13 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
Antibody recognition of plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the brazilian Amazon
In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infectionsthis presents an intriguing and underexplored area of research. In addition to the rapid access of infected p1095598601FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/17114-3576128/2008-2To Wolfgang Fischer and Márcio Yamamoto, for sequencing of DBLα tag sequences, and to Drs Mauro Shugiro Tada and Tony Katsuragawa, for help with data and sample collection at the site
Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon
Transcriptional memory and switching in the Plasmodium falciparum rif gene family
The human malaria parasite Plasmodium falciparum expresses erythrocyte-surface directed variant antigens which are important virulence factors Many are transcribed from multigene families and presumably their mode of expression is strictly controlled to guarantee immune evasion in the human host. In order to elucidate the dynamics of rif transcription and to investigate if rif switching is comparable to var switching we monitored rif variant gene expression in parasites with different cytoadhesive properties as well as after a number of reinvasions. We found identical transcripts in parasite lines with different adhesive phenotypes suggesting that rif genes do not have a critical role in determining the cytoadhesion specificity of infected erythrocytes. We show for the first time that rif genes may show a conserved mode of transcription, maintaining the previously dominant rif transcript in subsequent reinvasions, but also observed rapid switching at rates up to 45% per generation, much higher than for the var gene family. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CNPqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq
Proteomic, metabolic and immunological changes in biomphalaria glabrata infected with schistosoma mansoni
Mansonic schistosomiasis is a neglected disease transmitted by Biomphalaria spp. snails. Understanding what happens inside the intermediate host is important to develop more efficient ways of reducing schistosomiasis prevalence. Our purpose was to characterize metabolic and immunological changes in Biomphalaria glabrata 24 h after exposure to Schistosoma mansoni. For this purpose, proteins were extracted from snails' whole tissue with Tris-Urea buffer and digested with tripsin. Mass spectrometry was performed and analyzed with MaxQuant and Perseus software. Also, the hemolymph of five snails 24 h post exposure was collected, and the numbers of hemocytes, levels of urea, uric acid, nitric oxide, calcium, glycogen and alanine and aspartate aminotransferases activities were assessed. Snails were also dissected for measurement of glycogen content in the cephalopodal region and gonoda-digestive gland complex. Globin domain proteins were found to be up-regulated: also the number of circulating hemocytes was significantly higher after 24 h of exposure to the parasite. NO levels were higher 24 h post exposure. Several proteins associated with energy metabolism were found to be up-regulated. Glycogen analysis showed a significant decrease in the gonad-digestive gland complex glycogen content. We found several proteins which seem to be associated with the host immune response, most of which were up-regulated, however some were down-regulated, which may represent an important clue in understanding B. glabrata - S. mansoni compatibility4913-1410491060COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP001; 17426132016/07137-0; 2017/07364-9; 2009/54040-
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
Uncovering Notch pathway in the parasitic flatworm Schistosoma mansoni.
Several signaling molecules that govern development
in higher animals have been identified in the parasite
Schistosoma mansoni, including the transforming growth factor
?, protein tyrosine kinases, nuclear hormone receptors,
among others. The Notch pathway is a highly conserved signaling
mechanism which is involved in a wide variety of developmental
processes including embryogenesis and oogenesis
in worms and flies. Here we aimed to provide the molecular
reconstitution of the Notch pathway in S. mansoni using
the available transcriptome and genome databases. Our results
also revealed the presence of the transcripts coded for
SmNotch, SmSu(H), SmHes, and the gamma-secretase complex
(SmNicastrin, SmAph-1, and SmPen-2), throughout all
the life stages analyzed. Besides, it was observed that the
viability and separation of adult worm pairs were not affected
by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-
S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor.
Moreover, DAPT treatment decreased the production
of phenotypically normal eggs and arrested their development
in culture. Our results also showed a significant decrease in
SmHes transcript levels in both adult worms and eggs treated
withDAPT. These results provide, for the first time, functional
validation of the Notch pathway in S. mansoni and suggest its
involvement in parasite oogenesis and embryogenesis. Given
the complexity of the Notch pathway, further experiments
shall highlight the full repertoire of Notch-mediated cellular
processes throughout the S. mansoni life cycle
Molecular characterization of SUMO E2 conjugation enzyme : differential expression profile in Schistosoma mansoni.
SUMO-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
Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata
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Previous issue date: 2019Universidade Federal de Uberlândia. Laboratório de Bioinformática e Análises Moleculares. Patos de Minas, MG, Brasil.Universidade Federal de Uberlândia. Laboratório de Bioinformática e Análises Moleculares. Patos de Minas, MG, BrasilFundação Oswaldo Cruz. Instituto René Rachou. Grupo de Pesquisa em Biologia do Schistosoma mansoni e sua Interação com o Hospedeiro. Belo Horizonte, MG, Brasil.Universidade Federal de Lavras. Departamento de Biologia. Seção de Fisiologia de Plantas. Laboratório de Fisiologia Molecular de Plantas. Lavras, MG, Brasil.Universidade Federal de Ouro Preto. Escola de Farmácia. Departamento de Farmácia. Ouro Preto, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Grupo de Pesquisa em Biologia do Schistosoma mansoni e sua Interação com o Hospedeiro. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Grupo de Pesquisa em Biologia do Schistosoma mansoni e sua Interação com o Hospedeiro. Belo Horizonte, MG, Brasil.Universidade Federal de Uberlândia. Laboratório de Bioquímica e Biologia Molecular. Patos de Minas, MG, Brasil.Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Biologia Animal. Campinas, SP, Brasil.Fundação Oswaldo Cruz. Instituto René Rachou. Grupo de Pesquisa em Helmintologia e Malacologia Médica. Belo Horizonte, MG, Brasil.Universidade Federal de Uberlândia. Laboratório de Bioinformática e Análises Moleculares. Patos de Minas, MG, Brasil.BACKGROUND: Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses.
OBJECTIVE: The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata.
METHODS: The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM.
FINDINGS: 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance.
MAIN CONCLUSIONS: Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis