Parasitol Res (2015) 114:1747–1760 DOI 10.1007/s00436-015-4360-z
ORIGINAL PAPER
Proteasome stress responses in Schistosoma mansoni Renato Graciano de Paula & Alice Maria de Magalhães Ornelas & Enyara Rezende Morais & Matheus de Souza Gomes & Daniela de Paula Aguiar & Lizandra Guidi Magalhães & Vanderlei Rodrigues
Received: 6 September 2014 / Accepted: 30 January 2015 / Published online: 10 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The proteasome proteolytic system is the major ATPdependent protease in eukaryotic cells responsible for intracellular protein turnover. Schistosoma mansoni has been reported to contain an ubiquitin–proteasome proteolytic pathway, and many studies have suggested a biological role of proteasomes in the development of this parasite. Additionally, evidence has suggested diversity in proteasome composition under several cellular conditions, and this might contribute to the regulation of its function in this parasite. The proteasomal system has been considered important to support the protein homeostasis during cellular stress. In this study, we described in vitro effects of oxidative stress, heat shock, and chemical stress on S. mansoni adults. Our findings showed that chemical stress induced with curcumin, IBMX, and MG132 modified the gene expression of the proteasomal enzymes SmHul5 and SmUbp6. Likewise, the
Electronic supplementary material The online version of this article (doi:10.1007/s00436-015-4360-z) contains supplementary material, which is available to authorized users. R. G. de Paula (*) : V. Rodrigues Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida dos Bandeirantes, 3900, 14040-900 Ribeirão Preto, São Paulo, Brazil e-mail: [email protected]
expression of these genes was upregulated during oxidative stress and heat shock. Analyses of the S. mansoni life cycle showed differential gene expression in sporocysts, schistosomulae, and miracidia. These results suggested that proteasome accessory proteins participate in stress response during the parasite development. The expression level of SmHul5 and SmUbp6 was decreased by 16-fold and 9-fold, respectively, by the chemical stress induced with IBMX, which suggests proteasome disassembly. On the other hand, curcumin, MG132, oxidative stress, and heat shock increased the expression of these genes. Furthermore, the gene expression of maturation proteasome protein (SmPOMP) was increased in stress conditions induced by curcumin, MG132, and H2O2, which could be related to the synthesis of new proteasomes. S. mansoni adult worms were found to utilize similar mechanisms to respond to different conditions of stress. Our results demonstrated that oxidative stress, heat shock, and chemical stress modified the expression profile of genes related to the ubiquitin–proteasome system and suggested that the proteasome might be important in the cellular stress response in this parasite. Keywords S. mansoni . Proteasome . Stress response . E3 ligases . Deubiquitinases
A. M. de Magalhães Ornelas Instituto de Biologia, Centro de Ciências e Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21943-970 Rio de Janeiro, Rio de Janeiro, Brazil E. R. Morais : M. de Souza Gomes Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Campus Avançado Patos de Minas, Av. Getúlio Vargas, Palácio dos Cristais-Centro, 38700-126 Patos de Minas, Minas Gerais, Brazil D. de Paula Aguiar : L. G. Magalhães Grupo de Pesquisa em Produtos Naturais, Núcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca, 14404-600 Franca, SP, Brazil
Introduction Schistosoma mansoni is one of the causative agents of schistosomiasis, a severe disease affecting almost 240 million people worldwide in tropical and subtropical areas. Over 700 million individuals live in endemic areas (WHO 2013; Steinmann et al. 2006). Over the course of their life cycle, these parasites are exposed to different sources of stress (Coelho and Bezerra 2006; Ruelas et al. 2007).
