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1.
J Am Soc Mass Spectrom ; 34(4): 794-796, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-36947430

RESUMO

Complex protein mixtures typically generate many tandem mass spectra produced by different peptides coisolated in the gas phase. Widely adopted proteomic data analysis environments usually fail to identify most of these spectra, succeeding at best in identifying only one of the multiple cofragmenting peptides. We present PatternLab V (PLV), an updated version of PatternLab that integrates the YADA 3 deconvolution algorithm to handle such cases efficiently. In general, we expect an increase of 10% in spectral identifications when dealing with complex proteomic samples. PLV is freely available at http://patternlabforproteomics.org.


Assuntos
Peptídeos , Proteômica , Peptídeos/análise , Proteínas/análise , Algoritmos , Espectrometria de Massas em Tandem , Bases de Dados de Proteínas , Software
2.
J Fungi (Basel) ; 9(3)2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36983553

RESUMO

Histoplasmosis is a frequent mycosis in people living with HIV/AIDS and other immunocompromised hosts. Histoplasmosis has high rates of mortality in these patients if treatment is unsuccessful. Itraconazole and amphotericin B are used to treat histoplasmosis; however, both antifungals have potentially severe pharmacokinetic drug interactions and toxicity. The present study determined the minimal inhibitory and fungicidal concentrations of mebendazole, a drug present in the NIH Clinical Collection, to establish whether it has fungicidal or fungistatic activity against Histoplasma capsulatum. Protein extracts from H. capsulatum yeasts, treated or not with mebendazole, were analyzed by proteomics to understand the metabolic changes driven by this benzimidazole. Mebendazole inhibited the growth of 10 H. capsulatum strains, presenting minimal inhibitory concentrations ranging from 5.0 to 0.08 µM. Proteomics revealed 30 and 18 proteins exclusively detected in untreated and mebendazole-treated H. capsulatum yeast cells, respectively. Proteins related to the tricarboxylic acid cycle, cytoskeleton, and ribosomes were highly abundant in untreated cells. Proteins related to the nitrogen, sulfur, and pyrimidine metabolisms were enriched in mebendazole-treated cells. Furthermore, mebendazole was able to inhibit the oxidative metabolism, disrupt the cytoskeleton, and decrease ribosomal proteins in H. capsulatum. These results suggest mebendazole as a drug to be repurposed for histoplasmosis treatment.

3.
Biomolecules ; 11(10)2021 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-34680119

RESUMO

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Jatropha/enzimologia , Modelos Moleculares , Eletricidade Estática , Sequência de Aminoácidos , Análise de Variância , Hidrolases de Éster Carboxílico/química , Domínio Catalítico , Cátions Bivalentes/farmacologia , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Proteólise/efeitos dos fármacos , Proteômica , Solventes , Estereoisomerismo , Especificidade por Substrato/efeitos dos fármacos , Temperatura
4.
Sci Rep ; 11(1): 15149, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312428

RESUMO

Sepsis results from a dyshomeostatic response to infection, which may lead to hyper or hypoimmune states. Monocytes are central regulators of the inflammatory response, but our understanding of their role in the genesis and resolution of sepsis is still limited. Here, we report a comprehensive exploration of monocyte molecular responses in a cohort of patients with septic shock via proteomic profiling. The acute stage of septic shock was associated with an impaired inflammatory phenotype, indicated by the down-regulation of MHC class II molecules and proinflammatory cytokine pathways. Simultaneously, there was an up-regulation of glycolysis enzymes and a decrease in proteins related to the citric acid cycle and oxidative phosphorylation. On the other hand, the restoration of immunocompetence was the hallmark of recovering patients, in which an upregulation of interferon signaling pathways was a notable feature. Our results provide insights into the immunopathology of sepsis and propose that, pending future studies, immunometabolism pathway components could serve as therapeutic targets in septic patients.


Assuntos
Monócitos/imunologia , Monócitos/metabolismo , Choque Séptico/sangue , Choque Séptico/imunologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos de Coortes , Citocinas/sangue , Metabolismo Energético , Feminino , Antígenos de Histocompatibilidade Classe II/sangue , Humanos , Imunidade , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteômica
5.
J Proteomics ; 245: 104282, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34089898

RESUMO

In proteomics, the identification of peptides from mass spectral data can be mathematically described as the partitioning of mass spectra into clusters (i.e., groups of spectra derived from the same peptide). The way partitions are validated is just as important, having evolved side by side with the clustering algorithms themselves and given rise to many partition assessment measures. An assessment measure is said to have a selection bias if, and only if, the probability that a randomly chosen partition scoring a high value depends on the number of clusters in the partition. In the context of clustering mass spectra, this might mislead the validation process to favor clustering algorithms that generate too many (or few) spectral clusters, regardless of the underlying peptide sequence. A selection bias toward the number of peptides is desirable for proteomics as it estimates the number of peptides in a complex protein mixture. Here, we introduce an assessment measure that is purposely biased toward the number of peptide ion species. We also introduce a partition assessment framework for proteomics, called the Partition Assessment Tool, and demonstrate its importance by evaluating the performance of eight clustering algorithms on seven proteomics datasets while discussing the trade-offs involved. SIGNIFICANCE: Clustering algorithms are widely adopted in proteomics for undertaking several tasks such as speeding up search engines, generating consensus mass spectra, and to aid in the classification of proteomic profiles. Choosing which algorithm is most fit for the task at hand is not simple as each algorithm has advantages and disadvantages; furthermore, specifying clustering parameters is also a necessary and fundamental step. For example, deciding on whether to generate "pure clusters" or fewer clusters but accepting noise. With this as motivation, we verify the performance of several widely adopted algorithms on proteomic datasets and introduce a theoretical framework for drawing conclusions on which approach is suitable for the task at hand.


