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Phospholipid hydroperoxide glutathione peroxidase (also known as PHGPX or GPx4) is an essential mammalian protein that functions as both an oxidoreductase enzyme and a structural ...protein. It acts on a range of substrates from hydrogen peroxide to lipid hydroperoxides and has increased affinity for phospholipid hydroperoxides. To better elucidate the function of GPx4, we used RNAi technology to knockdown its expression in NIH3T3 cells. GPx4 knockdown cells grew more than 70% slower than control cells, developed a dendrite‐like structure and died abruptly after several passages providing evidence that GPx4 is essential for cell survival. The dendrite‐like phenotype suggests that the knockdown cells underwent significant changes in their plasma membrane structure. Since lipid hydroperoxides are known to be potent cytotoxins, we examined the cytoprotective mechanism of GPx4 against these compounds by investigating membrane lipid composition of GPx4 knockdown and control cells. The data provide important insights into GPx4 function beyond its role as a detoxification enzyme.
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Although dietary selenium (Se) deficiency results in phenotypes associated with selenoprotein depletion in various organs, the brain is protected from Se loss. To address the role of Se ...in brain function, we carried out comparative gene expression analyses for the complete selenoproteome and associated biosynthetic factors. Using the Allen Brain Atlas, we evaluated 159 regions of adult mouse brain and provided experimental analyses of selected selenoproteins. All 24 selenoprotein mRNAs were expressed in the mouse brain. Most strikingly, neurons in olfactory bulb, hippocampus, cerebral cortex and cerebellar cortex were exceptionally rich in selenoprotein gene expression, in particular in GPx4, SelK, SelM, SelW and Sep15. A contrasting pattern was observed for another highly expressed selenoprotein gene, SelP, which we suggest to provide neurons with Se. Cluster analysis of the expression data linked certain selenoproteins and selenocysteine machinery genes and suggested functional linkages among selenoproteins, such as that between SelM and Sep15. Overall, this study suggests that the key functions of selenium in mammals are confined to certain neurons in the brain.
The process of protein folding in the endoplasmic reticulum (ER) of mammalian cells is assisted by an extensive network of molecular chaperones and thiol‐disulfide oxidoreductases, enzymes that ...catalyze formation of disulfide bonds. Recently, a novel selenocysteine‐containing oxidoreductase, Sep15, has been reported to reside in the ER lumen, and a role in disulfide bond formation and quality control in the ER has been proposed for this selenoprotein. In order to address the possible role of Sep15 in protein folding, we analyzed whether expression of Sep15 is increased in response to accumulation of unfolded proteins within the ER. Herein, we demonstrate that Sep15 expression is increased in NIH3T3 cells in response to ER stress caused by tunicamycin and brefeldin A, whereas DTT stimulates specific degradation of Sep15 by proteasomes. We also tested whether Sep15 deficiency leads to accumulation of unfolded proteins and cause ER stress. Indeed, decreased levels of Sep15 caused by selenium deficiency combined with down‐regulation of a Sep15 homolog, SelM, by RNA interference activated unfolded protein response. Together, these data further support the role of Sep15 in protein folding and suggest that its function may be compensated by SelM.
Cysteine residues (Cys) often play critical roles in proteins, however, identification of their specific functions has been limited to case‐by‐case experimental approaches. We describe a procedure ...for large‐scale detection of catalytic redox‐active Cys through homology to sporadic selenocysteine (Sec)‐containing proteins. This method is not dependent on sequence motifs, structure and origin of the sequences and first identifies unique Cys/Sec pairs flanked by homologous sequences within the universe of translated nucleotide sequences; these pairs then serve as seeds for sequence analysis at the level of protein families and subfamilies. Application of this method identified majority of known proteins containing catalytic redox‐active Cys, while filtering out proteins in which conserved Cys are involved in other functions, such as non‐redox catalysis, structural disulfides, posttranslational modifications and binding of certain metals. Moreover, the identity of the attacking Cys could be identified. We predicted redox‐active Cys in several proteins, and directly verified the prediction in an S‐adenosyl methionine‐dependent methyltransferase family. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox‐active Cys in these proteins.