1748
An important system involved with stress response is the proteasome, a proteolytic system that controls many cellular processes (Schreiber and Peter 2014). The ubiquitin–proteasome pathway first processes the attachment of ubiquitin to a target protein, which involves three critical enzymes, the ubiquitin activating enzyme or E1 enzyme, the ubiquitin conjugating enzyme or E2 enzyme, and the ubiquitin ligase or E3 ligase (Pickart and Eddins 2004). Also, this multicatalytic system may associate with different accessory proteins that regulate both protein function and degradation (Hanna et al. 2006; Pickett 2007; Besche et al. 2009). The E3 ligase enzymes are crucial for the ubiquitination process and are involved in activation of E2 enzymes or acceptors of ubiquitin molecules, promoting the transfer of ubiquitin to protein substrates that are destined to proteasome degradation (Wolf 2004). However, the proteasome functionality is due to the interaction between different enzymatic systems. Therefore, both ubiquitin ligases and deubiquitinating enzymes are extremely important and play diverse functions in the regulation of ubiquitin–proteasome pathway (Komander et al. 2009; Reyes-Turcu et al. 2009). Interestingly, ubiquitin protein ligase H ul5 a nd deubiquitinating enzyme Ubp6 have been described as abundant components in the proteasome (Leggett et al. 2002). These enzymes have antagonistic cellular activities; in other words, Hul5 is responsible for the ubiquitin chain elongation whereas Ubp6 promotes the cleavage of ubiquitin chains extended by Hul5 (Kraut et al. 2007). Thus, the mutual action of these two enzymes might contribute as an additional mechanism to regulate the protein degradation, once Hul5 favors the proteolysis by increasing the time the substrates remain in the proteasome, and Ubp6 protects the proteasome from overload by promoting the substrate release (Kraut et al. 2007). Moreover, it was shown that direct association of Ubp6 with the proteasome delays the substrate degradation noncatalytically (Hanna et al. 2006; Lee et al. 2010). In addition, Ubp6 was also shown to regulate the opening of 20S gate by manipulating the interaction between the proteasome and ubiquitinated substrates (Peth et al. 2009; Lander et al. 2012). Hanna et al. (2003) revealed the crucial role of Ubp6 in ubiquitin turnover in a yeast mutant lacking the UBP6 gene. In the absence of this protein, the half-life of ubiquitin is dramatically reduced. Recently, Hul5 was shown to be required for the maintenance of cell fitness and increased ubiquitination of low solubility proteins after heat shock (Fang et al. 2011). Furthermore, Hul5 was described to be recruited to the proteasome in different stress conditions, including heat shock (Park et al. 2011). Nevertheless, the mechanism by which Hul5 promotes the ubiquitination increase and recognizes unfolded protein after heat shock remains unclear (Finley 2011). Fang and Mayor (2012) suggested that different pathways of protein quality control promote the degradation of unfolded proteins in the cytosol and Hul5 is crucial to degrade these proteins under heat shock.
Parasitol Res (2015) 114:1747–1760
Other important activity related to the proteasome and involved in stress response is played by the maturation of the proteasome protein (POMP). The overexpression of hUMP1/ POMP (maturation proteasome protein human homolog) in fibroblasts is associated with increased assembly and functionality of the proteasome as well as ability to respond quickly and effectively to oxidative stress (Chondrogianni and Gonos 2007). This protein is abundantly found in the proteasome precursor complex and degraded after full assembly of the 20S particle. Thus, POMP is a molecular marker that indicates the synthesis of new proteasomes (Griffin et al. 2000; Burri et al. 2000). Under these circumstances, proteasomes are multisubunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Guerra-Sá et al. (2005) reported for the first time biochemical evidence for the existence of an ubiquitin–proteasome proteolytic pathway in S. mansoni. Castro-Borges et al. (2007) identified different proteasome subunits in the transformation of cercaria to the skin schistosomulum stage, suggesting changes in the proteasome structure during parasite development. Likewise, Pereira-Júnior et al. (2013) demonstrated a differential expression profile for proteasome 19S Rpt subunits in cercariae and schistosomulae. Furthermore, Botelho-Machado et al. (2010) characterized the expression profile of SmPI31 through the S. mansoni life cycle. In this study, the first evidence presented that PI31 has a conserved structure and plays a role as a proteasome inhibitor in adult worms and that it is expressed throughout life cycle. Magalhaes et al. (2009) and Morais et al. (2013) demonstrated the schistosomicidal activity of curcumin against S. mansoni adult worms, suggesting that curcumin is a potential inhibitor of proteasome activity and is important to control the role of the proteasome in the development of this parasite. Here, we described the gene expression profile of SmHul5 and SmUbp6 during the S. mansoni life cycle and in adult worms exposed to different conditions of proteasome stress. Our results demonstrated that gene expression of SmHul5 and SmUbp6 was increased under oxidative stress, heat shock, curcumin-induced stress, MG132-induced stress, and decreased under IBMX-induced stress. Furthermore, the expression of SmPOMP was upregulated in response to the stress induced with H2O2, curcumin, and MG132. These results suggest that the proteasome is important to stress response in S. mansoni adult worms.