Assuntos
Proteômica , Software , Algoritmos , Análise por Conglomerados , Bases de Dados de Proteínas , Viés de Seleção , Espectrometria de Massas em Tandem
6.
J Fungi (Basel) ; 6(4)2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33271902

RESUMO

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen's survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well.

7.
Front Cell Infect Microbiol ; 10: 591121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33251160

RESUMO

Histoplasmosis is one of the most frequent systemic mycosis in HIV patients. In these patients, histoplasmosis has high rates of morbidity/mortality if diagnosis and treatment are delayed. Despite its relevance, there is a paucity of information concerning the interaction between Histoplasma capsulatum and the human host, especially regarding the B-cell response, which has a direct impact on the diagnosis. Culture-based "gold-standard" methods have limitations, making immunodiagnostic tests an attractive option for clinical decisions. Despite the continuous development of those tests, improving serological parameters is necessary to make these methods efficient tools for definitive diagnosis of histoplasmosis. This includes the determination of more specific and immunogenic antigens to improve specificity and sensitivity of assays. In this study, we performed a co-immunoprecipitation assay between a protein extract from the yeast form of H. capsulatum and pooled sera from patients with proven histoplasmosis, followed by shotgun mass spectrometry identification of antigenic targets. Sera from patients with other pulmonary infections or from healthy individuals living in endemic areas of histoplasmosis were also assayed to determine potentially cross-reactive proteins. The primary structures of H. capsulatum immunoprecipitated proteins were evaluated using the DNAStar Protean 7.0 software. In parallel, the online epitope prediction server, BCPREDS, was used to complement the B-epitope prediction analysis. Our approach detected 132 reactive proteins to antibodies present in histoplasmosis patients' sera. Among these antigens, 127 were recognized also by antibodies in heterologous patients' and/or normal healthy donors' sera. Therefore, the only three antigens specifically recognized by antibodies of histoplasmosis patients were mapped as potential antigenic targets: the M antigen, previously demonstrated in the diagnosis of histoplasmosis, and the catalase P and YPS-3 proteins, characterized as virulence factors of H. capsulatum, with antigenic properties still unclear. The other two proteins were fragments of the YPS-3 and M antigen. Overlapping results obtained from the two aforementioned bioinformatic tools, 16 regions from these three proteins are proposed as putative B-cell epitopes exclusive to H. capsulatum. These data reveal a new role for these proteins on H. capsulatum interactions with the immune system and indicate their possible use in new methods for the diagnosis of histoplasmosis.


Assuntos
Infecções por HIV , Histoplasmose , Antígenos de Fungos , Epitopos de Linfócito B , Histoplasma , Histoplasmose/diagnóstico , Humanos
8.
Free Radic Biol Med ; 146: 392-401, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31760093

RESUMO

During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H2O2-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H2O2 resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.


Assuntos
Antioxidantes , Trypanosomatina , Animais , Glicólise , Humanos , Peróxido de Hidrogênio , Simbiose
9.
BMC Cancer ; 19(1): 365, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30999875

RESUMO

BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method. RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fluido do Aspirado de Mamilo/metabolismo , Proteoma/análise , Proteômica/métodos , Microambiente Tumoral , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Prognóstico , Fluxo de Trabalho
10.
Biotechnol Biofuels ; 11: 226, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151054

