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Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum.

Song L, Li J, Xie S, Qian C, Wang J, Zhang W, Yin X, Hua Z, Yu C - PLoS ONE (2012)

Bottom Line: These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths.Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu, People's Republic of China.

ABSTRACT

Background: Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and findings: After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions: Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

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Analysis of isotope 75Se-cysteine incorporation.(A) Profile of expressed protein products on a 12% SDS-PAGE gel. (B) Autoradiogram profile of expressed products corresponding to the proteins on SDS-PAGE. Lane 1: Expression products of E.coli BL21 containing plasmid SjTGR-pET41a. Lane 2: Expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. MW: Protein molecular weight marker. The black dots on the SDS-PAGE gel are the expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. The expression products containing isotope 75Se-cysteine (SeCys) were dotted on the SDS-PAGE gel after electrophoresis as a positive control for autoradiography. The black arrow indicates the SjTGR selenoprotein developed by autoradiography.
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pone-0031456-g003: Analysis of isotope 75Se-cysteine incorporation.(A) Profile of expressed protein products on a 12% SDS-PAGE gel. (B) Autoradiogram profile of expressed products corresponding to the proteins on SDS-PAGE. Lane 1: Expression products of E.coli BL21 containing plasmid SjTGR-pET41a. Lane 2: Expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. MW: Protein molecular weight marker. The black dots on the SDS-PAGE gel are the expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. The expression products containing isotope 75Se-cysteine (SeCys) were dotted on the SDS-PAGE gel after electrophoresis as a positive control for autoradiography. The black arrow indicates the SjTGR selenoprotein developed by autoradiography.

Mentions: The selenocysteine insertion during selenoprotein translation in E. coli requires an E. coli-type SECIS element just following the UGA codon in the selenoprotein mRNA, as well as the products of the selA, selB, and selC and selD genes [31]. In this study, the recombinant SjTGR-pET41a plasmid was co-transformed into E. coli BL21 with the pSUABC vector expressing the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNASec, respectively) to aid in the production of SjTGR as a putative selenoprotein. When the cells were induced with 1 mM IPTG for 4 h at 37°C, the SjTGR proteins were expressed in inclusion bodies, which appeared in the precipitate of the bacterial lysate obtained by ultrasonication. Little soluble recombinant SjTGR was found in the supernatant of the bacterial lysate (Fig. 2A). However, when the growth temperature was reduced to 24°C and the bacteria were induced with IPTG for 24 h beginning at the stationary growth stage (OD600 nm of culture reaching 2.4) [38], soluble SjTGR proteins were produced successfully (Fig. 2B). The selenocysteine incorporation analysis using 75Se-labeling cysteine and autoradiograms showed that the products of selA, selB, and selC could increase the yield of SjTGR, suggesting that it is a selenoprotein (Fig. 3).


Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum.

Song L, Li J, Xie S, Qian C, Wang J, Zhang W, Yin X, Hua Z, Yu C - PLoS ONE (2012)

Analysis of isotope 75Se-cysteine incorporation.(A) Profile of expressed protein products on a 12% SDS-PAGE gel. (B) Autoradiogram profile of expressed products corresponding to the proteins on SDS-PAGE. Lane 1: Expression products of E.coli BL21 containing plasmid SjTGR-pET41a. Lane 2: Expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. MW: Protein molecular weight marker. The black dots on the SDS-PAGE gel are the expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. The expression products containing isotope 75Se-cysteine (SeCys) were dotted on the SDS-PAGE gel after electrophoresis as a positive control for autoradiography. The black arrow indicates the SjTGR selenoprotein developed by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285170&req=5

pone-0031456-g003: Analysis of isotope 75Se-cysteine incorporation.(A) Profile of expressed protein products on a 12% SDS-PAGE gel. (B) Autoradiogram profile of expressed products corresponding to the proteins on SDS-PAGE. Lane 1: Expression products of E.coli BL21 containing plasmid SjTGR-pET41a. Lane 2: Expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. MW: Protein molecular weight marker. The black dots on the SDS-PAGE gel are the expression products of E.coli BL21 containing plasmids SjTGR-pET41a and pSUABC. The expression products containing isotope 75Se-cysteine (SeCys) were dotted on the SDS-PAGE gel after electrophoresis as a positive control for autoradiography. The black arrow indicates the SjTGR selenoprotein developed by autoradiography.
Mentions: The selenocysteine insertion during selenoprotein translation in E. coli requires an E. coli-type SECIS element just following the UGA codon in the selenoprotein mRNA, as well as the products of the selA, selB, and selC and selD genes [31]. In this study, the recombinant SjTGR-pET41a plasmid was co-transformed into E. coli BL21 with the pSUABC vector expressing the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNASec, respectively) to aid in the production of SjTGR as a putative selenoprotein. When the cells were induced with 1 mM IPTG for 4 h at 37°C, the SjTGR proteins were expressed in inclusion bodies, which appeared in the precipitate of the bacterial lysate obtained by ultrasonication. Little soluble recombinant SjTGR was found in the supernatant of the bacterial lysate (Fig. 2A). However, when the growth temperature was reduced to 24°C and the bacteria were induced with IPTG for 24 h beginning at the stationary growth stage (OD600 nm of culture reaching 2.4) [38], soluble SjTGR proteins were produced successfully (Fig. 2B). The selenocysteine incorporation analysis using 75Se-labeling cysteine and autoradiograms showed that the products of selA, selB, and selC could increase the yield of SjTGR, suggesting that it is a selenoprotein (Fig. 3).

Bottom Line: These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths.Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu, People's Republic of China.

ABSTRACT

Background: Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and findings: After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions: Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

Show MeSH
Related in: MedlinePlus