title: Molecular and functional characterization of a putative PA28γ proteasome activator orthologue in Schistosoma mansoni.
abstract: PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28γ in larval growth/development of the S. mansoni.
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto - FMRP, Universidade de São Paulo - USP, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Cláudia Sossai
authors.initials: CS
authors.lastname: Soares
authors.forename: Enyara Rezende
authors.initials: ER
authors.lastname: Morais
authors.forename: Lizandra G
authors.initials: LG
authors.lastname: Magalhães
authors.forename: Carla Botelho
authors.initials: CB
authors.lastname: Machado
authors.forename: Érika Bueno de Carvalho
authors.initials: ÉB
authors.lastname: Moreira
authors.forename: Felipe Roberti
authors.initials: FR
authors.lastname: Teixeira
authors.forename: Vanderlei
authors.initials: V
authors.lastname: Rodrigues
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Yoshino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism*; D007814:Larva / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D016366:Open Reading Frames; D010802:Phylogeny; D046988:Proteasome Endopeptidase Complex / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics* / Q000254:growth & development*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D015801:Helminth Proteins; D009124:Muscle Proteins; C085047:PSME1 protein, human; D046988:Proteasome Endopeptidase Complex