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In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.
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Leishmania mexicana , Proteínas de Protozoários , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Leishmania mexicana/genética , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , ImunoprecipitaçãoRESUMO
We describe a strategy for the development of a rational approach of neoplastic disease therapy based on the demonstration that scale-free networks are susceptible to specific attacks directed against its connective hubs. This strategy involves the (i) selection of up-regulated hubs of connectivity in the tumors interactome, (ii) drug repurposing of these hubs, (iii) RNA silencing of non-druggable hubs, (iv) in vitro hub validation, (v) tumor-on-a-chip, (vi) in vivo validation, and (vii) clinical trial. Hubs are protein targets that are assessed as targets for rational therapy of cancer in the context of personalized oncology. We confirmed the existence of a negative correlation between malignant cell aggressivity and the target number needed for specific drugs or RNA interference (RNAi) to maximize the benefit to the patient's overall survival. Interestingly, we found that some additional proteins not generally targeted by drug treatments might justify the addition of inhibitors designed against them in order to improve therapeutic outcomes. However, many proteins are not druggable, or the available pharmacopeia for these targets is limited, which justifies a therapy based on encapsulated RNAi.
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Neoplasias , Mapeamento de Interação de Proteínas , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.
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Proteínas , Proteômica , Biotinilação , Poro Nuclear , Proteínas/química , Proteômica/métodos , Estreptavidina/químicaRESUMO
Lung cancer patients with COVID-19 present an increased risk of developing severe disease and, consequently, have poor outcomes. Determining SARS-CoV-2-host interactome in lung cancer cells and tissues, infected or uninfected with SARS-CoV-2, may reveal molecular mechanisms associated with COVID-19 development and severity in lung cancer patients. Here, we integrated transcriptome data of lung tumors from patients with small- or non-small cell lung cancer (SCLC and NSCLC) and normal lung and lung cancer cells infected with SARS-CoV-2. We aimed to characterize molecular mechanisms potentially associated with COVID-19 development and severity in lung cancer patients and to predict the SARS-CoV-2-host cell interactome. We found that the gene expression profiles of lung cell lines infected with SARS-CoV-2 resemble more primary lung tumors than non-malignant lung tissues. In addition, the transcriptomic-based interactome analysis of SCLC and NSCLC revealed increased expression of cancer genes BRCA1 and CENPF, whose proteins are known or predicted to interact with the SARS-CoV-2 spike glycoprotein and helicase, respectively. We also found that TRIB3, a gene coding a putative host-SARS-CoV-2 interacting protein associated with COVID-19 infection, is co-expressed with the up-regulated genes MTHFD2, ADM2, and GPT2 in all tested conditions. Our analysis identified biological processes such as amino acid metabolism and angiogenesis and 22 host mediators of SARS-CoV-2 infection and replication that may contribute to the development and severity of COVID-19 in lung cancers.
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COVID-19 , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Transcriptoma , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , COVID-19/genética , COVID-19/patologia , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , SARS-CoV-2RESUMO
BACKGROUND: Arylacetamide deacetylase (AADAC) is a lipolytic enzyme involved in xenobiotic metabolism. The characterization in terms of activity and substrate preference has been limited to a few mammalian species. The potential role and catalytic activities of AADAC from other organisms are still poorly understood. Therefore, in this work, the physicochemical properties, proteomic analysis, and protein-protein interactions from Gnathostomata organisms were investigated. RESULTS: The analysis were performed with 142 orthologue sequences with ~ 48-100% identity with human AADAC. The catalytic motif HGG[A/G] tetrapeptide block was conserved through all AADAC orthologues. Four variations were found in the consensus pentapeptide GXSXG sequence (GDSAG, GESAG, GDSSG, and GSSSG), and a novel motif YXLXP was found. The prediction of N-glycosylation sites projected 4, 1, 6, and 4 different patterns for amphibians, birds, mammals, and reptiles, respectively. The transmembrane regions of AADAC orthologues were not conserved among groups, and variations in the number and orientation of the active site and C-terminal carboxyl were observed among the sequences studied. The protein-protein interaction of AADAC orthologues were related to cancer, lipid, and xenobiotic metabolism genes. CONCLUSION: The findings from this computational analysis offer new insight into one of the main enzymes involved in xenobiotic metabolism from mammals, reptiles, amphibians, and birds and its potential use in medical and veterinarian biotechnological approaches.
