Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

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.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome.

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a 'conserved' proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

Sialic acid-containing glycans play a role in the activity of snake venom proteases.

Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

A snapshot of Bothrops jararaca snake venom gland subcellular proteome

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a ‘conserved’ proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

Profilings of subproteomes of lectin-binding proteins of nine Bothrops venoms reveal variability driven by different glycan types.

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

Sialic acid-containing glycans play a role in the activity of snake venom proteases

Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

Profilings of subproteomes of lectin-binding proteins of nine Bothrops venoms reveal variability driven by different glycan types

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

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.

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

Leishmaniases belong to the inglorious group of neglected tropical diseases, presenting different degrees of manifestations severity. It is caused by the transmission of more than 20 species of parasites of the Leishmania genus. Nevertheless, the disease remains on the priority list for developing new treatments, since it affects millions in a vast geographical area, especially low-income people. Molecular biology studies are pioneers in parasitic research with the aim of discovering potential targets for drug development. Among them are the telomeres, DNA-protein structures that play an important role in the long term in cell cycle/survival. Telomeres are the physical ends of eukaryotic chromosomes. Due to their multiple interactions with different proteins that confer a likewise complex dynamic, they have emerged as objects of interest in many medical studies, including studies on leishmaniases. This review aims to gather information and elucidate what we know about the phenomena behind Leishmania spp. telomere maintenance and how it impacts the parasite's cell cycle.

The distinct N-terminomes of Bothrops jararaca newborn and adult venoms.

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

Venom Profiling of the Insular Species Bothrops alcatraz: Characterization of Proteome, Glycoproteome, and N-Terminome Using Terminal Amine Isotopic Labeling of Substrates.

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

Community-based network analyses reveal emerging connectivity patterns of protein-protein interactions in murine melanoma secretome.

Protein-protein interaction networks (PPINs) are static representations of protein connections in which topological features such as subgraphs (communities) may contain proteins functionally related, revealing an additional layer of interactome complexity. We created two PPINs from the secretomes of a paired set of murine melanocytes (a normal melanocyte and its transformed phenotype). Community structures, identified by a graph clustering algorithm, resulted in the identification of subgraphs in both networks. Interestingly, the underlying structure of such communities revealed shared and exclusive proteins (core and exclusive nodes, respectively), in addition to proteins that changed their location within each community (rewired nodes). Functional enrichment analysis of core nodes revealed conserved biological functions in both networks whereas exclusive and rewired nodes in the tumoral phenotype network were enriched in cancer-related processes, including TGFß signaling. We found a remarkable shift in the tumoral interactome, resulting in an emerging pattern which was driven by the presence of exclusive nodes and may represent functional network motifs. Our findings suggest that the rearrangement in the tumoral interactome may be correlated with the malignant transformation of melanocytes associated with substrate adhesion impediment. The interactions found in core and new/rewired nodes might potentially be targeted for therapeutic intervention in melanoma treatment. SIGNIFICANCE: Malignant transformation is a result of synergistic action of multiple molecular factors in which genetic alterations as well as protein expression play paramount roles. During oncogenesis, cellular crosstalk through the secretion of soluble mediators modulates the phenotype of transformed cells which ultimately enables them to successfully disrupt important signaling pathways, including those related to cell growth and proliferation. Therefore, in this work we profiled the secretomes of a paired set of normal and transformed phenotypes of a murine melanocyte. After assembling the two interactomes, clusters of functionally related proteins (network communities) were observed as well as emerging patterns of network rewiring which may represent an interactome signature of transformed cells. In summary, the significance of this study relies on the understanding of the repertoire of 'normal' and 'tumoral' secretomes and, more importantly, the set of interacting proteins (the interactome) in both of these conditions, which may reveal key components that might be potentially targeted for therapeutic intervention.

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.

The distinct N-terminomes of Bothrops jararaca newborn and adult venoms

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

Venom profiling of the insular species Bothrops alcatraz: characterization of proteome, Glycoproteome, and N-Terminome using terminal amine isotopic labeling of substrates

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

Community-based network analyses reveal emerging connectivity patterns of protein-protein interactions in murine melanoma secretome