Material and methods Ethics statement All experiments involving animals were authorized by the Ethics Committee on Animal Experimentation (CETEA) of the University of São Paulo (protocol number 182/2011) and
Parasitol Res (2015) 114:1747–1760
were in accordance with the ethical principles in animal research adopted by the Brazilian School of Animal Experimentation (COBEA). The study protocols were in accordance with internationally accepted principles concerning the care and use of laboratory animals. Parasite maintenance The Luis Evangelista (LE) strain of S. mansoni was maintained by successive passage through Biomphalaria glabrata snails and BALB/c mice. After 56 days, S. mansoni adult worms were recovered under aseptic conditions from mice previously infected with 200 cercariae by perfusion of the livers and mesenteric veins (Smithers and Terry 1965). The paired adult worms were maintained in RPMI 1640 medium (Invitrogen) supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10 % bovine fetal serum (Gibco, Carlsbad, CA) at 37 °C and 5 % CO2, under dark conditions. After 24 h of incubation, S. mansoni adult worms were submitted to cellular stress assays. Cercariae were obtained from infected snails through exposure to artificial illumination at a 26 °C water bath for 1 h to induce larval shedding. To obtain schistosomulae, cercariae were mechanically transformed by vortexing (Ramalho-Pinto et al. 1974) followed by cultivation in sterile 24-well TC plates containing RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 25 mM HEPE S pH 7.5, antibiotics (100 UI/mL penicillin and 100 μg/mL streptomycin), and 10 % bovine fetal serum (Gibco, Carlsbad, CA) for 24 h at 37 °C in 5 % CO2. The liver of the infected mice was homogenized in extraction buffer (0.06 mol/L Na2HPO4, 0.0033 mol/L KH2PO4, pH 8.3) and digested with trypsin for 3 h at 37 °C. Then, the eggs were recovered by double sieving (0.30 mm upper sieve and 0.180 mm lower sieve) using isotonic saline (Ashton et al. 2001). Miracidia used in stage-associated gene expression studies were obtained from these eggs. Miracidia and sporocysts were prepared according to Yoshino and Laursen (1995). Proteasome stress assays Chemical stress was induced in the proteasome with curcumin (Diferulylmethane), MG132 (Z-Leu-Leu-Leu-al), and IBMX (3-Isobutyl-1-methylxanthine). These compounds were dissolved in dimethyl sulfoxide (DMSO). All the reagents used in chemical stress procedures were purchased from Sigma–Aldrich, St. Louis, MO, USA. As the control group, S. mansoni adult worms were maintained in RPMI 1640 culture medium with 1 % DMSO. Curcumin treatment was performed at doses of 5 and 10 μM during 24 h. Under these conditions, less than 20 % of worm pairs became separated (Magalhaes et al. 2009). MG132 and IBMX were used at concentrations of 50 μM (Guerra-Sá et al. 2005) and 80 and 200 μM (Fig. S1, in this concentration, no changes were observed in worm viability),
1749
respectively. MG132 treatment was conducted for 24, 48, 72, and 120 h, and IBMX treatment consisted of 24, 48, and 72 h. Oxidative stress was induced by the addition of H2O2 to the culture medium at an initial concentration of 200 μM (De Paula et al. 2014), and the worms were incubated for 0 (untreated), 30 min, 1 h, and 24 h at 37 °C and 5 % CO2. Finally, the heat shock was performed at 40 °C (Moreira, personal communication) and 42 °C (Aragon et al. 2008) in a water bath, and the worms were then harvested after 1 h of incubation. The control group was maintained at 37 °C and harvested after 1 h. Three biological replicates were assayed for each experimental condition (treated or untreated). Identification of genes encoding proteasome subunits related to stress response in S. mansoni The genomic structure of SmHul5 and SmUbp6 was identified by cDNA–genomic DNA alignments using the BSpidey^ software (Wheelan et al. 2001; Wheeler et al. 2004) (http://www. ncbi.nlm.nih.gov/spidey) and BLAST (bl2seq-NCBI). The putative S. mansoni ortholog sequences, Hul5-like (Smp_ 130650) and Ubp6-like (Smp_084740), were searched