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Viroids are the smallest pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling and hormone-related processes. Strikingly, a member of the bHLH-TF family has a specific potential role as a microprotein involved in the post-translational regulation of hormone signaling events. We found that ERF-MTRs are characteristic of severe symptoms, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.
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Solanum lycopersicum , Viroides , Hormônios , Solanum lycopersicum/metabolismo , Doenças das Plantas/genética , RNA Viral/genética , Fatores de Transcrição/genética , Viroides/genéticaRESUMO
Post-transcriptional gene regulation in Trypanosoma cruzi, the etiological agent of Chagas disease, plays a critical role in ensuring that the parasite successfully completes its life cycle in both of its obligate hosts: insect vector and mammals. This regulation is basically governed by RNA binding proteins (RBPs) through their interactions with cis-elements located in the UTRs of their mRNA targets. LYT1 gene, coding for a virulence factor of T. cruzi, is expressed into two isoforms: kLYT1 and mLYT1, which play different functions according to their cellular location and parasite life-cycle stages. Whereas kLYT1 exhibits a regulatory role during the epimastigote-to-metacyclic trypomastigote stage transition, mLYT1 acts as a pore-forming protein, relevant for host cell invasion and parasite intracellular survival. Considering the LYT1 biological relevance and the fact that this is a protein exclusive of T. cruzi, the protein and its mechanisms regulating the alternative gene expression products are promising targets for therapeutic intervention. In this work, an experimental approach consisting of pull-downs assays followed by proteomic analyzes was carried out to identify the proteins interacting with the different LYT1 mRNAs. The dataset presented here was obtained through three biological replicates using all the different UTRs characterized in the LYT1 mRNAs (i.e., 5´UTR kLYT1, 5´UTR mLYT1, and I and II-type 3´UTRs) as baits, and protein extracts from epimastigotes and trypomastigotes of the 058 PUJ (DTU I) strain. Bound proteins were analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). As a control of non-specificity, the same protein extracts were incubated with Leishmania braziliensis rRNA and the bound proteins also identified by LC/MS. In all, 1,557 proteins were identified, 313 of them were found in at least two replicates and 18 proteins were exclusively associated with the LYT1 baits. Of these, six proteins have motifs related to RNA binding, and seven remain annotated as hypothetical proteins. Remarkably, three of these hypothetical proteins also contain nucleic acid binding motifs. This knowledge, beside expanding the known T. cruzi proteome, gains insight into putative regulatory proteins responsible for alternative LYT1 mRNAs processing. Raw mass spectrometry data are available via MassIVE proteome Xchange with identifier PXD027371.
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Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare X-linked recessive disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which activates intracellular accumulation of nonmetabolized glycosaminoglycans such as heparan sulfate and dermatan sulfate. This accumulation causes severe damage to several tissues, principally the central nervous system. Previously, we identified 187 IDS-protein interactions in the mouse brain. To validate a subset of these interactions, we selected and cloned the coding regions of 10 candidate genes to perform a targeted yeast two-hybrid assay. The results allowed the identification of the physical interaction of IDS with LSAMP and SYT1. Although the physiological relevance of these complexes is unknown, recent advances allow us to point out that these interactions could be involved in vesicular trafficking of IDS through the interaction with SYT1, as well as to the ability to form a transcytosis module between the cellular components of the blood-brain-barrier (BBB) through its interaction with LSAMP. These results may shed light on the role of IDS on cellular homeostasis and may also contribute to the understanding of MPS II physiopathology and the development of novel therapeutic strategies to transport recombinant IDS through the brain endothelial cells toward the brain parenchyma.
