Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

Identification of Thioredoxin Glutathione Reductase Inhibitors That Kill Cestode and Trematode Parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites

Thioredoxin Glutathione Reductase as a Novel Drug Target: Evidence from Schistosoma japonicum

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 Trx

Användingen av olika brottspåföljder mot ungdomar under 21 år i Sverige under 1950-talet

See abstract in article.</jats:p

Brottslighetsutvecklingen i Sverige under 1950-talet

See abstract in article.</jats:p

Stretching the genetic code : Incorporation of selenocysteine at specific UGA codons in recombinant proteins produced in Escherichia coli

Selenocysteine (Sec) exists in all domains of life and represents the 21st naturally occurring amino acid. A Sec residue is co-translationally incorporated at a predefined opal (UGA) codon. UGA codons normally encode for translational stop via the protein release factor 2 (RF2). The incorporation mechanism of Sec into the selenoprotein involves complex machineries, dependent on several specific factors that differ between organisms. For both eukaryotes and prokaryotes, an mRNA secondary structure, called a Sec insertion sequence (SECIS) element, is required. Sec insertion systems for eukaryotes are different from that of bacteria. Due to the differences between species, recombinant expression of eukaryotic selenoproteins in E. coli is not a trivial task. However, our group has previously been able to overcome this species barrier and successfully expressed the mammalian selenoprotein thioredoxin reductase 1 (TrxR1) and other selenoproteins in E. coli. In this thesis, we have further developed this recombinant selenoprotein production system. We have also further characterized the recombinantly expressed rat TrxR1. We have studied growth conditions affecting yield of the recombinant selenoprotein when expressing rat TrxR1, using various levels of the selenoproteinencoding mRNA and growth in different types of medium. Guided by Principal Component Analysis (PCA), we discovered that the most efficient bacterial selenoprotein production conditions were obtained using high-transcription levels in the presence of the selA, selB and selC genes, with induction of production at late exponential phase. We also constructed an E. coli strain with the endogenous chromosomal promoter of the gene for relase factor 2 (RF2), prfB, replaced with the titrable PBAD promoter. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and on recombinant selenoprotein expression was studied, using rat TrxR1 as the model selenoprotein. This showed that lower levels of RF2 correlate directly to an increase of Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a slower growth rate. Recombinant rat TrxR is expressed as a mixture of full-length and twoamino acid truncated subunits. Phenylarsine oxide (PAO) Sepharose can be used to enrich the Sec-containing protein. We investigated the mechanism of this purification by extensively purifying recombinant rat TrxR1, which gave an enzyme with about 53 U/mg in specific activity, which was higher than ever reported. Surprisingly, onlyabout 65% of the subunits of this TrxR1 preparation contained Sec, which revealed a theoretical maximal specific activity of about 80 U/mg for TrxR with full Sec content. The high specific activity revealed that the inherent turnover capacity of rat TrxR1 must be revised, and that the efficiency of bacterial Sec incorporation may be lower than previously believed. We also discovered and characterized tetrameric forms of recombinant TrxR1, having about half the specific activity compared to the dimeric protein in relation to Sec content. In conclusion, this thesis describes limiting factors for recombinant selenoprotein production in E. coli and shows how this production system can be optimized for higher yield and specificity. The results may prove to be of importance for the further development of E. coli as a useful source for synthetic selenoproteins. Results are also presented and discussed regarding the catalytic capacity of rat TrxR1 and novel multimeric states of the protein, which could represent unknown regulatory features of TrxR having potential physiological importance