17 research outputs found
Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosoma mansoni
Small 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
NEDD8 conjugation in Schistosoma mansoni : genome analysis and expression profiles.
NEDD8 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
Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics
Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomicand metatranscriptomic-based analyses of a large composting operation in the Sao Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50 degrees C to 75 degrees C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the processand that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomass-degrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Provost's Office for Research of the University of Sao PauloCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, BrazilUniv Sao Paulo, Programa Pos Graduacao Interunidades Bioinformat, Sao Paulo, BrazilUniv Sao Paulo, Escola Artes Ciencias & Humanidades, Sao Paulo, Brazil|Fundacao Parque Zool Sao Paulo, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Ciencias Biol, Sao Paulo, BrazilBiocomplex Inst Virginia, Blacksburg, VA USADepartamento de Ciências Biológicas, Universidade Federal de São Paulo, São Paulo, BrazilFAPESP: 2011/50870-6Web of Scienc
Sumorilação e nedilação de proteínas em Schistosoma mansoni.
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.A 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.Schistosomiasis is the second most devastating parasitic disease according to the World Health Organization, afflicting over 240 million people worldwide. Although the Schistosoma mansoni genome has been extensively studied, the mechanisms involved in morphological remodeling and adaptation of the parasite in the mammalian host remain unknown. Our working hypothesis is that SUMO and NEDD8-dependent post-translational modifications regulate, in a stage - specific manner, the parasite´s proteome, contributing to a rapid adaptation to the mammalian host. To investigate this hypothesis, we initially used, domain and conserved residues similarity searches, followed by phylogenetic analysis to identify genes involved in the SUMOylation and NEDDylation pathways. In this step, the genes related to COP9 signalosome complex were also analyzed, due to their intrinsic deNEDDylating activity. The results suggest the involvement of 24 genes, of which 9 were related to SUMO modification, 7 to NEDD8 pathway and 8 involved in the assembly of the COP9 complex. In general, the predicted proteins showed a high degree of conservation compared to orthologue proteins. To investigate differential gene expression patterns in cercariae, adult worms and in-vitro schistosomula cultured for 3.5 hours and 7 days it was used the qRT-PCR technique. Concerning the SUMOylation pathway, it was observed a differential expression of E3 ligases: SmPIAS and SmRanBP2 and also for deSUMOylating enzymes: SmSENP1 and SmSENP7. Moreover, SmSENP1 protease showed a differential activity in adult worms and cercariae. As for the NEDDylation pathway, a positive correlation was observed comparing the expression profile of the NEDD8 pathway constituents and their classic targets p53 and p73 cullins 1-5. Subunits of the COP9 signalosome complex were also evaluated and showed to be up-regulated in the cercariae compared to adult worms, the expression profiles of such subunits were similar among the cultured schistosomula. It was also observed that both pathways are activated under stress and inhibited in the presence of MG132, a classic proteasome inhibitor. Proteomic techniques and gene silencing may further validate our hypothesis that the cercariae and early-schistosomula proteomes are extensively modified by SUMO and NEDD8, revealing the importance of these pathways for parasite installation and maintenance inside the vertebrate host
Modificação pós-traducional dependente de SUMO em Schistosoma mansoni : padrão de expressão diferencial durante a transição cercária a esquistossômulo.
A 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.Attachment of SUMO to target proteins occur through by similar mechanism to ubiquitination, with sequential action of three enzymes: E1 activating (Aos1-Uba2), E2 conjugation (Ubc9) and E3 ligases (PIAS, Polycomb2 and RanBP2). SUMO is synthesized as inactive precursors that require processing by specific proteases (SENPs). SUMO attachment is a reversible and transient modification. The same enzymes that convert SUMO to its mature form also catalyse the cleavage from their substrates. Reconstitution of SUMOylation pathway in Schistosoma mansoni, based on homology search using avaible databases, shown the existence of nine genes related with SUMOylation: two genes to SUMO (Smsmt3b/c), one to E1 (Smaos1-uba2), one to E2 (Smubc9), two to E3 (Smpias e Smranbp2) and two to SENPs (Smsenp1/7). In this work, the expression levels of these genes was quantified by qRT-PCR and total RNA obtained from cercariae, adult worm and mechanically transformed schistosomula cultivated from 3.5 h to 7 days in vitro (MTS). The results revealed differential gene expression profile to all analyzed genes. We also observed that the transcripts levels were 3 times higher in MTS-3,5h when compared with cercariae and adult worm. The SmUbc9 levels were analysed by Western blot, using specific polyclonal antibodies to S. mansoni protein produced using recombinant protein. The results suggest equivalent levels of SmUbc9 in the same evolutive stages analysed. The SUMOylated substrates profile was detected by Western blot using anti-SUMO. We observed a stage-specific profile of SUMOylated conjugated and an accumulation in initial stages of schistosomula development. These data demonstrate that SUMOylation pathway components are differentially expressed in S. mansoni, suggesting a stage-specific profile during cercariae-schistosomula transition. Whereas this transition is not controlled by real protein synthesis whole results suggest that posttranslational modification SUMO dependent could regulate important biological process during this period of parasite life-cycl
Characterisation of the COP9 signalosome in Schistosoma mansoni parasites.
The 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
Computational identification and evolutionary relationships of the MicroRNA gene cluster miR-71/2 in protostomes.
MicroRNAs (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
Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosoma mansoni
Small 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
Up-regulation of SUMO E3 ligases during lung schistosomula and adult worm stages.
Small 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
In silico analysis and developmental expression of ubiquitin-conjugating enzymes in Schistosoma mansoni.
Ubiquitin-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