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Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of 20 Leishmania deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCE Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.
Assuntos
Leishmania mexicana/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Transcriptoma , Leishmania mexicana/metabolismo , Espectrometria de Massas , Fases de Leitura Aberta , Ligação Proteica , Proteômica , Proteínas de Protozoários/metabolismo , RNA de Protozoário/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Noncoding RNAs (ncRNAs) play prominent roles in the regulation of gene expression via their interactions with other biological molecules such as proteins and nucleic acids. Although much of our knowledge about how these ncRNAs operate in different biological processes has been obtained from experimental findings, computational biology can also clearly substantially boost this knowledge by suggesting possible novel interactions of these ncRNAs with other molecules. Computational predictions are thus used as an alternative source of new insights through a process of mutual enrichment because the information obtained through experiments continuously feeds through into computational methods. The results of these predictions in turn shed light on possible interactions that are subsequently validated experimentally. This review describes the latest advances in databases, bioinformatic tools, and new in silico strategies that allow the establishment or prediction of biological interactions of ncRNAs, particularly miRNAs and lncRNAs. The ncRNA species described in this work have a special emphasis on those found in humans, but information on ncRNA of other species is also included.
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Biologia Computacional/métodos , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Animais , Bases de Dados Genéticas , Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/análise , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/análise , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Análise de Sequência de RNA/métodosRESUMO
The cellular and molecular mechanisms underlying neuropsychiatric and neurodevelopmental disorders show that most of them can be categorized as synaptopathies-or damage of synaptic function and plasticity. Synaptic formation and maintenance are orchestrated by protein complexes that are in turn regulated in space and time during neuronal development allowing synaptic plasticity. However, the exact mechanisms by which these processes are managed remain unknown. Large-scale genomic and proteomic projects led to the discovery of new molecules and their associated variants as disease risk factors. Neuronal glycoprotein M6a, encoded by the GPM6A gene is emerging as one of these molecules. M6a has been involved in neuron development and synapse formation and plasticity, and was also recently proposed as a gene-target in various neuropsychiatric disorders where it could also be used as a biomarker. In this review, we provide an overview of the structure and molecular mechanisms by which glycoprotein M6a participates in synapse formation and maintenance. We also review evidence collected from patients carrying mutations in the GPM6A gene; animal models, and in vitro studies that together emphasize the relevance of M6a, particularly in synapses and in neurological conditions.
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Microdeletion syndromes (MSs) are a heterogeneous group of genetic diseases that can virtually affect all functions and organs in humans. Although systems biology approaches integrating multiomics and database information into biological networks have expanded our knowledge of genetic disorders, cytogenomic network-based analysis has rarely been applied to study MSs. In this study, we analyzed data of 28 MSs, using network-based approaches, to investigate the associations between the critical chromosome regions and the respective underlying biological network systems. We identified MSs-associated proteins that were organized in a network of linked modules within the human interactome. Certain MSs formed highly interlinked self-contained disease modules. Furthermore, we observed disease modules involving proteins from other disease groups in the MSs interactome. Moreover, analysis of integrated data from 564 genes located in known chromosomal critical regions, including those contributing to topological parameters, shared pathways, and gene-disease associations, indicated that complex biological systems and cellular networks may underlie many genotype to phenotype associations in MSs. In conclusion, we used a network-based analysis to provide resources that may contribute to better understanding of the molecular pathways involved in MSs.
