The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

4-Hydroxynonenal impairs miRNA maturation in heart failure via Dicer post-translational modification.

BACKGROUND AND AIMS: Developing novel therapies to battle the global public health burden of heart failure remains challenging. This study investigates the underlying mechanisms and potential treatment for 4-hydroxynonenal (4-HNE) deleterious effects in heart failure. METHODS: Biochemical, functional, and histochemical measurements were applied to identify 4-HNE adducts in rat and human failing hearts. In vitro studies were performed to validate 4-HNE targets. RESULTS: 4-HNE, a reactive aldehyde by-product of mitochondrial dysfunction in heart failure, covalently inhibits Dicer, an RNase III endonuclease essential for microRNA (miRNA) biogenesis. 4-HNE inhibition of Dicer impairs miRNA processing. Mechanistically, 4-HNE binds to recombinant human Dicer through an intermolecular interaction that disrupts both activity and stability of Dicer in a concentration- and time-dependent manner. Dithiothreitol neutralization of 4-HNE or replacing 4-HNE-targeted residues in Dicer prevents 4-HNE inhibition of Dicer in vitro. Interestingly, end-stage human failing hearts from three different heart failure aetiologies display defective 4-HNE clearance, decreased Dicer activity, and miRNA biogenesis impairment. Notably, boosting 4-HNE clearance through pharmacological re-activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) using Alda-1 or its improved orally bioavailable derivative AD-9308 restores Dicer activity. ALDH2 is a major enzyme responsible for 4-HNE removal. Importantly, this response is accompanied by improved miRNA maturation and cardiac function/remodelling in a pre-clinical model of heart failure. CONCLUSIONS: 4-HNE inhibition of Dicer directly impairs miRNA biogenesis in heart failure. Strikingly, decreasing cardiac 4-HNE levels through pharmacological ALDH2 activation is sufficient to re-establish Dicer activity and miRNA biogenesis; thereby representing potential treatment for patients with heart failure.

Specificity and Reactivity of Mycobacterium tuberculosis Serine/Threonine Kinases PknG and PknB.

Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine protein kinases, which play essential roles in cell growth, signal transduction, and pathogenesis. Protein kinase G (PknG) regulates the carbon and nitrogen metabolism by phosphorylation of the glycogen accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB) is involved in cell wall synthesis and cell shape, as well as phosphorylates GarA but at Thr22. While PknG seems to be constitutively activated and recognition of GarA requires phosphorylation in its unstructured tail, PknB activation is triggered by phosphorylation of its activation loop, which allows binding of the forkhead-associated domain of GarA. In the present work, we used molecular dynamics and quantum-mechanics/molecular mechanics simulations of the catalytically competent complex and kinase activity assays to understand PknG/PknB specificity and reactivity toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem essential for PknG binding and allow specificity for Thr21 phosphorylation. On the other hand, phosphorylated residues in PknB bind Arg26 in GarA and regulate its specificity for Thr22. We also provide a detailed analysis of the free energy profile for the phospho-transfer reaction and show why PknG has a constitutively active conformation not requiring priming phosphorylation in contrast to PknB. Our results provide new insights into these two key enzymes relevant for Mtb and the mechanisms of serine/threonine phosphorylation in bacteria.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis.

A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys1pP0 and p64K-ßAla1pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys1pP0 showed a heterogeneous conjugate compared to p64K-ßAla1pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys1pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-ßAla1pP0 this ratio was 5-7. Cys1pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while ßAla1pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys1pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys1pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract.

Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE's DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.

Novel mechanistic insights into physiological signaling pathways mediated by mycobacterial Ser/Thr protein kinases.

