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A PUTATIVE CORRELATION AMONG TRANSCRIPT LEVELS OF 19S REGULATORY COMPLEX RPTS SUBUNITS AND PROTEOLYSIS CONTROL DURING THE LIFE CYCLE OF SCHISTOSOMA MANSONI
Pereira Júnior, O.S.1; Castro-Borges, W.1; Silva, S.H. 1; Silva, C.S. 3; Guerra-Sá, R.2; Macedo, E.1; Brigatto, O.M.1; Cabral, F. C.1; Andreolli, A.B.P.1; Magalhães, L.G.1; Soares, S.C. 1; Moreira, E.B.C.1; Botelho-Machado, C. 1; Momo, P.B. 1 ; Rodrigues,V. 1.
1 Department of Biochemistry and Immunology, School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil; 2 Department of Biological Science, ICEB, Federal University of Ouro Preto, MG, Brazil; 3 Department of Immunology, Microbiology and Parasitology, Federal University of Uberlândia, MG, Brazil.
The
ubiquitin–proteasome pathway is the principal route to degradation of
proteins in eukaryotic cells and it is thought to contribute for the
regulation of cellular homeostasis. The 26S proteasome is made up of
two 19S complexes and a proteolitic active 20S proteasome core. Objective: To analyze the gene expression profile of six 19S regulatory ATPases subunits throughout specific developmental stages of the S. mansoni life cycle. Material and Methods: RT-PCR was performed using total RNA (adult worms, eggs, hepatopancreas of infected and non-infected Biomphalaria
snails, cercariae, schistosomulae), in the presence of an oligo dT
primer and the thermoscript RT. For Northern Blot analysis, ten
micrograms of total RNA from cercariae and adult worms were transferred
to Hybond membrane and the hybridization procedures executed exactly as
described by Maniatis. Genomic DNA from S.mansoni adult worms was isolated by the CTAB method. Results:
Among the ATPase subunits investigated, the comparison of genomic and
mRNA sequences did not show the presence of introns. In addition, we
did not observe the amplification of the transcripts coding for the
SmRpt1, SmRpt2 and SmRpt6 in the cercariae stage. Northern Blot also
revealed a comparatively lower level of transcripts for SmRpt1, 2 and 6
in the cercariae stage, and SmRpt5b could only be detected in the
transforming schistosomula. Conclusion: The present study
provided, at the gene and transcript levels, interesting potential
alternatives for the existence of different 26S proteasome molecules
suggesting a mechanism to regulate proteolysis throughout the parasite
development. Supported by CNPq, FAPESP and FAEPA.
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