Navegando por Autor "Pereira, Roberta Verciano"
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Item Characterisation of major vault protein during the life cycle of the human parasite Schistosoma mansoni.(2014) Reis, Eneida Virgínia; Pereira, Roberta Verciano; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Babá, Élio Hideo; Coelho, Paulo Marcos Zech; Mattos, Ana Carolina Alves de; Couto, Flávia Fernanda Búbula; Borges, William de Castro; Cota, Renata Guerra de SáVaults are ribonucleoproteins (13MDa) highly conserved among lower and higher eukaryotes. Their association produces a complex composed of three proteins named Major Vault Protein (MVP), vault (PolyADP-ribose) polymerase (VPARP) and Telomerase-associated protein (TEP1), plus a small untranslated RNA. The exact function of this complex is unknown, although the biological role of vaults has been associated with multidrug resistance phenotypes and signal transduction pathways. Genomic analysis showed that model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, do not possess genes encoding vaults. However, we have found that vault-related genes are present in the Schistosoma mansoni genome. These observations raised questions on the involvement of vaults in mechanisms of adaptation of the parasite in its mammalian host. Therefore, molecular characterisation of the putative Major Vault Protein performed using bioinformatics tools showed that this vault component is highly conserved in S. mansoni. The MVP expression level was quantified by qRT-PCR using total RNA from susceptible (LE) and resistant (LE-PZQ) adult worm lineages, cercariae and mechanically transformed schistosomula(MTS) cultured for 3.5, 24, 48 and 72h in vitro. Our results suggest a stage-specific expression in all developmental stages analysed. Western blotting has shown upregulation of SmMVP in the MTS-3.5, 72 h and resistant adult worms, and similar levels in all other stages. Furthermore, SmMVPwas found differentially expressed in adult males and females fromthe susceptible lineage. Further studies should clarify whether SmMVP is somehow linked to drug resistance in S. mansoni.Item Characterisation of the COP9 signalosome in Schistosoma mansoni parasites.(2013) Pereira, Roberta Verciano; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáThe COP9 signalosome (CSN) is an eight-subunit complex found in all eukaryotes and shares structural features with both the 26S proteasome ‘lid’ and translation factor eIF3. Recent data have demonstrated that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). CSN controls substrate ubiquitination by cullin-RING Ub ligases, a step which determines substrate specificity of the UPS. Here, we reconstructed the CSN complex in Schistosoma mansoni and identified eight homologous components. Among these homologues, five subunits were predicted with their full-length sequences. Phylogenetic analysis confirmed the evolutionary conservation and the architecture of CSN, as well as the 26S proteasome ‘lid’. We performed quantitative reverse transcrip tion-polymerase chain reaction to detect the expression of the SmCSN transcripts. The Smcsn1, Smcsn2, Smcsn3, Smcsn4, Smcsn5, Smcsn6, Smcsn7 and Smcsn8 genes were upregulated in adult worms compared to cercariae, and the expression levels were similar to that of in vitro cultivated schistosomula. Taken together, these results suggest that the CSN complex may be important during cercariae, schistosome and adult worm development and might explain, at least in part, the differences among UPSs during the parasite life cycle.Item Characterization of export receptor exportins (XPOs) in the parasite Schistosoma mansoni.(2013) Abreu, Fabiano Carlos Pinto de; Pereira, Roberta Verciano; Oliveira, Victor Fernandes de; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáSeveral proteins and different species of RNA that are produced in the nucleus are exported through the nuclear pore complexes, which require a family of conserved nuclear export receptors called exportins (XPOs). It has been reported that the XPOs (XPO1, XPO5, and XPOT) are directly involved in the transport processes of noncoding RNAs from the nucleus to the cytoplasm and/or from cytoplasm to the nucleus. All three genes are present in fungi, plants, and deuterostome metazoans. However, protostome metazoan species lack one of the three genes across evolution. In this report, we have demonstrated that all three XPO