Protein phosphorylation is known to be one of the keystones of signal sensing and transduction in all living organisms. Once thought to be essentially confined to the eukaryotic kingdoms, reversible phosphorylation on serine, threonine, and tyrosine residues, has now been shown to play a major role in many prokaryotes, where the number of Ser/Thr protein kinases (STPKs) equals or even exceeds that of two-component systems. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most studied organisms for the role of STPK-mediated signaling in bacteria. Driven by the interest and tractability of these enzymes as potential therapeutic targets, extensive studies revealed the remarkable conservation of protein kinases and their cognate phosphatases across evolution, and their involvement in bacterial physiology and virulence. Here, we present an overview of the current knowledge of mycobacterial STPK structures and kinase activation mechanisms, and we then focus on PknB and PknG, two well-characterized STPKs that are essential for the intracellular survival of the bacillus. We summarize the mechanistic evidence that links PknB to the regulation of peptidoglycan synthesis in cell division and morphogenesis, and the major findings that establish PknG as a master regulator of central carbon and nitrogen metabolism. Two decades after the discovery of STPKs in M. tuberculosis, the emerging landscape of O-phosphosignaling is starting to unveil how eukaryotic-like kinases can be engaged in unique, non-eukaryotic-like, signaling mechanisms in mycobacteria.

Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection.

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed "clean linear B cell epitopes," and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels.

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.

S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.

Nitroalkylation of α-Synuclein by Nitro-Oleic Acid: Implications for Parkinson's Disease.

α-Synuclein (α-syn) represents the main component of the amyloid aggregates present in Parkinson's disease and other neurodegenerative disorders, collectively named synucleinopathies. Although α-syn is considered a natively unfolded protein, it shows great structural flexibility which allows the protein to adopt highly rich beta-sheet structures like protofibrils, oligomers and fibrils. In addition, this protein can adopt alpha-helix rich structures when interacts with fatty acids or acidic phospholipid vesicle membranes. When analyzing the toxicity of α-syn, protein oligomers are thought to be the main neurotoxic species by mechanisms that involve modification of intracellular calcium levels, mitochondrial and lysosomal function. Extracellular fibrillar α-syn promotes intracellular protein aggregation and shows many toxic effects as well. Nitro-fatty acids (nitroalkenes) represent novel pleiotropic anti-inflammatory signaling mediators that could interact with α-syn to exert unraveling actions. Herein, we demonstrated that nitro-oleic acid (NO2-OA) nitroalkylate α-syn, forming a covalent adduct at histidine-50. The nitroalkylated-α-syn exhibited strong affinity for phospholipid vesicles, moving the protein to the membrane compartment independent of composition of the membrane phospholipids. Moreover, NO2-OA-modified α-syn showed a reduced capacity to induce α-syn fibrillization compared to the non-nitrated oleic acid. From this data we hypothesize that nitroalkenes, in particular NO2-OA, may inhibit α-syn fibril formation exerting protective actions in Parkinson's disease.

DiagnoProt: a tool for discovery of new molecules by mass spectrometry.

MOTIVATION: Around 75% of all mass spectra remain unidentified by widely adopted proteomic strategies. We present DiagnoProt, an integrated computational environment that can efficiently cluster millions of spectra and use machine learning to shortlist high-quality unidentified mass spectra that are discriminative of different biological conditions. RESULTS: We exemplify the use of DiagnoProt by shortlisting 4366 high-quality unidentified tandem mass spectra that are discriminative of different types of the Aspergillus fungus. AVAILABILITY AND IMPLEMENTATION: DiagnoProt, a demonstration video and a user tutorial are available at http://patternlabforproteomics.org/diagnoprot . CONTACT: andrerfsilva@gmail.com or paulo@pcarvalho.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2.

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.

Characterization of prophages containing "evolved" Dit/Tal modules in the genome of Lactobacillus casei BL23.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.

Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation.

Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases.

Trypanosoma cruzi chemical proteomics using immobilized benznidazole.

Benznidazole (Bzn) is a nitroimidazole drug currently used as first line treatment against Chagas disease, a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. Although the drug has been used since the late 1960s, its mechanism of action is not fully understood. In an attempt to study Bzn mode of action, a structurally modified derivative of the drug was synthesized and immobilized into a solid matrix. This allowed enrichment of T. cruzi proteins capable of binding immobilized Bzn, which were subsequently analysed by mass spectrometry. The proteins identified as specific non-covalent Bzn interactors were a homologue of the bacterial YjeF proteins, a Sec23A orthologue and the aldo-ketoreductase family member TcAKR. TcAKR is closely related to other enzymes previously associated with Bzn reductive activation such as NTRI and TcOYE. Thus, our untargeted search for Bzn binding partners allowed us to encounter proteins that could be related to drug reductive activation and/or resistance mechanisms.