Kinetics of Bovine leukemia virus aspartic protease reveals its dimerization and conformational change.

The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.

Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34.

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.

Functional and Mass Spectrometric Evaluation of an Anti-Tick Antigen Based on the P0 Peptide Conjugated to Bm86 Protein.

A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from Megathura crenulata and used as an immunogen against Rhipicephalus microplus and Rhipicephalus sanguineus s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against Amblyomma mixtum and Ixodes ricinus species, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein. SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0-Bm86 conjugate and challenged with R. sanguineus s.l. and R. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate.

Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

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.

Natural Multi-Target Inhibitors of Cholinesterases and Monoamine Oxidase Enzymes with Antioxidant Potential from Skin Extracts of Hypsiboas cordobae and Pseudis minuta (Anura: Hylidae).

Alzheimer's disease (AD) is the most common cause of dementia, characterized by loss of selective neuronal and normal brain functions. Every year, ten million new cases are diagnosed worldwide. AD is a complex disease associated with all kind of different pathways, making their simultaneous modulation necessary. Nowadays anti-AD treatments are focused on enzymatic inhibitors. The study of the amphibians' skin had acquired great importance in the fields of biology and human health and represents an attractive and novel source for natural compounds with high potential in the development of new drugs. The present work exhibits the power of amphibian skins as a source of bioactive compounds. Herein we report the activity of extracts of two species from Hylidae family (H. cordobae and P. minuta) as reversible inhibitors of acetylcholinesterase and butyrylcholinesterase enzymes. Furthermore, the extracts inhibit MAO-B enzyme and showed antioxidant activities, acting on four important pathways of AD.

Protein content of the Hylesia metabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism.

Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SIGNIFICANCE: The present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.

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.

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.