Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites' tegument. We identified 1,919 genes with a statistically significant (q-value <= 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG132 caused important changes in the worm tegumentpeeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis.Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, Ribeirao Preto, SP, BrazilUniv Sao Paulo, Dept Bioquim, Inst Quim, Sao Paulo, SP, BrazilAdolfo Lutz Inst, Ctr Parasitol & Micol, Nucleo Enteroparasitas, Sao Paulo, SP, BrazilUniv Franca, Nucleo Pesquisa Ciencias Exatas & Tecnol, Grp Pesquisa Prod Nat, Franca, SP, BrazilInst Butantan, Lab Expressao Genica Eucariotos, Sao Paulo, SP, BrazilUniv Fed Uberlandia, Inst Genet Bioquim, Campus Patos de Minas, Patos De Minas, MG, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Discipline Parasitol, Sao Paulo, SP, BrazilUniv Fed Rio de Janeiro, Inst Biol, Ctr Ciencias & Saude, Rio De Janeiro, RJ, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Discipline Parasitol, Sao Paulo, SP, BrazilWeb of Scienc

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

Effects of (-)-6,6'-dinitrohinokinin on adult worms of Schistosoma mansoni: a proteomic analyses

Abstract Schistosomiasis, a chronic disease that affects million people worldwide, is caused by trematode flukes of the genus Schistosoma. The lack of an anti-schistosomiasis vaccine and massive monotherapy with praziquantel reinforces the need for search and development of new therapeutic drugs. Recently, we demonstrated that the essential oil of Piper cubeba L., Piperaceae, and their derivative dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin, presents in vitro and in vivo activities against Schistosoma mansoni. Here, we identified changes in the protein expression after exposure to dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin. We applied two-dimensional gel electrophoresis (2-DE) to S. mansoni soluble protein extracts and observed at least 38 spots to be affected by dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin. We further identified 25 differentially expressed proteins by mass spectrometry. Enrichment for biological processes and predictive analyses of protein-protein interactions suggest that dibenzylbutyrolactolic (-)-6,6'-dinitrohinokinin targets proteins involved mainly in metabolic processes, especially carbohydrate metabolism. In summary, this study provides an interesting approach to understand the anti-parasitic activity of semi-synthetic (-)-6,6'-dinitrohinokinin a derivative compound from lignan and for the development of new therapy strategies

Effect of MG-132 on gene expression profile in <i>Schistosoma mansoni</i> adult worms.

<p>Adult worm pairs were treated for 24 h with 50 μM MG-132. Microarrays were used to measure gene expression on a large scale. The figure shows a group of 1,919 genes with a statistically significant (<i>q-value</i> ≤ 0.025) differential expression in adult worms treated with MG-132 <i>versus</i> controls. Each horizontal line represents a gene and each column represents an experimental replicate. There are two technical replicates for each one of four biological replicates. Genes with transcription induced by treatment are shown in red, genes with repressed transcription are in green, and the color intensity is proportional to the log2 ratio (treated/control), as indicated by the color scale at the bottom.</p

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 ?-helix and non-structured regions. Inhibition assays, using the SucLeu-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

Scanning electron microscopy of the tegument of <i>S</i>. <i>mansoni</i> adult worms.

<p>(A, x600) Normal morphology of adult worm tegument. (B, x1200) and (C, x3000) magnification of normal morphology of <i>S</i>. <i>mansoni</i> tegument showing the large number of tubercles (tu) and spines (sp). (D, x5000) Magnification of normal morphology of <i>S</i>. <i>mansoni</i> tegument showing spines (sp). (E, x600) Morphology of <i>S</i>. <i>mansoni</i> tegument after treatment with 50 μM MG-132 for 24 h. (F, x1200) Magnification showing tegumental changes in treated male adult worms: peeling (p), swelling (s), outbreak (o). (G, x3000) and (H, x5000) Magnification showing peeling (p) and bubbles (b) in the tegument promoted by MG-132.</p

Microarray results validation by real-time PCR.

<p>Validation is shown for a group of selected differentially expressed genes in <i>S</i>. <i>mansoni</i> adult worms treated with MG-132 compared with control parasites. Real time PCR data, expressed as Fold Change (normalized to the control group) are displayed as a bar graph while the corresponding data from the microarray (fold change) are shown below in numbers. The asterisk (*) indicates a statistically significant change (p < 0.05, t-test) when comparing treated with control samples.</p

Representative list of 30 induced genes in <i>S</i>. <i>mansoni</i> adult worms in response to MG-132 treatment.

<p>Representative list of 30 induced genes in <i>S</i>. <i>mansoni</i> adult worms in response to MG-132 treatment.</p

Number of genes with expression detected in each category generated by the analysis of microarray experiments.

<p>Number of genes with expression detected in each category generated by the analysis of microarray experiments.</p

Scanning electron microscopy of the tegument of <i>S</i>. <i>mansoni</i> adult worms.

<p>(A, x600) Normal morphology of adult worm tegument. (B, x1200) and (C, x3000) magnification of normal morphology of <i>S</i>. <i>mansoni</i> tegument showing the large number of tubercles (tu) and spines (sp). (D, x5000) Magnification of normal morphology of <i>S</i>. <i>mansoni</i> tegument showing spines (sp). (E, x600) Morphology of <i>S</i>. <i>mansoni</i> tegument after treatment with 50 μM MG-132 for 24 h. (F, x1200) Magnification showing tegumental changes in treated male adult worms: peeling (p), swelling (s), outbreak (o). (G, x3000) and (H, x5000) Magnification showing peeling (p) and bubbles (b) in the tegument promoted by MG-132.</p