Browsing by Author "Pereira Junior, Olavo dos Santos"

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    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, Vanderlei
    The 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.
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    Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins.
    (2008) Cabral, Fernanda Janku; Pereira Junior, Olavo dos Santos; Silva, Camila Siqueira; Cota, Renata Guerra de Sá; Rodrigues, Vanderlei
    The sumoylation pathway is a post-translational modification of nuclear proteins widespread among several organisms. SMT3C is the main protein involved in this process and it is covalently conjugated to a diverse assortment of nuclear protein targets. To date, 3 SUMO paralogues (SMT3C, A/B) have been characterized in mammals and plants. In this work we characterized two SUMO related genes, named SMT3B and SMT3C throughout Schistosoma mansoni life cycle. The SmSMTB/C encodes for proteins sharing significant amino acid homology with SMT3. Phylogenetical analyses revealed that both SmSMT3B/C are distinct proteins. Additionally, SmSMT3B and C are expressed in cercariae, adult worms, eggs and schistosomula however SmSMT3C gene showed an expression level 7 to 9 fold higher than SmSMT3B in eggs, schistosomula and adult worms. The comparison between the SmSMT3C genomic and cDNA sequences established that the encoding sequence is interrupted by 3 introns of 70, 37 and 36 bp. Western Blot has shown SMT3 conjugates are present in nuclear and total protein fractions of adults and cercariae. Therefore our results suggest a functional sumoylation pathway, and the presence of two paralogues also suggests the specificity of substrates for SMT3 in S. mansoni.
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    The ubiquitin proteasome system in Strongyloididae. Biochemical evidence for developmentally regulated proteolysis in Strongyloides venezuelensis.
    (2009) Paula, Fabiana Martins de; Borges, William de Castro; Pereira Junior, Olavo dos Santos; Gomes, Matheus de Souza; Ueta, Marlene Tiduko; Rodrigues, Vanderlei
    Nematode parasites from the genus Strongyloides spp. are important pathogens of the intestinal mucosa of animals and humans. Their complex life cycles involve alternating developmental adaptations between larvae stages and the adult parthenogenetic female. Here, we report, primarily through homology-based searching, the existence of the major components of the ubiquitin– proteasome system in this genus, using the available EST data from S. ratti, S. stercoralis, and Parastrongyloides trichosuri. In this study, S. venezuelensis was used as our model organism for detection of proteasome activity and ubiquitinated substrates in cytosolic preparations from the L3 larvae and the adult female. Marked differences in proteasome capabilities were found when these two stages were compared. A preference for degradation of chymotryptic synthetic peptides was found in both stages with the adult exhibiting a higher rate of hydrolysis compared to the larvae. Due to the high evolutionary conservation of proteasome alpha subunits, an anti-human proteasome antibody was able to recognize proteasome subunits in these preparations by Western blotting, supporting the proposal that the activity of the ubiqutin–proteasome system is developmentally regulated in this nematode.