at the S. mansoni genome database GeneDB (http://www.genedb. org/genedb/smansoni/) using query sequences retrieved from the National Center for Biotechnology Information (NCBI) database (http://www.Ncbi.nlm.nih.gov) and from Homo sapiens species (NP 055486.2–Hul5-like and NP 005142.1– Ubp6-like) and Drosophila melanogaster species (NP 611896.1–Hul5-like and NP 609377.1–Ubp6-like). The BLASTp algorithm, underpinned by the PFAM (v 26.0) database, was used for searches of conserved protein domains or motifs of S. mansoni sequences (Finn et al. 2010). Multiple alignments of Hul5 and Ubp6 were performed using ClustalX 2.0 with default settings (Larkin et al. 2007). The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei 1987). The bootstrap consensus tree inferred from 2000 replicates represents the evolutionary history of the taxa analyzed (Felsenstein 1985). The tree is drawn to scale, with the same units used for both branch length and the evolutionary distances used to infer the phylogenetic tree. Branches with bootstrap values lower than 30 were hidden. The evolutionary distances were computed using the JTT matrix-based method (Jones et al. 1992) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA 5 (Tamura et al. 2011). Gene expression analyses Total RNA from different S. mansoni stages (LE strain eggs, miracidia, sporocysts, cercariae, schistosomulae, paired and unpaired adult worms) was extracted and isolated using
1750
Trizol® reagent (Invitrogen, Carlsbad, CA). The isolated RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water and subjected to DNAse treatment using the DNAse I (Promega, Madison, WI) to eliminate genomic DNA contamination. RNA samples were quantified and their purity assessed through Nanodrop (Spectometer ND-1000, Thermo Scientific). One microgram of DNAse-treated RNA was used as a template to synthesize cDNA using the ThermoScript™ RT-PCR System (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. S. mansoni-specific primers were designed using Primer3 software (Rozen and Skaletsky 2000). The sequence accession numbers and the respective pair of primers used in this study were as follows: SmHul5 (Smp_130650) forward, 5′-CAACTGGCTTAGCTGAAG TTGG-3′; reverse 5′-GCAGATGCTTGTGGATTTGG-3′, SmUbp6 (Smp_084740) forward 5′-ACCTGGCCTCGTAA ATCTTG-3′, reverse 5′-GCGATATTTCGTCGAGCTTC-3′, SmPOMP (Smp_074160), forward 5′-TCCTGAATTCCC TGTTCGAC-3′, reverse 5′-TTCGCTTTTTCAGCTGCTTT3′. Reverse-transcribed cDNA samples were used as templates for PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7500 System (Applied Biosystems, Rio de Janeiro, Brazil). Specific primers for S. mansoni GAPDH were used as endogenous control (Smp_056970.1 forward, 5′-TCGTTGAGTCTACTGGAG TCTTTACG-3′, reverse 5′-AATATGAGCCTGAGCTTTAT CAATGG-3′) (Mourão et al. 2009). The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied Biosystems application (cDNA dilutions were 1:10, 1:100, and 1:1000). For all investigated transcripts, three biological replicates were performed. To PCR amplification, the samples were incubated at 95 °C for 10 min and submitted to 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The gene expression of SmHul5 and SmUbp6 during the S. mansoni life cycle was normalized to the GAPDH transcript using the Applied Biosystems 7500 software according to the 2−ΔCt method (Livak and Schmittgen 2001). Moreover, the gene expression of SmHul5 and SmUbp6 was analyzed in the parasites under oxidative stress, heat shock, and chemical stress. Additionally, the SmPOMP gene expression was investigated in S. mansoni adult worms exposed to curcumininduced stress, MG132-induced stress, and oxidative stress. In this case, relative expression of RNA in each sample was calculated. The comparative Ct method (2−ΔΔCT method) was used for gene expression analysis. Data were normalized relative to an endogenous standard gene (GAPDH) and calculated as the fold change in expression levels relative to the untreated group (calibrator) (Livak and Schmittgen 2001). Statistical analysis Statistical tests were performed using GraphPad Prism® software v.5.0 (GraphPad Software incorporation, San Diego,
