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.

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 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.

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.

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-a 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

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-a 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

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

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.(AU)

Proteomic and interactome approaches reveal PAK4, PHB-2, and 14-3-3n 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 14-3-3?, 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.

Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (<1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy.

A first step towards building spectral libraries as complementary tools for snake venom proteome/peptidome studies

Snake venoms are complex mixtures of a large number of distinct proteins and peptides with biological activity. Peptide spectral libraries are compilations of previously identified MS/MS spectra obtained from proteomics experiments. Here we present the generation and use of a Venom Peptidome and a Venom Proteome spectral library for the analysis of venom proteomes and peptidomes from distinct snake species.

Early response of C2C12 myotubes to a sub-cytotoxic dose of hemorrhagic metallo proteinase HF3 from Bothrops jararaca venom

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.

Comparative analysis of the high molecular mass subproteomes of eight Bothrops snake venoms

Snake venoms are extremely active biological secretions composed primarily of various classes of enzymes. The genusBothropscomprises various pit viper speciesthat represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes inthe region. Venom proteomes of manyBothropsspecies have been well-characterized but investigations have focused almost exclusively on proteins smaller than100 kDa despite expression of larger components being documented in severalBothropsvenoms. This study sought to achieve detailed identification of majorcomponents in the high molecular mass subproteome of venoms from eightBothropsspecies (B.brazili,B.cotiara,B.insularis,B.jararaca,B.jararacussu,B.leucurus,B.moojeniandB. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusionchromatography fractions of these venoms. Minor components also identified in the first peaks included 5'-nucleotidase, aminopeptidase, phosphodiesterase, andphospholipases A2and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of morethan one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating thatthey may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecificvariability among these venom components.

Naja annulifera snake: new insights into the venom components and pathogenesis of envenomation

Background Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed. Methodology/Principal findings Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity. Conclusions/Significance The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom.

Proteomic profiling of the proteolytic events in the secretome of the transformed phenotype of melanocyte-derived cells using terminal amine isotopic labeling of substrates

The comprehensive profiling of the repertoire of secreted proteins from cancer cells samples provides information on the signaling events in oncogenesis as well as on the cross-talk between normal and tumoral cells. Moreover, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In this context, we used Terminal Amine Isotopic Labeling of Substrates (TAILS) to perform a system-wide investigation on the N-terminome of the secretomes derived from a paired set of mouse cell lines: Melan-a (a normal melanocyte) and Tm1 (its transformed phenotype). Evaluation of the amino acid identities at the scissile bond in internal peptides revealed significant differences, suggesting distinct proteolytic processes acting in the normal and tumoral secretomes. The mapping and annotation of cleavage sites in the tumoral secretome suggested functional roles of active proteases in central biological processes related to oncogenesis, such as the processing of growth factors, cleavage of extracellular matrix proteins and the shedding of ectopic domains from the cell surface, some of which may represent novel processed forms of the corresponding proteins. In the context of the tumor microenvironment, these results suggest important biological roles of proteolytic processing in murine melanoma secreted proteins.

Proteomic and interactome approaches reveal PAK4, PHB-2, and 14-3-3n 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 14-3-3?, 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.

Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (<1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy.

A first step towards building spectral libraries as complementary tools for snake venom proteome/peptidome studies

Snake venoms are complex mixtures of a large number of distinct proteins and peptides with biological activity. Peptide spectral libraries are compilations of previously identified MS/MS spectra obtained from proteomics experiments. Here we present the generation and use of a Venom Peptidome and a Venom Proteome spectral library for the analysis of venom proteomes and peptidomes from distinct snake species.