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Cromossomos , Redes Reguladoras de Genes , Genótipo , Humanos , Fenótipo , SíndromeRESUMO
Leishmania are protozoan parasites responsible for leishmaniasis. These parasites present a precise gene regulation that allows them to survive different environmental conditions during their digenetic life cycle. This adaptation depends on the regulation of the expression of a wide variety of genes, which occurs, mainly at the post-transcriptional level. This differential gene expression is achieved by mechanisms based mainly in RNA binding proteins that regulate the translation and/or stability of mRNA targets by interaction with cis elements principally located in the untranslated regions (UTR). In recent studies, our group identified and characterized two proteins, SCD6 and RBP42, as RNA binding proteins in Leishmania braziliensis. To find clues about the cellular processes in which these proteins are involved, this work was aimed to determine the SCD6- and RBP42-interacting proteins (interactome) in L. braziliensis promastigotes. For this purpose, after an in vivo UV cross-linking, cellular extracts were used to immunoprecipitated, by specific antibodies, protein complexes in which SCD6 or RBP42 were present. Protein mass spectrometry analysis of the immunoprecipitated proteins identified 96 proteins presumably associated with SCD6 and 173 proteins associated with RBP42. Notably, a significant proportion of the identified proteins were shared in both interactomes, indicating a possible functional relationship between SCD6 and RBP42. Remarkably, many of the proteins identified in the SCD6 and RBP42 interactomes are related to RNA metabolism and translation processes, and many of them have been described as components of ribonucleoprotein (RNP) granules in Leishmania and related trypanosomatids. Thus, these results support a role of SCD6 and RBP42 in the assembly and/or function of mRNA-protein complexes, participating in the fate (decay/accumulation/translation) of L. braziliensis transcripts. SIGNIFICANCE: Parasites of the Leishmania genus present a particular regulation of gene expression, operating mainly at the post-transcriptional level, surely aimed to modulate quickly both mRNA and protein levels to survive the sudden environmental changes that occur during a parasite's life cycle as it moves from one host to another. This regulation of gene expression processes would be governed by the interaction of mRNA with RNA binding proteins. Nevertheless, the entirety of protein networks involved in these regulatory processes is far from being understood. In this regard, our work is contributing to stablish protein networks in which the L. braziliensis SCD6 and RBP42 proteins are involved; these proteins, in previous works, have been described as RNA binding proteins and found to participate in gene regulation in different cells and organisms. Additionally, our data point out a possible functional relationship between SCD6 and RBP42 proteins as constituents of mRNA granules, like processing bodies or stress granules, which are essential structures in the regulation of gene expression. This knowledge could provide a new approach for the development of therapeutic targets to control Leishmania infections.
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Leishmania braziliensis , Regulação da Expressão Gênica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Neurological and neuropsychiatric disorders are mediated by several pathophysiological mechanisms, including developmental and degenerative abnormalities caused primarily by disturbances in cell migration, structural plasticity of the synapse, and blood-vessel barrier function. In this context, critical pathways involved in the pathogenesis of these diseases are related to structural, scaffolding, and enzymatic activity-bearing proteins, which participate in Ca2+- and Ras Homologs (Rho) GTPases-mediated signaling. Rho GTPases are GDP/GTP binding proteins that regulate the cytoskeletal structure, cellular protrusion, and migration. These proteins cycle between GTP-bound (active) and GDP-bound (inactive) states due to their intrinsic GTPase activity and their dynamic regulation by GEFs, GAPs, and GDIs. One of the most important upstream inputs that modulate Rho GTPases activity is Ca2+ signaling, positioning ion channels as pivotal molecular entities for Rho GTPases regulation. Multiple non-selective cationic channels belonging to the Transient Receptor Potential (TRP) family participate in cytoskeletal-dependent processes through Ca2+-mediated modulation of Rho GTPases. Moreover, these ion channels have a role in several neuropathological events such as neuronal cell death, brain tumor progression and strokes. Although Rho GTPases-dependent pathways have been extensively studied, how they converge with TRP channels in the development or progression of neuropathologies is poorly understood. Herein, we review recent evidence and insights that link TRP channels activity to downstream Rho GTPase signaling or modulation. Moreover, using the TRIP database, we establish associations between possible mediators of Rho GTPase signaling with TRP ion channels. As such, we propose mechanisms that might explain the TRP-dependent modulation of Rho GTPases as possible pathways participating in the emergence or maintenance of neuropathological conditions.