Parasitol Res (2015) 114:1747–1760
CA). Significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Significance level was set at P
ORIGINAL PAPER
Proteasome stress responses in Schistosoma mansoni Renato Graciano de Paula & Alice Maria de Magalhães Ornelas & Enyara Rezende Morais & Matheus de Souza Gomes & Daniela de Paula Aguiar & Lizandra Guidi Magalhães & Vanderlei Rodrigues
Received: 6 September 2014 / Accepted: 30 January 2015 / Published online: 10 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The proteasome proteolytic system is the major ATPdependent protease in eukaryotic cells responsible for intracellular protein turnover. Schistosoma mansoni has been reported to contain an ubiquitin–proteasome proteolytic pathway, and many studies have suggested a biological role of proteasomes in the development of this parasite. Additionally, evidence has suggested diversity in proteasome composition under several cellular conditions, and this might contribute to the regulation of its function in this parasite. The proteasomal system has been considered important to support the protein homeostasis during cellular stress. In this study, we described in vitro effects of oxidative stress, heat shock, and chemical stress on S. mansoni adults. Our findings showed that chemical stress induced with curcumin, IBMX, and MG132 modified the gene expression of the proteasomal enzymes SmHul5 and SmUbp6. Likewise, the
Electronic supplementary material The online version of this article (doi:10.1007/s00436-015-4360-z) contains supplementary material, which is available to authorized users. R. G. de Paula (*) : V. Rodrigues Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida dos Bandeirantes, 3900, 14040-900 Ribeirão Preto, São Paulo, Brazil e-mail: [email protected]
expression of these genes was upregulated during oxidative stress and heat shock. Analyses of the S. mansoni life cycle showed differential gene expression in sporocysts, schistosomulae, and miracidia. These results suggested that proteasome accessory proteins participate in stress response during the parasite development. The expression level of SmHul5 and SmUbp6 was decreased by 16-fold and 9-fold, respectively, by the chemical stress induced with IBMX, which suggests proteasome disassembly. On the other hand, curcumin, MG132, oxidative stress, and heat shock increased the expression of these genes. Furthermore, the gene expression of maturation proteasome protein (SmPOMP) was increased in stress conditions induced by curcumin, MG132, and H2O2, which could be related to the synthesis of new proteasomes. S. mansoni adult worms were found to utilize similar mechanisms to respond to different conditions of stress. Our results demonstrated that oxidative stress, heat shock, and chemical stress modified the expression profile of genes related to the ubiquitin–proteasome system and suggested that the proteasome might be important in the cellular stress response in this parasite. Keywords S. mansoni . Proteasome . Stress response . E3 ligases . Deubiquitinases
A. M. de Magalhães Ornelas Instituto de Biologia, Centro de Ciências e Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, 21943-970 Rio de Janeiro, Rio de Janeiro, Brazil E. R. Morais : M. de Souza Gomes Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Campus Avançado Patos de Minas, Av. Getúlio Vargas, Palácio dos Cristais-Centro, 38700-126 Patos de Minas, Minas Gerais, Brazil D. de Paula Aguiar : L. G. Magalhães Grupo de Pesquisa em Produtos Naturais, Núcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca, 14404-600 Franca, SP, Brazil
Introduction Schistosoma mansoni is one of the causative agents of schistosomiasis, a severe disease affecting almost 240 million people worldwide in tropical and subtropical areas. Over 700 million individuals live in endemic areas (WHO 2013; Steinmann et al. 2006). Over the course of their life cycle, these parasites are exposed to different sources of stress (Coelho and Bezerra 2006; Ruelas et al. 2007).
