3β. The interaction of SteE with GSK3β and the sequential SteE phosphorylation by GSK3β is hypothesised to stimulate a conformational change that increases the binding affinity of SteE with GSK3β and alters the substrate amino acid specificity of GSK3β from a Ser/Thr kinase to a Tyr kinase. The subsequent phosphorylation of Y143 in SteE by GSK3β forms the ‘pYxxQ’ SH2 binding motif that enables the recruitment of STAT3 via its SH2 domain for the phosphorylation of STAT3 at Y705. Overall, this work conveyed that SteE acts as an adaptor protein, recruiting a host kinase to a non-canonical substrate, and as a functional regulator of the well-known host Ser/Thr kinase GSK3, enabling Tyr phosphorylation on substrates. This unique and distinct function of SteE was also evident in the putative SteE homologs from diverse bacteria.Open Acces
Structural and functional characterisation of Class IV TRIM E3 ligases
Get PDFUbiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes. Target ubiquitination is achieved through the action of three separate enzymes; the E1, ubiquitin-activating enzyme, the E2, ubiquitin-conjugating enzyme and the E3, ubiquitin ligase. TRIM E3 ligases are the largest family of RING-type E3 ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain, one or two B-box domains, B1 and B2, and a coiled-coil domain. In addition, most TRIMs possess a C-terminal substrate-binding domain, which classifies them into one of eleven TRIM classes. The PRYSPRY domain is the most common substrate-binding domain in humans and links class IV TRIMs to roles in cellular innate immunity. TRIM22 and TRIM6, are Class IV TRIMs that share high sequence identity with the well-studied HIV restriction factor, TRIM5, and have also been implicated in the anti-viral response. TRIM22 is reported to function directly as a viral restriction factor against RNA viruses such as, IAV, HCV and EMCV. While TRIM6 functions to activate the innate immune signalling pathway through activation of the immune signalling factor, IKK. Aspects of TRIM22 and TRIM6 function remain understudied, including their biochemical and biophysical properties and this is the focus of this study. The results described herein outline key differences in the self-association properties of these proteins in comparison to TRIM5. Furthermore, they highlight discrepancies between the ubiquitination profiles of TRIM22 and TRIM6 presented in the literature and the activity observed in this study. Overall, this emphasizes the need for further study of the roles of TRIM22 and TRIM6, to verify current proposed functions, as well as identify potential additional functions within the cell.Open Acces
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
Studying the mechanism of Salmonella effector protein, SteC
No full textSteC, a Salmonella effector kinase translocated across the Salmonella-containing vacuole (SCV) into the host cytosol, causes actin polymerisation in host cells. However, its mechanism of activity is poorly understood. The goal of this thesis was to structurally and biophysically characterise SteC using bioinformatic and experimental techniques; determine its interaction with substrates and molecular requirements for its kinase activity; and interrogate the role of kinase activity in host cells using ectopic expression and infection of mammalian cells with Salmonella. SteC has a C-terminal kinase domain (KD) that shares some sequence and predicted structural features with eukaryotic protein kinases, whilst lacking others. The purified kinase domain shows conformational flexibility and is monomeric in solution. One proposed substrate, FMNL1, was analysed using mass-spectrometry to determine SteC-dependent phosphorylation sites. SteC KD bound a peptide of FMNL1 spanning 3 phosphorylation sites. SteC recombinantly expressed in Sf9 cells was phosphorylated at S379. In vitro kinase assays showed that SteC KD was active and this activity was dependent on S379 phosphorylation. S379 phosphorylation was efficiently achieved in vitro by the mammalian kinase STK38, a reported binding partner of SteC. Phosphorylation on S379 was required for ATP binding. In Salmonella infection of 3T3 cells, S379 phosphorylation was required for dense focal actin polymerisation. In addition, a second capsular F-actin pattern was observed, which was SteC- dependent and kinase activity independent. SteC localised to one end of the SCV in a cap pattern, and in vitro lipid binding assays showed that the N-terminal domain bound phosphatidic acid.
In summary this thesis determined that SteC is structurally flexible and lacks some essential eukaryotic protein kinase features. Furthermore, the activating mechanism of the kinase domain was uncovered and its importance in Salmonella-infected mammalian cells was confirmed. Finally, a second kinase-independent actin polymerisation activity of SteC was described.Open Acces
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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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