Protein-protein interaction networks (PPINs) are static representations of protein connections in which topological features such as subgraphs (communities) may contain proteins functionally related, revealing an additional layer of interactome complexity. We created two PPINs from the secretomes of a paired set of murine melanocytes (a normal melanocyte and its transformed phenotype). Community structures, identified by a graph clustering algorithm, resulted in the identification of subgraphs in both networks. Interestingly, the underlying structure of such communities revealed shared and exclusive proteins (core and exclusive nodes, respectively), in addition to proteins that changed their location within each community (rewired nodes). Functional enrichment analysis of core nodes revealed conserved biological functions in both networks whereas exclusive and rewired nodes in the tumoral phenotype network were enriched in cancer-related processes, including TGFβ signaling. We found a remarkable shift in the tumoral interactome, resulting in an emerging pattern which was driven by the presence of exclusive nodes and may represent functional network motifs. Our findings suggest that the rearrangement in the tumoral interactome may be correlated with the malignant transformation of melanocytes associated with substrate adhesion impediment. The interactions found in core and new/rewired nodes might potentially be targeted for therapeutic intervention in melanoma treatment. Significance: Malignant transformation is a result of synergistic action of multiple molecular factors in which genetic alterations as well as protein expression play paramount roles. During oncogenesis, cellular crosstalk through the secretion of soluble mediators modulates the phenotype of transformed cells which ultimately enables them to successfully disrupt important signaling pathways, including those related to cell growth and proliferation. Therefore, in this work we profiled the secretomes of a paired set of normal and transformed phenotypes of a murine melanocyte. After assembling the two interactomes, clusters of functionally related proteins (network communities) were observed as well as emerging patterns of network rewiring which may represent an interactome signature of transformed cells. In summary, the significance of this study relies on the understanding of the repertoire of ‘normal’ and ‘tumoral’ secretomes and, more importantly, the set of interacting proteins (the interactome) in both of these conditions, which may reveal key components that might be potentially targeted for therapeutic intervention.

Heterotypic signaling between dermal fibroblasts and melanoma cells induces phenotypic plasticity and proteome rearrangement in malignant cells.

The signaling events triggered by soluble mediators released from both transformed and stromal cells shape the phenotype of tumoral cells and have significant implications in cancer development and progression. In this study we performed an in vitro heterotypic signaling assays by evaluating the proteome diversity of human dermal fibroblasts after stimulation with the conditioned media obtained from malignant melanoma cells. In addition, we also evaluated the changes in the proteome of melanoma cells after stimulation with their own conditioned media as well as with the conditioned medium from melanoma-stimulated fibroblasts. Our results revealed a clear rearrangement in the proteome of stromal and malignant cells upon crosstalk of soluble mediators. The main proteome signature of fibroblasts stimulated with melanoma conditioned medium was related to protein synthesis, which indicates that this process might be an early response of stromal cells. In addition, the conditioned medium derived from 'primed' stromal cells (melanoma-stimulated fibroblasts) was more effective in altering the functional phenotype (cell migration) of malignant cells than the conditioned medium from non-stimulated fibroblasts. Collectively, self- and cross-stimulation may play a key role in shaping the tumor microenvironment and enable tumoral cells to succeed in the process of melanoma progression and metastasis. Although the proteome landscape of cells participating in such a heterotypic signaling represents a snapshot of a highly dynamic state, understanding the diversity of proteins and enriched biological pathways resulting from stimulated cell states may allow for targeting specific cell regulatory motifs involved in melanoma progression and metastasis.

Heterotypic signaling between dermal fibroblasts and melanoma cells induces phenotypic plasticity and proteome rearrangement in malignant cells

The signaling events triggered by soluble mediators released from both transformed and stromal cells shape the phenotype of tumoral cells and have significant implications in cancer development and progression. In this study we performed an in vitro heterotypic signaling assays by evaluating the proteome diversity of human dermal fibroblasts after stimulation with the conditioned media obtained from malignant melanoma cells. In addition, we also evaluated the changes in the proteome of melanoma cells after stimulation with their own conditioned media as well as with the conditioned medium from melanoma-stimulated fibroblasts. Our results revealed a clear rearrangement in the proteome of stromal and malignant cells upon crosstalk of soluble mediators. The main proteome signature of fibroblasts stimulated with melanoma conditioned medium was related to protein synthesis, which indicates that this process might be an early response of stromal cells. In addition, the conditioned medium derived from ‘primed’ stromal cells (melanoma-stimulated fibroblasts) was more effective in altering the functional phenotype (cell migration) of malignant cells than the conditioned medium from non-stimulated fibroblasts. Collectively, self- and cross-stimulation may play a key role in shaping the tumor microenvironment and enable tumoral cells to succeed in the process of melanoma progression and metastasis. Although the proteome landscape of cells participating in such a heterotypic signaling represents a snapshot of a highly dynamic state, understanding the diversity of proteins and enriched biological pathways resulting from stimulated cell states may allow for targeting specific cell regulatory motifs involved in melanoma progression and metastasis.

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 genus Bothrops comprises various pit viper species that represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes in the region. Venom proteomes of many Bothrops species have been well-characterized but investigations have focused almost exclusively on proteins smaller than 100 kDa despite expression of larger components being documented in several Bothrops venoms. This study sought to achieve detailed identification of major components in the high molecular mass subproteome of venoms from eight Bothrops species (B. brazili, B. cotiara, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni and B. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusion chromatography fractions of these venoms. Minor components also identified in the first peaks included 5'-nucleotidase, aminopeptidase, phosphodiesterase, and phospholipases A2 and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of more than one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating that they may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecific variability among these venom components.

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.