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Chagas disease, a zoonosis caused by the flagellate protozoan Trypanosoma cruzi, is a chronic and systemic parasitic infection that affects ~5-7 million people worldwide, mainly in Latin America. Chagas disease is an emerging public health problem due to the lack of vaccines and effective treatments. According to recent studies, several T. cruzi secreted proteins interact with the human host during cell invasion. Moreover, some comparative studies with T. rangeli, which is non-pathogenic in humans, have been performed to identify proteins directly involved in the pathogenesis of the disease. In this study, we present an integrated analysis of canonical putative secreted proteins (PSPs) from both species. Additionally, we propose an interactome with human host and gene family clusters, and a phylogenetic inference of a selected protein. In total, we identified 322 exclusively PSPs in T. cruzi and 202 in T. rangeli. Among the PSPs identified in T. cruzi, we found several trans-sialidases, mucins, MASPs, proteins with phospholipase 2 domains (PLA2-like), and proteins with Hsp70 domains (Hsp70-like) which have been previously characterized and demonstrated to be related to T. cruzi virulence. PSPs found in T. rangeli were related to protozoan metabolism, specifically carboxylases and phosphatases. Furthermore, we also identified PSPs that may interact with the human immune system, including heat shock and MASP proteins, but in a lower number compared to T. cruzi. Interestingly, we describe a hypothetical hybrid interactome of PSPs which reveals that T. cruzi secreted molecules may be down-regulating IL-17 whilst T. rangeli may enhance the production of IL-15. These results will pave the way for a better understanding of the pathophysiology of Chagas disease and may ultimately lead to the identification of molecular targets, such as key PSPs, that could be used to minimize the health outcomes of Chagas disease by modulating the immune response triggered by T. cruzi infection.
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Doença de Chagas/parasitologia , Proteoma , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma rangeli/metabolismo , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Biologia Computacional , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes , Genômica , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Mapas de Interação de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Via Secretória , Transdução de Sinais , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Trypanosoma rangeli/genética , Trypanosoma rangeli/imunologiaRESUMO
Echinococcus granulosus sensu lato (s.l.) is a cestode parasite affecting both human and livestock health. Recombinant ectodomains of human scavenger receptors CD5 (rshCD5) and CD6 (rshCD6) were previously reported to bind its tegumental antigens and to exert prophylactic effects in a murine model of infection. Although the properties of mammalian scavenger receptors include the binding to diverse pathogen-derived structures, their interaction with helminth parasites has been scarcely explored. Therefore, we report here a search for CD5 and CD6 interactors within E. granulosus s.l. antigens. Mass spectrometry analysis of pull-downs from soluble tegumental components with biotinylated rshCD5 and rshCD6 resulted in 17 and 11 overrepresented interactors, respectively, 8 of which were shared. The interactors included previously reported protective molecules against E. granulosus s.l. and/or other helminths. Similar studies performed with 11-mer peptides mapping to each of the three extracellular scavenger domains of CD5 and CD6 allowed an estimated molecular topology of the interactions. In conclusion, the fact that most helminth interactors identified for rshCD5 and rshCD6 were already reported as vaccine candidates or pharmacological targets against different helminthiases, supports the view that their beneficial effects in experimental infection results from binding to multiple relevant tegumental antigens.
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Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos CD5/genética , Equinococose/parasitologia , Echinococcus granulosus/genética , Animais , Equinococose/genética , Echinococcus granulosus/patogenicidade , Genótipo , Helmintos/genética , Helmintos/parasitologia , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas/genéticaRESUMO
Knowledge about the molecular basis of SARS-CoV-2 infection is incipient. However, recent experimental results about the virus interactome have shown that this single-positive stranded RNA virus produces a set of about 28 specific proteins grouped into 16 non-structural proteins (Nsp1 to Nsp16), four structural proteins (E, M, N, and S), and eight accessory proteins (orf3a, orf6, orf7a, orf7b, orf8, orf9b, orf9c, and orf10). In this brief communication, the network model of the interactome of these viral proteins with the host proteins is analyzed. The statistical analysis of this network shows that it has a modular scale-free topology in which the virus proteins orf8, M, and Nsp7 are the three nodes with the most connections (links). This result suggests the possibility that a simultaneous pharmacological attack on these hubs could assure the destruction of the network and the elimination of the virus.
