Molecular and functional characterization of a putative PA28? proteasome activator orthologue in Schistosoma mansoni.

ABSTRACT: PA28? 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 PA28? of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28?, this transcript has a 753bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28? with multiple PA28? orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28? from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28? to its sister group that contains well-characterized PA28? sequences from Drosophila spp., as well as sharing the same branch with PA28? from S. japonicum. Gene expression profiling of SmPA28? 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-SmPA28? antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28? from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28? transcript and protein in these stages may be correlated with an important role of the PA28? 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 SmPA28? double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28? 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 SmPA28? in larval growth/development of the S. mansoni.

SUBMITTER: Soares CS 

PROVIDER: S-EPMC3712533 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature