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Published on: December 4, 2015
Characterization and mRNA expression analysis of PI31, an endogenous proteasome inhibitor from Schistosoma mansoni
Carla Botelho-Machado1, F J Cabral, C S Soares
1Department of Biochemistry and Immunology, FMRP-USP, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, Brazil 14040-900. carlabmachado@usp.br
Abstract:
The proline-rich inhibitor of 31 kDa (PI31) is highly conserved through metazoan evolution, and its activity in the proteasome inhibition is well-established although the precise mechanism of inhibition is unclear. The coding DNA sequence of Schistosoma mansoni PI31 (SmPI31) was cloned, and the recombinant protein was expressed in bacterial system. The correct amino acid sequence was confirmed by mass spectrometry and circular dichroism suggests that SmPI31 contains both α-helix and non-structured regions. Inhibition assays, using the Suc-Leu-Leu-Val-Tyr-4-MCA substrate for proteasome degradation, showed that the S. mansoni proteasome may be regulated by the inhibitory activity of SmPI31. A gene expression assay using qRT-PCR at various stages during the S. mansoni life cycle has shown that SmPI31 transcripts are expressed in all studied stages, suggesting that PI31 plays an important role during the developmental processes of the parasite. In this study first evidence is presented that PI31 has a conserved structure and plays a role as proteasome inhibitor in adult worms and it is expressed through life cycle.
Insights
The proline-rich inhibitor of 31 kDa (PI31) from Schistosoma mansoni acts as a proteasome inhibitor. This study confirms its conserved structure and expression throughout the parasite's life cycle.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- The proline-rich inhibitor of 31 kDa (PI31) is a conserved protein across metazoans.
- Its role in proteasome inhibition is known, but the exact mechanism remains unclear.
- Understanding PI31 in parasites like Schistosoma mansoni is crucial for potential therapeutic targets.
Purpose of the Study:
- To clone and characterize Schistosoma mansoni PI31 (SmPI31).
- To investigate the inhibitory activity of SmPI31 on the S. mansoni proteasome.
- To determine the expression pattern of SmPI31 throughout the S. mansoni life cycle.
Main Methods:
- Cloning of SmPI31 coding DNA sequence and recombinant protein expression in bacteria.
- Mass spectrometry and circular dichroism for protein characterization.
- Proteasome inhibition assays using Suc-Leu-Leu-Val-Tyr-4-MCA substrate.
- Quantitative reverse transcription PCR (qRT-PCR) for gene expression analysis.
Main Results:
- Recombinant SmPI31 was successfully expressed and confirmed by mass spectrometry.
- Circular dichroism indicated SmPI31 possesses both α-helix and non-structured regions.
- Inhibition assays demonstrated SmPI31's inhibitory effect on the S. mansoni proteasome.
- qRT-PCR revealed SmPI31 transcripts are present in all life cycle stages of S. mansoni.
Conclusions:
- SmPI31 exhibits a conserved structure and functions as a proteasome inhibitor in adult S. mansoni worms.
- SmPI31 is expressed throughout the parasite's life cycle, suggesting a vital role in its development.
- This study provides the first evidence for PI31's role as a proteasome inhibitor in S. mansoni.