proteins are present in the parasite protostome Schistosoma mansoni . As this parasite has a complex life cycle presenting several stages in different hosts and environments, implying a differential gene regulation, we proposed a genomic analysis of XPOs to validate their annotation. The results showed the conservation of exportin family members and gene duplication events in S. mansoni .We performed quantitative RT-PCR, which revealed an upregulation of SmXPO1 in 24 h schistosomula (sixfold when compared with cercariae), and similar transcription levels were observed for SmXPO5 and SmXPOT in all the analyzed stages. These three XPO proteins have been identified for the first time in the protostome clade, which suggests a higher complexity in RNA transport in the parasite S. mansoni. Taken together, these results suggest that RNA transport by exportins might control cellular processes during cercariae, schistosomula, and adult worm development.Item Computational identification and evolutionary relationships of the MicroRNA gene cluster miR-71/2 in protostomes.(2013) Gomes, Matheus de Souza; Donoghue, Mark T. A.; Muniyappa, Mohan Kumar; Pereira, Roberta Verciano; Cota, Renata Guerra de Sá; Spillane, CharlesMicroRNAs (miRNAs) are small noncoding RNA molecules which are processed into *20–24 nt molecules that can regulate the gene expression posttranscriptionally. MiRNA gene clusters have been identified in a range of species, where in miRNAs are often processed from polycistronic transcripts. In this study, a computational approach is used to investigate the extent of evolutionary conservation of the miR-71/2 cluster in animals, and to identify novel miRNAs in the miRNA cluster miR-71/2. The miR-71/2 cluster, consisting of copies of the miR-71 and miR-2 (including miR-13) families, was found to be Protostome-specific. Although, this cluster is highly conserved across the Protostomia, the miR-2 family is completely absent from the Deuterostomia species, while miR-71 is absent from the Vertebrata and Urochordata. The evolutionary conservation and clustering propensity of the miR-71/2 family across the Protostomes could indicate the common functional roles across the member species of the Protostomia.Item Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni.(2014) Pereira, Roberta Verciano; Vieira, Helaine Graziele Santos; Oliveira, Victor Fernandes de; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáSeveral genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiq¬uitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an im¬portant role in Ub-dependent processes, little is known about their role in S. mansoni. In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana-lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, SmUCH-L3, SmUCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We per¬formed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.Item In silico analysis and developmental expression of ubiquitin-conjugating enzymes in Schistosoma mansoni.(2015) Costa, Marcela Pereira; Oliveira, Victor Fernandes de; Pereira, Roberta Verciano; Abreu, Fabiano Carlos Pinto de; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáUbiquitin-conjugating enzymes (Ub-E2) perform the second step of ubiquitination and, consequently, are essential for regulating proteolysis and for modulating protein function, interactions and trafficking. Previously, our group demonstrated the crucial role of ubiquitination and the Ubproteasome pathway during the Schistosoma mansoni life cycle. In the present investigation, we used a homology-based genome-wide bioinformatics approach to identify and molecularly characterise the Ub-E2 enzymes in S. mansoni. The putative functions were further investigated through molecular phylogenetic and expression profile analyses using cercariae, adult worms, eggs and mechanically transformed schistosomula (MTS) cultured in vitro for 3.5 h or 1 or 3 days. We identified, via in silico analysis, 17 Ub-E2 enzymes with conserved structural characteristics: the beta-sheet and the helix-2 form a central core bordered by helix-1 at one side and helix-3 and helix-4 at the other. The observed quantitative differences in the steady-state transcript levels between the cercariae and adult worms may contribute to the differential protein ubiquitination observed during the parasite’s life cycle. This study is the first to identify and characterise the E2 ubiquitin conjugation family in S. mansoni and provides fundamental information regarding their molecular phylogenetics and developmental expression during intra-mammalian stages.Item Investigation on the 19S ATPase proteasome subunits (Rpt1 6) conservation and their differential gene expression in Schistosoma mansoni.