Characterization and mRNA expression analysis of PI31, an endogenous proteasome inhibitor from Schistosoma mansoni.
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.
Botelho-Machado
Carla
C
Department of Biochemistry and Immunology, FMRP-USP, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, Brazil 14040-900. carlabmachado@usp.br
Cabral
F J
FJ
Soares
C S
CS
Moreira
E B C
EB
Morais
E R
ER
Magalhães
L G
LG
Gomes
M S
MS
Guerra-Sá
R
R
Rosa
J C
JC
Ruller
R
R
Ward
R J
RJ
Rodrigues
V
V
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D051379:Mice; D008807:Mice, Inbred BALB C; D061988:Proteasome Inhibitors; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics
D015853:Cysteine Proteinase Inhibitors; D061988:Proteasome Inhibitors; D015800:Protozoan Proteins; D012333:RNA, Messenger