RESUMO

BACKGROUND: ß-Glucosidases are components of the cellulase system, a family of enzymes that hydrolyze the ß-1,4 linkages of cellulose. These proteins have been extensively studied due to the possibility of their use in various biotechnological processes. They have different affinities for substrates (depending on their source) and their activities can be used for saccharification of different types of biomass. In this context, the properties and the synergistic capacity of ß-glucosidases from different organisms, to supplement the available commercial cellulase cocktails, need a comprehensive evaluation. RESULTS: Two ß-glucosidases belonging to GH3 family were secreted by Penicillium citrinum UFV. PcßGlu1 (241 kDa) and PcßGlu2 (95 kDa) presented acidic and thermo-tolerant characteristics. PcßGlu1 showed Michaelis-Menten kinetics for all substrates tested with Km values ranging from 0.09 ± 0.01 (laminarin) to 1.7 ± 0.1 mM (cellobiose, C2) and kcat values ranging from 0.143 ± 0.005 (laminarin) to 8.0 ± 0.2 s-1 (laminaribiose, Lb). PcßGlu2 showed substrate inhibition for 4-methylumbelliferyl-ß-d-glucopyranoside (MUßGlu), p-nitrophenyl-ß-d-glucopyranoside (pNPßGlu), cellodextrins (C3, C4, and C5), N-octil-ß-d-glucopyranoside, and laminaribiose, with Km values ranging from 0.014 ± 0.001 (MUßGlu) to 0.64 ± 0.06 mM (C2) and kcat values ranging from 0.49 ± 0.01 (gentiobiose) to 1.5 ± 0.2 s-1 (C4). Inhibition constants (Ki) for PcßGlu2 substrate inhibition ranged from 0.69 ± 0.07 (MUßGlu) to 10 ± 1 mM (Lb). Glucose and cellobiose are competitive inhibitors of PcßGlu1 and PcßGlu2 when pNPßGlu is used as a substrate. For PcßGlu1 inhibition, Ki = 1.89 ± 0.08 mM (glucose) and Ki = 3.8 ± 0.1 mM (cellobiose); for PcßGlu2, Ki = 0.83 ± 0.05 mM (glucose) and Ki = 0.95 ± 0.07 mM (cellobiose). The enzymes were tested for saccharification of different biomasses, individually or supplementing a Trichoderma reesei commercial cellulose preparation. PcßGlu2 was able to hydrolyze banana pseudostem and coconut fiber with the same efficiency as the T. reesei cocktail, showing significant synergistic properties with T. reesei enzymes in the hydrolysis of these alternative biomasses. CONCLUSIONS: The ß-glucosidases from P. citrinum UFV1 present different enzymatic properties from each other and might have potential application in several biotechnological processes, such as hydrolysis of different types of biomass.

11.
Toxins (Basel) ; 10(3)2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29533989

RESUMO

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.


Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Glândulas Exócrinas/metabolismo , Animais , Bothrops/anatomia & histologia , Glândulas Exócrinas/anatomia & histologia , Glândulas Exócrinas/ultraestrutura , Feminino , Microscopia Eletrônica de Transmissão , Proteômica , Proteínas de Répteis/metabolismo
12.
Toxins (Basel) ; 10(2)2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29415440

RESUMO

Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson's disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents.


Assuntos
Venenos de Crotalídeos/farmacologia , Descoberta de Drogas , Animais , Bothrops , Venenos de Crotalídeos/uso terapêutico , Humanos , Células MCF-7 , Transcriptoma/efeitos dos fármacos
13.
J Proteomics ; 177: 137-147, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29325991

RESUMO

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.


Assuntos
Bothrops/fisiologia , Venenos de Crotalídeos/biossíntese , Proteoma/análise , Animais , Venenos de Crotalídeos/antagonistas & inibidores , Glândulas Exócrinas/química , Perfilação da Expressão Gênica , Metaloproteases/antagonistas & inibidores , Metaloproteases/biossíntese , Metaloproteases/metabolismo , Inibidores de Fosfolipase A2/metabolismo , Fosfolipases A2/biossíntese , Fosfolipases A2/metabolismo
14.
J Proteomics, v. 177, p. 137-147, abr. 2018
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2506

RESUMO

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

15.
Toxins, v. 10, n. 3, 121, mar. 2018
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2479

RESUMO

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct’s epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins—mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct’s toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

16.
J. Proteomics ; 177: p. 137-147, 2018.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15262

RESUMO

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

17.
Toxins ; 10(3): 121, 2018.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15149

RESUMO

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

18.
Toxicon ; 133: 1-9, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28414035

RESUMO

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.


Assuntos
Bothrops , Venenos de Crotalídeos/biossíntese , Norepinefrina/farmacologia , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Animais , Células Cultivadas , Venenos de Crotalídeos/metabolismo , Feminino , Metaloproteases , Proteômica , Glândulas Salivares/metabolismo
19.
J. Proteomics ; 151: 214-231, 2017.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib13586

RESUMO

A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents. Biological Significance: Our proteopeptidomic strategy yielded unprecedented insights into the remarkable diversity of B. jararaca venom composition, both at the peptide and protein levels. These results bring a substantial contribution to the actual pursuit of large-scale protein-level assignment in snake venomics. The detection of typical elapidic venom components, in a Viperidae venom, reinforces our view that the use of this approach (hand in-hand with transcriptomic and genomic data) for venom proteomic analysis, at the specimen-level, can greatly contribute for venom toxin evolution studies. Furthermore, data were generated in support of a previous hypothesis that venom gland secretory vesicles are specialized forms of lysosomes. Two testable hypotheses also emerge from the results of this work. The first is that a nucleobindin-2-derived protein could lead to prey disorientation during envenomation, aiding in its capture by the snake. The other being that the venom's peptidome might contain a population of cryptides, whose biological activities could lead to the development of new therapeutical agents.

20.
Toxicon ; 133: 1-9, 2017.
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15129

RESUMO

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.

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