1748
An important system involved with stress response is the proteasome, a proteolytic system that controls many cellular processes (Schreiber and Peter 2014). The ubiquitin–proteasome pathway first processes the attachment of ubiquitin to a target protein, which involves three critical enzymes, the ubiquitin activating enzyme or E1 enzyme, the ubiquitin conjugating enzyme or E2 enzyme, and the ubiquitin ligase or E3 ligase (Pickart and Eddins 2004). Also, this multicatalytic system may associate with different accessory proteins that regulate both protein function and degradation (Hanna et al. 2006; Pickett 2007; Besche et al. 2009). The E3 ligase enzymes are crucial for the ubiquitination process and are involved in activation of E2 enzymes or acceptors of ubiquitin molecules, promoting the transfer of ubiquitin to protein substrates that are destined to proteasome degradation (Wolf 2004). However, the proteasome functionality is due to the interaction between different enzymatic systems. Therefore, both ubiquitin ligases and deubiquitinating enzymes are extremely important and play diverse functions in the regulation of ubiquitin–proteasome pathway (Komander et al. 2009; Reyes-Turcu et al. 2009). Interestingly, ubiquitin protein ligase H ul5 a nd deubiquitinating enzyme Ubp6 have been described as abundant components in the proteasome (Leggett et al. 2002). These enzymes have antagonistic cellular activities; in other words, Hul5 is responsible for the ubiquitin chain elongation whereas Ubp6 promotes the cleavage of ubiquitin chains extended by Hul5 (Kraut et al. 2007). Thus, the mutual action of these two enzymes might contribute as an additional mechanism to regulate the protein degradation, once Hul5 favors the proteolysis by increasing the time the substrates remain in the proteasome, and Ubp6 protects the proteasome from overload by promoting the substrate release (Kraut et al. 2007). Moreover, it was shown that direct association of Ubp6 with the proteasome delays the substrate degradation noncatalytically (Hanna et al. 2006; Lee et al. 2010). In addition, Ubp6 was also shown to regulate the opening of 20S gate by manipulating the interaction between the proteasome and ubiquitinated substrates (Peth et al. 2009; Lander et al. 2012). Hanna et al. (2003) revealed the crucial role of Ubp6 in ubiquitin turnover in a yeast mutant lacking the UBP6 gene. In the absence of this protein, the half-life of ubiquitin is dramatically reduced. Recently, Hul5 was shown to be required for the maintenance of cell fitness and increased ubiquitination of low solubility proteins after heat shock (Fang et al. 2011). Furthermore, Hul5 was described to be recruited to the proteasome in different stress conditions, including heat shock (Park et al. 2011). Nevertheless, the mechanism by which Hul5 promotes the ubiquitination increase and recognizes unfolded protein after heat shock remains unclear (Finley 2011). Fang and Mayor (2012) suggested that different pathways of protein quality control promote the degradation of unfolded proteins in the cytosol and Hul5 is crucial to degrade these proteins under heat shock.
Parasitol Res (2015) 114:1747–1760
Other important activity related to the proteasome and involved in stress response is played by the maturation of the proteasome protein (POMP). The overexpression of hUMP1/ POMP (maturation proteasome protein human homolog) in fibroblasts is associated with increased assembly and functionality of the proteasome as well as ability to respond quickly and effectively to oxidative stress (Chondrogianni and Gonos 2007). This protein is abundantly found in the proteasome precursor complex and degraded after full assembly of the 20S particle. Thus, POMP is a molecular marker that indicates the synthesis of new proteasomes (Griffin et al. 2000; Burri et al. 2000). Under these circumstances, proteasomes are multisubunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Guerra-Sá et al. (2005) reported for the first time biochemical evidence for the existence of an ubiquitin–proteasome proteolytic pathway in S. mansoni. Castro-Borges et al. (2007) identified different proteasome subunits in the transformation of cercaria to the skin schistosomulum stage, suggesting changes in the proteasome structure during parasite development. Likewise, Pereira-Júnior et al. (2013) demonstrated a differential expression profile for proteasome 19S Rpt subunits in cercariae and schistosomulae. Furthermore, Botelho-Machado et al. (2010) characterized the expression profile of SmPI31 through the S. mansoni life cycle. In this study, the first evidence presented that PI31 has a conserved structure and plays a role as a proteasome inhibitor in adult worms and that it is expressed throughout life cycle. Magalhaes et al. (2009) and Morais et al. (2013) demonstrated the schistosomicidal activity of curcumin against S. mansoni adult worms, suggesting that curcumin is a potential inhibitor of proteasome activity and is important to control the role of the proteasome in the development of this parasite. Here, we described the gene expression profile of SmHul5 and SmUbp6 during the S. mansoni life cycle and in adult worms exposed to different conditions of proteasome stress. Our results demonstrated that gene expression of SmHul5 and SmUbp6 was increased under oxidative stress, heat shock, curcumin-induced stress, MG132-induced stress, and decreased under IBMX-induced stress. Furthermore, the expression of SmPOMP was upregulated in response to the stress induced with H2O2, curcumin, and MG132. These results suggest that the proteasome is important to stress response in S. mansoni adult worms.