Thioredoxin reductase 1 (TR1) is a major antioxidant and redox regulator in mammals. This selenium‐containing oxidoreductase is enriched in many malignant cells and has been proposed as a target for ...cancer therapy. To assess the role of TR1 in the malignancy process, we used RNAi technology to knockdown its expression in a lung cancer cell line whereby reversing many of the malignant phenotypes (Yoo et al. JBC, 281: , 2006). To further elucidate the role of TR1 in cancer therapy, we targeted the removal of TR1 expression in DT cells that overexpress oncogenic ras and manifest numerous malignant phenotypes. We found that DT cells overexpress TR1 compared with its normal control, NIH3T3 cells. Knockdown of TR1 resulted in a significant reduction in its anchorage‐independent growth properties. We examined gene expression in normal (NIH3T3), cancer (DT) and TR1 knockdown (DT/siTR1) cells by microarray analysis and found several mRNAs that are characteristically elevated in cancer cells were down‐regulated in the DT/siTR cells. We are expanding these findings and examining additional properties of DT/siTR1 cells to better evaluate TR1 as a target for cancer therapy.
This research was supported by the Intramural Research Program of the NIH, NCI, CCR.
Thioredoxin reductase (TR) and thioredoxin (Trx) constitute a major cellular redox system. Besides Trx, TR is able to reduce many other proteins and small non‐protein molecules in in vitro assays. In ...the present study, we report the characterization of cellular targets for cytosolic TR1 in different rodent tissues. The targets were identified in a proteomic method using affinity columns containing various mutant forms of TR1 differing in C‐terminal sequences. We found that cytosolic Trx1 was the major target of TR1 in rat and mouse liver cytosol as well as in rat brain cytosol and mouse serum. The truncated form of TR1 lacking selenocysteine was most efficient in binding Trx1. To examine the roles of TRs in redox homeostasis, we used knockout mice, in which selenocysteine tRNA gene was disrupted specifically in the liver. We found that the levels of cytosolic TR1 and mitochondrial TR3 were significantly lower in the knockout samples compared to wild type controls, whereas the levels of cytosolic Trx1 and mitochondrial Trx2 were elevated. However, despite increased expression levels, Trx1 was mostly in the oxidized state in the knockout samples. Overall, these data provide further evidence for the key roles of TRs in redox homeostasis.
Selenium, an essential micronutrient in the diet of mammals, is incorporated into proteins (selenoproteins) as the 21st amino acid, selenocysteine (Sec). The targeted removal of the Sec tRNA gene ...(trsp) in mice hepatocytes demonstrated the importance of selenoprotein expression in liver function (Carlson et al., J. Biol. Chem., 279: , 2004). Analysis of serum proteins of the hepatocyte trsp knockout and wild type mice revealed elevated levels of apolipoprotein E (ApoE) in the knockout mice. The increase in ApoE amount was accompanied by an increase in cholesterol and a decrease in triglyceride levels. Comparative gene analysis of liver from knockout and wild type mice detected enhanced expression of genes involved in cholesterol biosynthesis and decreased expression of genes involved in cholesterol metabolism/transport. In another mouse model, the trsp gene in the knockout mice was rescued with a mutant of trsp (A34), thus selectively restoring some selenoproteins (Carlson et al., unpublished data). Immunodetection assays showed that the levels of ApoE were not affected in these mice. The data indicate a novel relationship between selenoproteins and lipoproteins, wherein certain selenoproteins regulate lipoprotein biosynthesis and metabolism.
This research was supported by the Intramural Research Program of the NIH, NCI, CCR.
Phenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best-known within gene families, where an important shared function may ...only be revealed by mutating all family members. Here we describe the ‘Green Monster’ technology enabling the precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein (
GFP
) reporter gene, is subjected to repeated rounds of mating, meiosis, and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci within single cells. The Green Monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To demonstrate the technology, we generated a single broadly drug-sensitive strain of
Saccharomyces cerevisiae
bearing precise deletions of all 16 adenosine triphosphate-binding cassette transporters within clades associated with multi-drug resistance.
The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA
SerSec
, ...is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by
O
-phosphoseryl-tRNA kinase to form
O
-phosphoseryl-tRNA
SerSec
. Sec synthase (SecS) then uses
O
-phosphoseryl-tRNA
SerSec
and the active donor of selenium, selenophosphate, to form Sec-tRNA
SerSec
. Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with
O
-phosphoseryl-tRNA
SerSec
in the presence of SecS to form cysteine-(Cys-)tRNA
SerSec
. This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.
Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure ...for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redox-active Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins. PUBLICATION ABSTRACT
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