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Structural and functional characterization of A-B toxins: diphtheria toxin and clostridial neurotoxins
Full text linkI performed my doctorate research activity studying three important human pathogens that are A-B toxins: Diphtheria Toxin (DT), Tetanus Neurotoxin (TeNT) and Botulinum neurotoxins (BoNTs), the etiologic agents of diphtheria, tetanus and botulism respectively. In terms of structural organization these toxins consist of three domains, which are termed L chain (the N-terminal catalytic domain), HN (the transmembrane domain), and HC (the C-terminal binding domain). These domains are closely related to the common four step mechanism of action: membrane binding mediated by HC, endocytosis, membrane translocation mediated by HN and L-chain mediated substrate modification.
I studied the conformational change of diphtheria toxin at acidic pH. DT includes a T domain which is known to mediate the pH-dependent membrane translocation, by forming a channel through which the catalytic domain crosses the endocytic vesicle membrane. To date no structural data are available about the pore/channel formed by the T domain, nor is known if it is monomeric or oligomeric. I have performed biochemical and structural studies to characterize the T domain of DT. The T domain is also considered a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to cancer cells. I obtained the crystal structure of DT in the presence of lipid bicelles (which simulate the endocytic vesicle membrane) and grown at pH 5.5, pH that mimics the acidic environment where translocation takes place. The reported structure throws lights on the initial event of this process, the destabilization of the three α-helices present at the bottom of the toxin (Leka et al., 2014).
I then worked on a project which aimed to unravel the three dimensional structure of tetanus neurotoxin by crystallization studies. Because TeNT is considered “uncrystallizable” I focused on the use of antibody fragments (Fabs) as crystallization chaperons to aid the structural determination. Native gel analysis and size exclusion chromatography showed the formation of a stable complex in vitro between TeNT and the relative Fabs. Several crystallization experiments were carried out by high throughput crystallization screens.
Further, I performed functional analysis on the trafficking of botulinum neurotoxin at the neuromuscular junction (NMJ). I expressed the binding domains of different BoNT serotypes, which are both necessary and sufficient for binding to the neuronal surface and internalization. The two step purifications, chromatography and gel filtration, were sufficient to yield purifications of each binding domain to >90% purity. Using cerebellum granular neurons (CGNs), I tested their functionality and specificity. I performed also in vivo assays in order to analyze their distribution along the NMJ. The data from fluorescence analysis show high specificity of these binding domains at the NMJ, and a different staining between different BoNT serotypes, reflecting their different time of intoxication, and perhaps a different pathway of vesicular trafficking
On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments
No full textDiphtheria Toxin conformational switching at acidic pH
No full textDiphtheria toxin (DT), the etiological agent of the homonymous disease, like other bacterial toxins, has to undergo a dramatic structural change in order to be internalized into the cytosol, where it finally performs its function. The molecular mechanism of toxin transit across the membrane is not well known, but the available experimental evidence indicates that one of the three domains of the toxin, called the central alpha-helical domain, inserts into the lipid bilayer, so favoring the translocation of the catalytic domain. This process is driven by the acidic pH of the endosomal lumen. Here, we describe the crystal structure of DT grown at acidic pH in the presence of bicelles. We were unable to freeze the moment of DT insertion into the lipid bilayer, but our crystal structure indicates that the low pH causes the unfolding of the TH2, TH3 and TH4 alpha-helices. This event gives rise to the exposure of a hydrophobic surface that includes the TH5 and TH8 alpha-helices, and the loop region connecting the TH8 and TH9 alpha-helices. Their exposure is probably favored by the presence of lipid bilayers in the crystallization solution, and they appear to be ready to insert into the membrane
The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain
Full text linkThe genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo
Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals
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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
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
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