Material and methods Ethics statement All experiments involving animals were authorized by the Ethics Committee on Animal Experimentation (CETEA) of the University of São Paulo (protocol number 182/2011) and
Parasitol Res (2015) 114:1747–1760
were in accordance with the ethical principles in animal research adopted by the Brazilian School of Animal Experimentation (COBEA). The study protocols were in accordance with internationally accepted principles concerning the care and use of laboratory animals. Parasite maintenance The Luis Evangelista (LE) strain of S. mansoni was maintained by successive passage through Biomphalaria glabrata snails and BALB/c mice. After 56 days, S. mansoni adult worms were recovered under aseptic conditions from mice previously infected with 200 cercariae by perfusion of the livers and mesenteric veins (Smithers and Terry 1965). The paired adult worms were maintained in RPMI 1640 medium (Invitrogen) supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10 % bovine fetal serum (Gibco, Carlsbad, CA) at 37 °C and 5 % CO2, under dark conditions. After 24 h of incubation, S. mansoni adult worms were submitted to cellular stress assays. Cercariae were obtained from infected snails through exposure to artificial illumination at a 26 °C water bath for 1 h to induce larval shedding. To obtain schistosomulae, cercariae were mechanically transformed by vortexing (Ramalho-Pinto et al. 1974) followed by cultivation in sterile 24-well TC plates containing RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 25 mM HEPE S pH 7.5, antibiotics (100 UI/mL penicillin and 100 μg/mL streptomycin), and 10 % bovine fetal serum (Gibco, Carlsbad, CA) for 24 h at 37 °C in 5 % CO2. The liver of the infected mice was homogenized in extraction buffer (0.06 mol/L Na2HPO4, 0.0033 mol/L KH2PO4, pH 8.3) and digested with trypsin for 3 h at 37 °C. Then, the eggs were recovered by double sieving (0.30 mm upper sieve and 0.180 mm lower sieve) using isotonic saline (Ashton et al. 2001). Miracidia used in stage-associated gene expression studies were obtained from these eggs. Miracidia and sporocysts were prepared according to Yoshino and Laursen (1995). Proteasome stress assays Chemical stress was induced in the proteasome with curcumin (Diferulylmethane), MG132 (Z-Leu-Leu-Leu-al), and IBMX (3-Isobutyl-1-methylxanthine). These compounds were dissolved in dimethyl sulfoxide (DMSO). All the reagents used in chemical stress procedures were purchased from Sigma–Aldrich, St. Louis, MO, USA. As the control group, S. mansoni adult worms were maintained in RPMI 1640 culture medium with 1 % DMSO. Curcumin treatment was performed at doses of 5 and 10 μM during 24 h. Under these conditions, less than 20 % of worm pairs became separated (Magalhaes et al. 2009). MG132 and IBMX were used at concentrations of 50 μM (Guerra-Sá et al. 2005) and 80 and 200 μM (Fig. S1, in this concentration, no changes were observed in worm viability),
1749
respectively. MG132 treatment was conducted for 24, 48, 72, and 120 h, and IBMX treatment consisted of 24, 48, and 72 h. Oxidative stress was induced by the addition of H2O2 to the culture medium at an initial concentration of 200 μM (De Paula et al. 2014), and the worms were incubated for 0 (untreated), 30 min, 1 h, and 24 h at 37 °C and 5 % CO2. Finally, the heat shock was performed at 40 °C (Moreira, personal communication) and 42 °C (Aragon et al. 2008) in a water bath, and the worms were then harvested after 1 h of incubation. The control group was maintained at 37 °C and harvested after 1 h. Three biological replicates were assayed for each experimental condition (treated or untreated). Identification of genes encoding proteasome subunits related to stress response in S. mansoni The genomic structure of SmHul5 and SmUbp6 was identified by cDNA–genomic DNA alignments using the BSpidey^ software (Wheelan et al. 2001; Wheeler et al. 2004) (http://www. ncbi.nlm.nih.gov/spidey) and BLAST (bl2seq-NCBI). The putative S. mansoni ortholog sequences, Hul5-like (Smp_ 130650) and Ubp6-like (Smp_084740), were searched at the S. mansoni genome database GeneDB (http://www.genedb. org/genedb/smansoni/) using query