(2013) Pereira Junior, Olavo dos Santos; Pereira, Roberta Verciano; Silva, Camila Siqueira; Borges, William de Castro; Cota, Renata Guerra de Sá; Cabral, Fernanda Janku; Silva, Sérgio Henrique da; Soares, Cláudia Sossai; Morais, Enyara Rezende; Moreira, Érika Bueno de Carvalho; Magalhães, Lizandra Guidi; Paula, Fabiana Martins de; Rodrigues, VanderleiThe ubiquitin-proteasome system is responsible for degradation of the majority of intracellular proteins in eukaryotic cells. The 26S proteasome proteolytic complex is composed of a 20S core particle responsible for protein degradation and the 19S lid which plays a role in the recognition of polyubiquitinated substrates. The 19S regulatory particle (Rps) is composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. In this study, we analyzed the expression profile of 19S Rpt subunits in the larvae and adult stage of the Schistosoma mansoni life cycle. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the majority of the 19S Rpt subunits amplified at the expected molecular masses for various investigated stages. In addition, SmRpt1, SmRpt2, and SmRpt6 transcript levels were increased in 3 h-cultured schistosomula and reasonably maintained until 5 h in culture, as revealed by qRT-PCR. Phylogenetic analysis of 19S Rpt subunits showed high structural conservation in comparison to other Rpt orthologues. The mRNA expression profile of 19S Rpt subunits did not correlate with 26S proteasome proteolytic activity as judged by a 14C-casein-degrading assay, in the early cultured schistosomula. Taken together, these results revealed a differential expression profile for 19S Rpt subunits whose transcript levels could not be directly associated to 26S proteasome activity.Item Medicamentos anti-hipertensivos utilizados na área rural de Ouro Preto, MG.(2007) Silva, Adriana Carneiro; Pereira, Roberta Verciano; Figueiredo, Bárbara de Castro Pimentel; Couto, Wagner Faria; Gramigna, Luísa Leite; Sana, Dandara Emery Morais; Gomes, Rodrigo Saar; Andrade, Graziela de Fátima; Brant, João Francisco de Avelar Caldeira; Breguez, Gustavo Silveira; Silva, Mariana Augusta Resende; Barbeitos, Priscila Oliveira; Junior, Luiz Carlos Pereira; Reis, Levi Eduardo Soares; Jayme, Marisa Ferrareto; Camargo, Rúbia Santos; Franco, Marcelo Nóbile; Amaral, Murilo Sena; Carneiro, Cláudia Martins; Guimarães, Andrea GrabeItem Modificação pós-traducional dependente de SUMO em Schistosoma mansoni : padrão de expressão diferencial durante a transição cercária a esquistossômulo.(Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2010) Pereira, Roberta Verciano; Cota, Renata Guerra de SáA conjugação de SUMO aos substatos alvo ocorre através de um mecanismo análogo ao da ubiquitina, pela ação sequencial de três classes de enzimas: E1 ativadora (Aos1-Uba2), E2 conjugadora (Ubc9) e E3 ligases (PIAS, Polycomb2 e RanBP2). SUMO é sintetizada como um precursor inativo, que requer processamento por proteases específicas (SENPs). A conjugação de SUMO é uma modificação reversível e transitória. As mesmas enzimas que convertem SUMO em sua forma madura também catalisam a clivagem dos seus substratos. A reconstituição da via de sumorilação em Schistosoma mansoni, baseado em busca por homologia utilizando banco de dados disponíveis, mostra a existência de 9 genes envolvidos com esta via: dois genes para SUMO (Smsmt3b/c), um para E1 (Smaos1-uba2), um para E2 (Smubc9), dois para E3 (Smpias e Smranbp2) e dois para SENPs (Smsenp1/7). Neste trabalho, os níveis de expressão destes genes foram quantificados utilizando o método de qRT-PCR e RNA total obtidos a partir de cercária, verme adulto e esquistossômulos de 3,5 horas a 7 dias de cultivo in vitro (MTS). Os resultados evidenciaram um padrão de expressão gênica diferencial para todos os genes analisados. Vale ressaltar que os níveis de transcritos foram 3 vezes maiores em MTS-3,5h quando comparado com cercária e verme adulto. Os níveis de SmUbc9 foram analisados por Western blot, utilizando anticorpos policlonais específicos para a proteína de S. mansoni produzidos utilizando a proteína recombinante. Os resultados sugerem níveis equivalentes da SmUbc9 