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(1) Background: voltage-gated sodium channels (Navs) are integral membrane proteins that allow the sodium ion flux into the excitable cells and initiate the action potential. They comprise an α (Navα) subunit that forms the channel pore and are coupled to one or more auxiliary ß (Navß) subunits that modulate the gating to a variable extent. (2) Methods: after performing homology in silico modeling for all nine isoforms (Nav1.1α to Nav1.9α), the Navα and Navß protein-protein interaction (PPI) was analyzed chemometrically based on the primary and secondary structures as well as topological or spatial mapping. (3) Results: our findings reveal a unique isoform-specific correspondence between certain segments of the extracellular loops of the Navα subunits. Precisely, loop S5 in domain I forms part of the PPI and assists Navß1 or Navß3 on all nine mammalian isoforms. The implied molecular movements resemble macroscopic springs, all of which explains published voltage sensor effects on sodium channel fast inactivation in gating. (4) Conclusions: currently, the specific functions exerted by the Navß1 or Navß3 subunits on the modulation of Navα gating remain unknown. Our work determined functional interaction in the extracellular domains on theoretical grounds and we propose a schematic model of the gating mechanism of fast channel sodium current inactivation by educated guessing.
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Aminoácidos/química , Modelos Moleculares , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/químicaRESUMO
BACKGROUND: Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby inducing apoptosis via mitochondrial pathway activation through the modulation in the expression of the Bcl-2 family. METHODS: To describe topological features of the MTAs networks associated to intrinsic apoptosis induction in p53-null prostate cancer cells, we predicted and compared the interactomes and topological properties of Paclitaxel and Vincristine, and thus, the essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics were subjected to experimental analysis in PC-3 cell line. RESULTS: The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX, and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells exposed to both treatments. Immunoprecipitation analysis showed that treatments induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the role of BimEL as a de-repressor from sequestering complexes, in addition, new protein complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing data to the Vincristine network for p53-null cells in response to MTAs. CONCLUSION: The differences in sensitivities, protein profiles, and protein complex kinetics observed between the drugs confirmed that the selectivity and stimulation of the apoptotic system vary depending on the cell's genotype, the drug used and its exposure period.
Assuntos
Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Vincristina/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células PC-3/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Traditional approaches to cancer therapy seek common molecular targets in tumors from different patients. However, molecular profiles differ between patients, and most tumors exhibit inherent heterogeneity. Hence, imprecise targeting commonly results in side effects, reduced efficacy, and drug resistance. By contrast, personalized medicine aims to establish a molecular diagnosis specific to each patient, which is currently feasible due to the progress achieved with high-throughput technologies. In this report, we explored data from human RNA-seq and protein-protein interaction (PPI) networks using bioinformatics to investigate the relationship between tumor entropy and aggressiveness. To compare PPI subnetworks of different sizes, we calculated the Shannon entropy associated with vertex connections of differentially expressed genes comparing tumor samples with their paired control tissues. We found that the inhibition of up-regulated connectivity hubs led to a higher reduction of subnetwork entropy compared to that obtained with the inhibition of targets selected at random. Furthermore, these hubs were described to be participating in tumor processes. We also found a significant negative correlation between subnetwork entropies of tumors and the respective 5-year survival rates of the corresponding cancer types. This correlation was also observed considering patients with lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) based on the clinical data from The Cancer Genome Atlas database (TCGA). Thus, network entropy increases in parallel with tumor aggressiveness but does not correlate with PPI subnetwork size. This correlation is consistent with previous reports and allowed us to assess the number of hubs to be inhibited for therapy to be effective, in the context of precision medicine, by reference to the 100% patient survival rate 5 years after diagnosis. Large standard deviations of subnetwork entropies and variations in target numbers per patient among tumor types characterize tumor heterogeneity.