sequences retrieved from the National Center for Biotechnology Information (NCBI) database (http://www.Ncbi.nlm.nih.gov) and from Homo sapiens species (NP 055486.2–Hul5-like and NP 005142.1– Ubp6-like) and Drosophila melanogaster species (NP 611896.1–Hul5-like and NP 609377.1–Ubp6-like). The BLASTp algorithm, underpinned by the PFAM (v 26.0) database, was used for searches of conserved protein domains or motifs of S. mansoni sequences (Finn et al. 2010). Multiple alignments of Hul5 and Ubp6 were performed using ClustalX 2.0 with default settings (Larkin et al. 2007). The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei 1987). The bootstrap consensus tree inferred from 2000 replicates represents the evolutionary history of the taxa analyzed (Felsenstein 1985). The tree is drawn to scale, with the same units used for both branch length and the evolutionary distances used to infer the phylogenetic tree. Branches with bootstrap values lower than 30 were hidden. The evolutionary distances were computed using the JTT matrix-based method (Jones et al. 1992) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA 5 (Tamura et al. 2011). Gene expression analyses Total RNA from different S. mansoni stages (LE strain eggs, miracidia, sporocysts, cercariae, schistosomulae, paired and unpaired adult worms) was extracted and isolated using
1750
Trizol® reagent (Invitrogen, Carlsbad, CA). The isolated RNA was resuspended in diethylpyrocarbonate (DEPC)-treated water and subjected to DNAse treatment using the DNAse I (Promega, Madison, WI) to eliminate genomic DNA contamination. RNA samples were quantified and their purity assessed through Nanodrop (Spectometer ND-1000, Thermo Scientific). One microgram of DNAse-treated RNA was used as a template to synthesize cDNA using the ThermoScript™ RT-PCR System (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. S. mansoni-specific primers were designed using Primer3 software (Rozen and Skaletsky 2000). The sequence accession numbers and the respective pair of primers used in this study were as follows: SmHul5 (Smp_130650) forward, 5′-CAACTGGCTTAGCTGAAG TTGG-3′; reverse 5′-GCAGATGCTTGTGGATTTGG-3′, SmUbp6 (Smp_084740) forward 5′-ACCTGGCCTCGTAA ATCTTG-3′, reverse 5′-GCGATATTTCGTCGAGCTTC-3′, SmPOMP (Smp_074160), forward 5′-TCCTGAATTCCC TGTTCGAC-3′, reverse 5′-TTCGCTTTTTCAGCTGCTTT3′. Reverse-transcribed cDNA samples were used as templates for PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7500 System (Applied Biosystems, Rio de Janeiro, Brazil). Specific primers for S. mansoni GAPDH were used as endogenous control (Smp_056970.1 forward, 5′-TCGTTGAGTCTACTGGAG TCTTTACG-3′, reverse 5′-AATATGAGCCTGAGCTTTAT CAATGG-3′) (Mourão et al. 2009). The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied Biosystems application (cDNA dilutions were 1:10, 1:100, and 1:1000). For all investigated transcripts, three biological replicates were performed. To PCR amplification, the samples were incubated at 95 °C for 10 min and submitted to 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The gene expression of SmHul5 and SmUbp6 during the S. mansoni life cycle was normalized to the GAPDH transcript using the Applied Biosystems 7500 software according to the 2−ΔCt method (Livak and Schmittgen 2001). Moreover, the gene expression of SmHul5 and SmUbp6 was analyzed in the parasites under oxidative stress, heat shock, and chemical stress. Additionally, the SmPOMP gene expression was investigated in S. mansoni adult worms exposed to curcumininduced stress, MG132-induced stress, and oxidative stress. In this case, relative expression of RNA in each sample was calculated. The comparative Ct method (2−ΔΔCT method) was used for gene expression analysis. Data were normalized relative to an endogenous standard gene (GAPDH) and calculated as the fold change in expression levels relative to the untreated group (calibrator) (Livak and Schmittgen 2001). Statistical analysis Statistical tests were performed using GraphPad Prism® software v.5.0 (GraphPad Software incorporation, San Diego,
Parasitol Res (2015) 114:1747–1760
CA). Significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Significance level was set at P