nos estágios analisados. O perfil de substratos sumorilados foi determinado utilizando a técnica de Western blot usando anti-SUMO. Foi observado um perfil estágio-específico de conjugados sumorilados e um aumento nos estágios iniciais do desenvolvimento dos esquistossômulos. Estes dados demonstram que os constituintes da via de sumorilação são diferencialmente expressos, sugerindo um perfil estágio-específico durante a transição cercária-esquistossômulo. Considerando que esta transição não é controlada por uma síntese real de proteínas, esses resultados em conjunto, sugerem que a via de modificação pós-traducional dependente de SUMO pode regular processos biológicos importantes durante esse período do ciclo biológico do parasito.Item Molecular characterization of SUMO E2 conjugation enzyme : differential expression profile in Schistosoma mansoni.(2011) Pereira, Roberta Verciano; Cabral, Fernanda Janku; Gomes, Matheus de Souza; Babá, Élio Hideo; Passos, Liana Konovaloff Jannotti; Carvalho, Omar; Rodrigues, Vanderlei; Afonso, Robson José de Cássia Franco; Borges, William de Castro; Cota, Renata Guerra de SáSUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.Item NEDD8 conjugation in Schistosoma mansoni : genome analysis and expression profiles.(2013) Pereira, Roberta Verciano; Gomes, Matheus de Souza; Olmo, Roenick Proveti; Sousa, Daniel M.; Passos, Liana Konovaloff Jannotti; Babá, Élio Hideo; Borges, William de Castro; Cota, Renata Guerra de SáNEDD8 is an ubiquitin-like molecule that covalently binds to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to bind to p53 and p73, as well as all Cullin family proteins, which are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes. Here, we focused on a genomic analysis of the genes involved in the NEDD8 conjugation pathway in Schistosoma mansoni. The results revealed seven genes related to NEDD8 conjugation that are conserved in Schistosoma japonicum, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We performed quantitative RT-PCR (qRT-PCR), which showed differential profiles for Smnedd8, Smapp1, Smuba3, Smube2f, Smdcn1, Smrbx and Smsenp8 throughout the life cycle of S. mansoni. Upregulation was observed in 3-day-old schistosomula and adult worms for all analysed genes. We also analysed the transcription levels of Cullin family members Smp63 and Smp73, and observed upregulation in early schistosomula, while cercariae and adult worms showed expression levels similar to one another. Taken together, these results suggest that the NEDDylation/ DeNEDDylation pathway controls important cellular regulators during worm development from cercariae to schistosomula and, finally, to adult.Item Sumorilação e nedilação de proteínas em Schistosoma mansoni.(2014) Pereira, Roberta Verciano; Cota, Renata Guerra de SáA esquistosomose mansônica é a segunda parasitose mais devastadora segundo a Organização Mundial da Saúde, afligindo mais de 240 milhões de pessoas no mundo. Contudo, apesar do genoma do Schistosoma mansoni ser extensivamente estudado, os mecanismos envolvidos no remodelamento morfológico e adaptação do parasito após instalação no hospedeiro mamífero permanecem desconhecidos. A hipótese deste trabalho é que modificações pós-traducionais dependentes de SUMO e NEDD8 regulam o proteoma de maneira estágio-específica, contribuindo para uma adaptação rápida ao hospedeiro mamífero. Para investigar essa hipótese, inicialmente foi utilizado busca por similaridade, pesquisa de domínio e resíduos conservados, seguido de análises filogenéticas para identificar os genes relacionados com a via de sumorilação e nedilação de proteínas. Nesta etapa, também foram analisados genes do complexo COP9 signalossoma, devido a sua atividade denediladora intrínseca. Os resultados sugerem o envolvimento de 24 genes, sendo 9 com a modificação dependente de SUMO, 7 para a via dependente de NEDD8 e 8 com a montagem do complexo COP9. Em geral, as proteínas preditas apresentam um alto grau de conservação em relação às proteínas ortólogas. Para investigar padrões de expressão gênica diferencial ou estágio-específica nos estágios de cercária, verme adulto e esquistossômulos de 3,5 horas a 7 dias de cultivo in vitro, foi utilizada a técnica de qRT-PCR. Com relação à via de sumorilação, foi verificada uma expressão diferencial das E3 ligases: SmPIAS e SmRanBP2 e das enzimas desumoriladoras: SmSENP1 e SmSENP7. Além disso, a protease SmSENP1 mostrou uma atividade