Caracterização funcional da proteína SmRbx: um aproteína de Schistosoma mansoni similar à proteína RING box envolvida no processo de Ubiquitinação

Abstract

Ubiquitination is part of the process that leads to proteolysis, in which the polyubiquitinated protein is directed to degradation by 26S proteasome. Ubiquitin is transferred to a target protein by an E3 ligase enzyme. One of the members of E3 class is the SCF complex. This complex consists of the proteins Cul1, Skp1, an F-box family member and a RING box family member. The latter is denominated Rbx1 in humans, and its yeast homolog is HRT1. The human protein was reported to complement and to revert the lethality of HRT1 deletion in yeast, suggesting that both have the same biochemical function. We isolated in our laboratory the cDNA coding for the SmRbx protein, the Schistosoma mansoni ortholog of Rbx1 and HRT1. In silico studies demonstrated that SmRbx is a single copy gene in S. mansoni genome, presenting three introns and that the transcript is present in stages of the parasite life cycle in both hosts. In order to investigate SmRbx involvement in the ubiquitination process, a heterologous functional complementation study in yeast was conducted. To obtain a HRT1 null mutant yeast strain, Saccharomyces cerevisiae diploid cells were used to disrupt one of the gene alleles. It is essential to use diploid yeast cells due to the fact that the haploid HRT1 null mutant is lethal. The disruption of HRT1 gene from diploid yeast cells was done by homologous recombination, introducing the HIS3 gene into the HRT1 coding region. Diploid yeast cells knockout for one allele of HRT1 and containing the SmRbx cDNA cloned into a plasmid were sporulated. Haploid yeast cells were obtained by micromanipulation. Functional complementation was confirmed by the growth reestablishment of the haploid yeast cells deleted for HRT1 and containing the SmRbx cDNA. It was verified that the knockout yeast cells containing the S. mansoni gene presented an elongated morphology and were not able to grow at 23°C and 37°C, but showed normal growth at 30°C, in comparison with wild type yeast cells. These results indicate that the complementation with the parasite gene is not perfect. In order to identify SmRbx protein regions essential for its function, gene mutants were produced by various deletions in the protein N-terminal and C-terminal coding regions by PCR. Regions involved in the interaction with cullin are essential for protein function, because mutants lacking the first 24 residues in the N-terminal region and mutants lacking 18 residues in the C-terminal region were no longer capable to complement the yeast HRT1 null mutant. The interaction between SmRbx and yeast cullin was tested by two-hybrid system. For this assay, it was used the S. cerevisiae Cul1 protein portion that interacts with the human Rbx1, and SmRbx or SmRbx24 (lacking 24 N-terminal residues). Yeast cells that activated the reporter genes HIS3 and b-galactosidase demonstrated that SmRbx, but not SmRbx24, is capable of interacting with Cul1. These results suggest that the SmRbx protein is probably involved in the ubiquitination process, as its human and yeast orthologues. The recombinant protein was produced in a bacterial system and used to immunize BALB/c mice. After the fifth immunization, mouse serum did not present anti-SmRbx polyclonal antibodies, indicating that the high similarity between the S. mansoni protein and its murine counterpart probably prevented antibody production.