RESUMEN
Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor ß (TGF-ß) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.
Asunto(s)
Venenos de Crotálidos , Mordeduras de Serpientes , Animales , Ratones , Antivenenos , Mordeduras de Serpientes/genética , Venenos de Serpiente , Venenos de Crotálidos/farmacología , Músculos , ColágenoRESUMEN
Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.
RESUMEN
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.
RESUMEN
Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.
RESUMEN
Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.
RESUMEN
Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.
RESUMEN
Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.
RESUMEN
Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1′ position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1′. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.
RESUMEN
Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs. Significance One of the main features of viperid snake envenomation is myotoxicity at the bite site, which, in turn is often associated with edema, blistering and hemorrhage, composing a complex pattern of local tissue damage. In this scenario, besides muscle cells, other types of cells, components of the extracellular matrix and blood vessels may also be affected, resulting in an outcome of deficient muscle regeneration. The main venom components participating in this pathology are metalloproteinases and phospholipases A2. Muscle necrosis induced by metalloproteinases is considered as an indirect effect related to ischemia, due to hemorrhage resulted from damage to the microvasculature. The pathogenesis of local effects induced by Bothrops venoms or isolated toxins has been studied by traditional methodologies. More recently, proteomic and peptidomic approaches have been used to study venom-induced pathogenesis. Here, in order to investigate the role of metalloproteinase activity in local tissue damage, we asked whether the hemorrhagic metalloproteinase HF3, at sub-cytotoxic levels, could alter the proteome of C2C12 myotubes in culture, thereby providing an insight into the mechanisms for the development of myonecrosis. Our results from mass spectrometric analyses showed subtle, early changes in the cells, including differential abundance of some proteins and proteolysis in the culture supernatant. The data illustrate the potential ability of metalloproteinases to trigger early systemic responses progressing from local cells and up to tissues.
RESUMEN
Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.
RESUMEN
Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs. Significance One of the main features of viperid snake envenomation is myotoxicity at the bite site, which, in turn is often associated with edema, blistering and hemorrhage, composing a complex pattern of local tissue damage. In this scenario, besides muscle cells, other types of cells, components of the extracellular matrix and blood vessels may also be affected, resulting in an outcome of deficient muscle regeneration. The main venom components participating in this pathology are metalloproteinases and phospholipases A2. Muscle necrosis induced by metalloproteinases is considered as an indirect effect related to ischemia, due to hemorrhage resulted from damage to the microvasculature. The pathogenesis of local effects induced by Bothrops venoms or isolated toxins has been studied by traditional methodologies. More recently, proteomic and peptidomic approaches have been used to study venom-induced pathogenesis. Here, in order to investigate the role of metalloproteinase activity in local tissue damage, we asked whether the hemorrhagic metalloproteinase HF3, at sub-cytotoxic levels, could alter the proteome of C2C12 myotubes in culture, thereby providing an insight into the mechanisms for the development of myonecrosis. Our results from mass spectrometric analyses showed subtle, early changes in the cells, including differential abundance of some proteins and proteolysis in the culture supernatant. The data illustrate the potential ability of metalloproteinases to trigger early systemic responses progressing from local cells and up to tissues.
RESUMEN
Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress, which are regulated by phosphatases and kinases activities. Furthermore, we present additional evidence for a functional two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.
RESUMEN
Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress, which are regulated by phosphatases and kinases activities. Furthermore, we present additional evidence for a functional two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.
RESUMEN
Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.