diferencial em verme adulto e em cercária. Já em relação à via de nedilação, foi observada uma correlação positiva entre o perfil de expressão dos constituintes da via de NEDD8 e seus alvos clássicos: culinas 1-5; p53 e p73. Subunidades do complexo COP9 signalossoma também foram avaliadas e mostraram ser mais expressas em cercária do que em verme adulto, o perfil de expressão entre os esquistossômulos foi semelhante. Também foram observadas que ambas as vias de modificação são ativadas em condições de estresse e inibidas na presença de MG132, um inibidor clássico do proteassoma. A continuação desta linha de investigação, utilizando técnicas proteômicas e silenciamento gênico, poderá confirmar a hipótese de que o proteoma de cercária e esquistossômulos jovens são extensivamente modificados por SUMO e NEDD8, sugerindo uma importância dessas vias de modificação para a instalação do parasitismo.Item Transcriptional profile and structural conservation of SUMO-Specific proteases in schistosoma mansoni.(2012) Pereira, Roberta Verciano; Cabral, Fernanda Janku; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáSmall ubiquitin-related modifier (SUMO) is involved in numerous cellular processes including protein localization, transcription, and cell cycle control. SUMOylation is a dynamic process, catalyzed by three SUMO-specific enzymes and reversed by Sentrin/SUMO-specific proteases (SENPs). Here we report the characterization of these proteases in Schistosoma mansoni. Using in silico analysis, we identified two SENPs sequences, orthologs of mammalian SENP1 and SENP7, confirming their identities and conservation through phylogenetic analysis. In addition, the transcript levels of Smsenp1/7 in cercariae, adult worms, and in vitro cultivated schistosomula were measured by qRT-PCR. Our data revealed upregulation of the Smsenp1/7 transcripts in cercariae and early schistosomula, followed by a marked differential gene expression in the other analyzed stages. However, no significant difference in expression profile between the paralogs was observed for the analyzed stages. Furthermore, in order to detect deSUMOylating capabilities in crude parasite extracts, SmSENP1 enzymatic activity was evaluated using SUMO-1- AMC substrate. The endopeptidase activity related to SUMO-1 precursor processing did not differ significantly between cercariae and adult worms. Taken together, these results support the developmentally regulated expression of SUMO-specific proteases in S. mansoni.Item Up-regulation of SUMO E3 ligases during lung schistosomula and adult worm stages.(2014) Pereira, Roberta Verciano; Gomes, Matheus de Souza; Cabral, Fernanda Janku; Passos, Liana Konovaloff Jannotti; Rodrigues, Vanderlei; Borges, William de Castro; Cota, Renata Guerra de SáSmall ubiquitin-like modifier (SUMO) conjugation of proteins occurs through a concert action of enzymes using a similar ubiquitination mechanism. After a C-terminal peptide is cleaved from the SUMO precursor by a protease to reveal a di-glycine motif, SUMO is activated by an E1 enzyme (Aos1/Uba2) and conjugated to target proteins by the sole E2 enzyme (Ubc9) guided to the appropriate substrates by the SUMO E3 ligase. Previous reports from our group showed that Schistosoma mansoni has two paralogs of SUMO: one E2 conjugation Ubc9 and two SUMO-specific proteases (SENPs). The differential gene expression profile observed for SUMO pathway genes throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development particularly during the cercariae to schistosomula transition. To continue this investigation, we here analysed the repertoire of SUMO E3 ligases and their expression profiles during cercariae/ schistosomula transition. In silico analysis through S. mansoni databases showed two conserved SUMO E3 ligases: protein inhibitor of activated STAT (PIAS) and Ran-binding protein 2 (RanBP2). Furthermore, expression levels of the SUMO E3 ligases were measured by qRT-PCR using total RNA from cercariae, adult worms and mechanically transformed schistosomula. Our data showed an up-regulation of expression in lung schistosomula and adult worm stages. In conclusion, the differential expression of SmPIAS and SmRanBP2 during schistosomula development was similar to the expression levels of all genes related to SUMO conjugation, thereby suggesting that the control of protein activity, localisation or stability during cercariae to schistosomula transition is SUMO-dependent.