Analysis of the Interactions Between Thioredoxin and 20 Selenoproteins in Chicken

Thioredoxin (Trx) is a small molecular protein with complicated functions in a number of processes, including inflammation, apoptosis, embryogenesis, cardiovascular disease, and redox regulation. Some selenoproteins, such as glutathione peroxidase (Gpx), iodothyronine deiodinase (Dio), and thioredoxin reductase (TR), are involved in redox regulation. However, whether there are interactions between Trx and selenoproteins is still not known. In the present paper, we used a Modeller, Hex 8.0.0, and the KFC2 Server to predict the interactions between Trx and selenoproteins. We used the Modeller to predict the target protein in objective format and assess the accuracy of the results. Molecular interaction studies with Trx and selenoproteins were performed using the molecular docking tools in Hex 8.0.0. Next, we used the KFC2 Server to further test the protein binding sites. In addition to the selenoprotein physiological functions, we also explored potential relationships between Trx and selenoproteins beyond all the results we got. The results demonstrate that Trx has the potential to interact with 19 selenoproteins, including iodothyronine deiodinase 1 (Dio1), iodothyronine deiodinase 3 (Dio3), glutathione peroxidase 1 (Gpx1), glutathione peroxidase 2 (Gpx2), glutathione peroxidase 3 (Gpx3), glutathione peroxidase 4 (Gpx4), selenoprotein H (SelH), selenoprotein I (SelI), selenoprotein M (SelM), selenoprotein N (SelN), selenoprotein T (SelT), selenoprotein U (SelU), selenoprotein W (SelW), selenoprotein 15 (Sep15), methionine sulfoxide reductase B (Sepx1), selenophosphate synthetase 1 (SPS1), TR1, TR2, and TR3, among which TR1, TR2, TR3, SPS1, Sep15, SelN, SelM, SelI, Gpx2, Gpx3, Gpx4, and Dio3 exhibited intense correlations with Trx. However, additional experiments are needed to verify them.     

Targeting thioredoxin reductase 1 reduction in cancer cells inhibits self-sufficient growth and DNA replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase alpha, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.     

Decreased Expression of Selenoproteins as a Poor Prognosticator of Gastric Cancer in Humans

The aim of the present study was to analyze the selenoprotein expression levels in gastric cancer patients. We enrolled 40 patients (29 males, 11 females) who were recently diagnosed with gastric cancer and 50 healthy people (30 males, 20 females) as controls. The expression of 25 selenoprotein genes (Dio1, Dio2, Dio3, Gpx1, Gpx2, Gpx3, Gpx4, Gpx6, SelH, SelI, SelK, SelM, SelN, SelO, SelP, SelS, SelT, SelV, SelW, SelX, Sel15, Sps2, TR1, TR2, and TR3) in human gastric cancer tissues, para-carcinoma tissues, adjacent normal gastric tissues, erythrocytes, and lymphocytes in the gastric cancer group and healthy control group was analyzed by qRT-PCR. Here, we showed that among the 25 selenoproteins, 13 selenoproteins in erythrocytes (Gpx1, Gpx4, Sel15, TR1, TR2, SelH, SelK, SelM, SelO, SelS, SelV, SelW, and Sps2), 15 selenoproteins in lymphocytes (Gpx1, Gpx4, Sel15, TR1, TR2, SelH, SelK, SelN, SelO, SelS, SelT, SelV, SelX, SelW, and Sps2) and 13 selenoproteins in gastric cancer and para-carcinoma tissues (Dio1, Dio2, Dio3, Gpx1, Gpx4, Sel15, SelH, SelK, SelM, SelS, SelT, SelW, and Sps2) were significantly decreased (P < 0.05) in the gastric cancer group compared to the control group. In summary, the decreasing expression of selenoprotein genes in gastric cancer patients play an important role in the gastric cancer, although further studies are needed to better understand our findings.     

High Error Rates in Selenocysteine Insertion in Mammalian Cells Treated with the Antibiotic Doxycycline,Chloramphenicol, or Geneticin

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.     

Titration and conditional knockdown of the prfB gene in Escherichia coli: effects on growth and overproduction of the recombinant mammalian selenoprotein thioredoxin reductase

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P(BAD) promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P(BAD)-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.     

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.     

Selenoprotein expression in endothelial cells from different human vasculature and species

Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.     

Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.     

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.     

Regulation of selenoprotein mRNA expression by hormones and retinoic acid in bovine mammary cells

Selenium is essential for maintaining many body functions through the actions of selenoproteins. To find factors regulating selenoprotein biosynthesis in the bovine mammary cell line MAC-T, the effects of supplementation with selenite and also with retinoic acid, insulin, hydrocortisone and prolactin on the mRNA expression of a number of selenoproteins were investigated. It was found that MAC-T cells express glutathione peroxidase (GPx) 1 and 4, thioredoxin reductase 1 and selenoprotein P, but not GPx 3, which is interesting considering that GPx 3 is one of the only few selenoproteins detected in milk so far. Addition of selenite to the cell culture resulted in a large increase in GPx 1 expression and an increase in selenoprotein P expression, which is similar to the findings made in other systems investigated. Increased mRNA levels of GPx 1 were also observed in cells treated with insulin and hydrocortisone or with retinoic acid. The expression of thioredoxin reductase 1 was increased in cells treated with retinoic acid, whereas that of selenoprotein P was decreased in cells exposed to insulin. The results indicate that several hormones, selenium, and retinoic acid regulate the biosynthesis of various selenoproteins differently in the bovine mammary cell. The possible implications of the findings for processes related to milk formation and mammary carcinogenesis will need additional investigation. Further study of the detailed mechanisms involved is also necessary.     

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.     

Selenoprotein expression is regulated at multiple levels in prostate cells

Selenium supplementation in a population with low basal blood selenium levels has been reported to decrease the incidence of several cancers including prostate cancer. Based on the clinical findings, it is likely that the antioxidant function of one or more selenoproteins is responsible for the chemopreventive effect, although low molecular weight seleno-compounds have also been posited to selectively induce apoptosis in transformed cells. To address the effects of selenium supplementation on selenoprotein expression in prostate cells, we have undertaken an analysis of antioxidant selenoprotein expression as well as selenium toxicity in non-tumorigenic prostate epithelial cells （RWPE- 1 ） and prostate cancer cells （LNCaP and PC-3）. Our results show that two of the glutathione peroxidase family members （GPX1 and GPX4） are highly induced by supplemental selenium in prostate cancer cells but only slightly induced in RWPE-1 cells. In addition, GPX 1 levels are dramatically lower in PC-3 cells as compared to RWPE- 1 or LNCaP cells. GPX2 protein and mRNA, however, are only detectable in RWPE-1 cells. Of the three selenium compounds tested （sodium selenite, sodium selenate and selenomethionine）, only sodium selenite shows toxicity in a physiological range of selenium concentrations. Notably and in contrast to previous studies, RWPE-1 cells were significantly more sensitive to selenite than either of the prostate cancer cell lines. These results demonstrate that selenoproteins and selenium metabolism are regulated at multiple levels in prostate cells.     

The 15kDa Selenoprotein and Thioredoxin Reductase 1 Promote Colon Cancer by Different Pathways

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.     

Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P_BAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P_BAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.     

Selenoproteins and selenocysteine insertion system in the model plant cell system,Chlamydomonas reinhardtii

Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.     

Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of > 20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.