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1.
J Proteome Res ; 21(11): 2783-2797, 2022 11 04.
Article in English | MEDLINE | ID: mdl-36260604

ABSTRACT

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.


Subject(s)
Spider Venoms , Spiders , Animals , Male , Female , Spiders/genetics , Spiders/metabolism , Spider Venoms/genetics , Spider Venoms/chemistry , Spider Venoms/metabolism , Cysteine/metabolism , Proteomics/methods , Mass Spectrometry/methods , Proteome/genetics , Proteome/metabolism , Peptides/analysis
2.
São Paulo; 2022. 119 p.
Thesis in Portuguese | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4870

ABSTRACT

The process of discovering and developing new drugs costs billions of dollars and can take more than ten years. The search for new drugs begins with some form of screening of libraries of chemical compounds that often contain a variety of natural products. Venoms contain toxins with extremely specific functions, therefore being a secretion of great potential in the search for new bioactive compounds. In the era of Next Generation Sequencing and shotgun proteomics, a large number of data of toxins and venom protein sequences from venomous animals have been generated. Thus, there is great interest in applying computational methodologies to facilitate parts of the screening of peptide and proteins for biological activity. To assist in the characterization and search for new toxins and antiviral peptides in arachnid venoms, two computational methods were developed: ArachnoFamTox and EnAVPClass. ArachnoFamTox performs the prediction and classification of arachnid toxins and venom proteins with an approach that uses evolutionary conservation information with Position Specific Scoring Matrices and Hidden Markov Models and is the most specific predictor of toxins among the existing methods and one of the few that classify toxins in families. EnAVPClass predicts antiviral peptides and classifies them according to their mechanism of action. The method uses an approach based on Supervised Machine Learning with Random Forest and Support Vector Machines models and Deep Learning with Long Short Term Memory neural networks. As an example of application of the developed tools, transcriptomes of seven tick species were processed and assembled de novo and the ArachnoFamTox and EnAVPClass tools were used for the prediction and classification of toxins and prediction of Antivirals. A total of 2.069 toxins and venom proteins classified in 13 different families were identified and 47.160 peptides were generated, of which 3.559 antiviral peptides (7.54%) were predicted. Of these, 1.765 were identified as Antivirals that act on virus membranes, 755 on viral replication and 64 on viral assembly. The results show the importance of ticks as promising organisms in the discovery of new antiviral compounds based on their saliva and venom proteins. The tools developed in this work will be available as packages open source in https://github.com/yutakajr after publication and will help in the process of discovering new drugs in silico, classifying families of toxins in arachnids and in the development of new tools.


O processo de descoberta e desenvolvimento de novas drogas tem um custo de bilhões de dólares e pode durar décadas. A procura por novas drogas começa com alguma forma de screening de bibliotecas de compostos químicos que geralmente contém uma variedade de produtos naturais. Os venenos contém toxinas com funções extremamente específicas sendo uma secreção de grande potencial na procura por novos compostos bioativos. Na era do Sequenciamento de Nova Geração e proteômica shotgun, é gerado um grande número de dados de sequências de toxinas e proteínas de veneno de animais venenosos. Assim há um grande interesse em aplicar metodologias computacionais para agilizar partes do processo de screening de sequências de peptídeos e proteínas que possam ter atividade biológica e potencial para serem desenvolvidos novos medicamentos. Para auxiliar na caracterização e busca por novas toxinas e peptídeos antivirais em venenos de aracnídeos, foram desenvolvidos dois métodos computacionais: o ArachnoFamTox e o EnAVPClass. O ArachnoFamTox realiza a predição e classificação de toxinas e proteínas de veneno de aracnídeos com uma abordagem que utiliza informações da conservação evolutiva com Position Specific Scoring Matrices e Hidden Markov Models e é o preditor de toxinas com maior especificidade dentre os métodos existentes e um dos únicos a classificar em famílias de toxinas. O EnAVPClass faz a predição de peptídeos antivirais e classifica-os de acordo com seu mecanismo de ação. O método utiliza uma abordagem baseada em Aprendizagem de Máquina Clássico com modelos de Random Forest e Support Vector Machines e Aprendizagem Profunda com redes neurais do tipo Long Short Term Memory. Como exemplo de aplicação das ferramentas desenvolvidas, foram processados e montados de novo transcriptomas de sete espécies de carrapatos e utilizadas as ferramentas ArachnoFamTox e EnAVPClass para a predição e classificação de toxinas e predição de Antivirais. Foram identificadas 2.069 toxinas e proteínas de veneno classificadas em 13 diferentes famílias e gerados 47.160 peptídeos dos quais foram preditos 3.559 peptídeos antivirais (7.54%). Desses, 1.765 foram identificados como Antivirais que agem nas membranas dos vírus, 755 na replicação viral e 64 na montagem viral. Os resultados evidenciam a importância dos carrapatos como organismos promissores na descoberta de novos compostos antivirais baseados em suas proteínas de saliva e veneno. As ferramentas desenvolvidas neste trabalho ficarão disponíveis como pacotes open source https://github.com/yutakajr após publicação e auxiliarão no processo de descoberta de novas drogas in silico, classificação de famílias de toxinas em aracnídeos e no desenvolvimento de novas ferramentas.

3.
J Proteome Res, v. 21, p. 2783-2797, out. 2022
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4567

ABSTRACT

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.

4.
Cell Microbiol ; 23(4): e13295, 2021 04.
Article in English | MEDLINE | ID: mdl-33222354

ABSTRACT

Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.


Subject(s)
Cyclophilins/metabolism , Cytosol/metabolism , Host-Parasite Interactions , Reactive Oxygen Species/metabolism , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/metabolism , Animals , Cyclophilins/biosynthesis , Cyclophilins/genetics , Cytosol/chemistry , Myoblasts/parasitology , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rats , Trypanosoma cruzi/genetics
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