Repurposed 3D Printer Allows Economical and Programmable Fraction Collection for Proteomics of Nanogram Scale Samples.

In this work, we describe the construction and application of a repurposed 3D-printer as a fraction collector. We utilize a nano-LC to ensure minimal volumes and surfaces although any LC can be coupled. The setup operates as a high-pH fractionation system capable of effectively working with nanogram scales of lysate digests. The 2D RP-RP system demonstrated superior proteome coverage over single-shot data-dependent acquisition (DDA) analysis using only 5 ng of human cell lysate digest with performance increasing with increasing amounts of material. We found that the fractionation system allowed over 60% signal recovery at the peptide level and, more importantly, we observed improved protein level intensity coverage, which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of data-independent acquisition (DIA) and wide window acquisition (WWA) to fractionated samples allowed nearly 8000 proteins to be identified from 50 ng of the material. The utility of the 2D system was further investigated for phosphoproteomics (>21 000 phosphosites from 50 µg starting material) and pull-down type experiments and showed substantial improvements over single-shot experiments. We show that the 2D RP-RP system is a highly versatile and powerful tool for many proteomics workflows.

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Multiomics Profiling of Toxins in the Venom of the Amazonian Spider Acanthoscurria juruenicola.

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Proteomic and Interactome Approaches Reveal PAK4, PHB-2, and 14-3-3η as Targets of Overactivated Cdc42 in Cellular Responses to Genomic Instability.

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein-protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3η, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.

Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into Bothrops jararaca Intraspecific Venom Variation.

We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

BACKGROUND: The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. METHODS: The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. RESULTS: The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. CONCLUSION: We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. GENERAL SIGNIFICANCE: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues.

Proteoforms of the platelet-aggregating enzyme PA-BJ, a serine proteinase from Bothrops jararaca venom.

Snake venoms contain serine proteinases that are functionally similar to thrombin and specifically cleave fibrinogen to convert it into fibrin or activate platelets to aggregation. PA-BJ is a serine proteinase from Bothrops jararaca venom that promotes platelet aggregation and this effect is mediated by the G-coupled protein receptors PAR1 and PAR4. In this study we describe an improved procedure to obtain PA-BJ from B. jararaca venom that uses less chromatographic steps, and, interestingly, results in the isolation of eight proteoforms showing slightly different pIs and molecular masses due to variations in their glycosylation levels. The identity of the isolated PA-BJ forms (1-8) was confirmed by mass spectrometry, and they showed similar platelet-activating activity on washed platelet suspensions. N- and O-deglycosylation of PA-BJ 1-8 under denaturing conditions generated variable electrophoretic profiles and showed that some forms were resistant to complete deglycosylation. Furthermore, N- and O-deglycosylation under non-denaturing conditions also showed different electrophoretic profiles between the PA-BJ forms and caused partial loss of their ability to cleave a recombinant exodomain of PAR1 receptor. In parallel, three cDNAs encoding PA-BJ-like enzymes were identified by pyrosequencing of a B. jararaca venom gland library constructed with RNA from a single specimen. Taken together, our results suggest that PA-BJ occurs in the B. jararaca venom in multiple proteoforms displaying similar properties upon platelets regardless of their variable isoelectric points, molecular masses, carbohydrate moieties and susceptibility to the activity of glycosidases, and highlight that variability of specific venom components contributes to venom proteome complexity.

Exploring potential virulence regulators in Paracoccidioides brasiliensis isolates of varying virulence through quantitative proteomics.

Few virulence factors have been identified for Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in P. brasiliensis. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how Paracoccidioides modulates the host-pathogen interaction to its advantage.

Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes.

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

Analysis of the Snake Venom Peptidome.

Snake venom peptidomes are known to be a large source of molecules with different pharmacological properties. The complexity and variability of snake venoms, the presence of proteinases, and the lack of complete species-specific genome sequences make snake venom peptidome profiling a challenging task that requires especial technical strategies for sample processing and mass spectrometric analysis. Here, we describe a method for assessing the content of snake venom peptides and highlight the importance of sampling procedures, as they substantially influence the peptidomic complexity of snake venoms.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity.

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 µM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

Individual variability in the venom proteome of juvenile Bothrops jararaca specimens.

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

Proteomic profiling of hydatid fluid from pulmonary cystic echinococcosis.

BACKGROUND: Most cystic echinococcosis cases in Southern Brazil are caused by Echinococcus granulosus and Echinococcus ortleppi. Proteomic studies of helminths have increased our knowledge about the molecular survival strategies that are used by parasites. Here, we surveyed the protein content of the hydatid fluid compartment in E. granulosus and E. ortleppi pulmonary bovine cysts to better describe and compare their molecular arsenal at the host-parasite interface. METHODS: Hydatid fluid samples from three isolates of each species were analyzed using mass spectrometry-based proteomics (LC-MS/MS). In silico functional analyses of the identified proteins were performed to examine parasite survival strategies. RESULTS: The identified hydatid fluid protein profiles showed a predominance of parasite proteins compared to host proteins that infiltrate the cysts. We identified 280 parasitic proteins from E. granulosus and 251 from E. ortleppi, including 52 parasitic proteins that were common to all hydatid fluid samples. The in silico functional analysis revealed important molecular functions and processes that are active in pulmonary cystic echinococcosis, such as adhesion, extracellular structures organization, development regulation, signaling transduction, and enzyme activity. CONCLUSIONS: The protein profiles described here provide evidence of important mechanisms related to basic cellular processes and functions that act at the host-parasite interface in cystic echinococcosis. The molecular tools used by E. granulosus and E. ortleppi for survival within the host are potential targets for new therapeutic approaches to treat cystic echinococcosis and other larval cestodiases.

Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

Comparative gender peptidomics of Bothrops atrox venoms: are there differences between them?

BACKGROUND: Bothrops atrox is known to be the pit viper responsible for most snakebites and human fatalities in the Amazon region. It can be found in a wide geographical area including northern South America, the east of Andes and the Amazon basin. Possibly, due to its wide distribution and generalist feeding, intraspecific venom variation was reported by previous proteomics studies. Sex-based and ontogenetic variations on venom compositions of Bothrops snakes were also subject of proteomic and peptidomic analysis. However, the venom peptidome of B. atrox remains unknown. METHODS: We conducted a mass spectrometry-based analysis of the venom peptides of individual male and female specimens combining bottom-up and top-down approaches. RESULTS: We identified in B. atrox a total of 105 native peptides in the mass range of 0.4 to 13.9 kDa. Quantitative analysis showed that phospholipase A2 and bradykinin potentiating peptides were the most abundant peptide families in both genders, whereas disintegrin levels were significantly increased in the venoms of females. Known peptides processed at non-canonical sites and new peptides as the Ba1a, which contains the SVMP BATXSVMPII1 catalytic site, were also revealed in this work. CONCLUSION: The venom peptidomes of male and female specimens of B. atrox were analyzed by mass spectrometry-based approaches in this work. The study points to differences in disintegrin levels in the venoms of females that may result in distinct pathophysiology of envenomation. Further research is required to explore the potential biological implications of this finding.

Inflammatory Reaction Induced by Two Metalloproteinases Isolated from Bothrops atrox Venom and by Fragments Generated from the Hydrolysis of Basement Membrane Components.

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 µg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 106 with ATXL and 5 × 106 with BATXH. TNF-α was identified in the supernatant of BATXH-or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC-MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4-5 × 106) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways.

A Multiomics Approach Unravels New Toxins With Possible In Silico Antimicrobial, Antiviral, and Antitumoral Activities in the Venom of Acanthoscurria rondoniae.

The Araneae order is considered one of the most successful groups among venomous animals in the world. An important factor for this success is the production of venoms, a refined biological fluid rich in proteins, short peptides and cysteine-rich peptides (CRPs). These toxins may present pharmacologically relevant biological actions, as antimicrobial, antiviral and anticancer activities, for instance. Therefore, there is an increasing interest in the exploration of venom toxins for therapeutic reasons, such as drug development. However, the process of peptide sequencing and mainly the evaluation of potential biological activities of these peptides are laborious, considering the low yield of venom extraction and the high variability of toxins present in spider venoms. Here we show a robust methodology for identification, sequencing, and initial screening of potential bioactive peptides found in the venom of Acanthoscurria rondoniae. This methodology consists in a multiomics approach involving proteomics, peptidomics and transcriptomics analyses allied to in silico predictions of antibacterial, antifungal, antiviral, and anticancer activities. Through the application of this strategy, a total of 92,889 venom gland transcripts were assembled and 84 novel toxins were identified at the protein level, including seven short peptides and 10 fully sequenced CRPs (belonging to seven toxin families). In silico analysis suggests that seven CRPs families may have potential antimicrobial or antiviral activities, while two CRPs and four short peptides are potentially anticancer. Taken together, our results demonstrate an effective multiomics strategy for the discovery of new toxins and in silico screening of potential bioactivities. This strategy may be useful in toxin discovery, as well as in the screening of possible activities for the vast diversity of molecules produced by venomous animals.

Analysis of the subproteomes of proteinases and heparin-binding toxins of eight Bothrops venoms.

Viperid snakes show the most complex snake-venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2-DE, and their subproteomes of proteinases were explored by 2-D immunostaining and 2-D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti-coagulant and anti-inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high-affinity for heparin as composed of mainly serine proteinases and C-type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin-binding toxins. Only the non-bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin-bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.