Dolph L. Hatfield

Redox Regulation of Cell Signaling by Selenocysteine in Mammalian Thioredoxin Reductases

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function.

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA[Ser]Sec and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.

Structure-Expression Relationships of the 15-kDa Selenoprotein Gene POSSIBLE ROLE OF THE PROTEIN IN CANCER ETIOLOGY

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3′-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3′-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.

Transfer RNA in protein synthesis

Preface. tRNA in the Molecular Biology of Retroviruses (S. Weilson and J. Abbotts). The Role of Modified Nucleosides in tRNA Interactions (G. Bj rk). Correlation between Codon Usage and tRNA Population in Microorganisms (T. Ikemura). Aminoacyl-tRNA (Anticodon): Codon Adaptation in Reticulocytes and Other Cells of Higher Eucaryotes (D. Hatfield, J.-E. Jung, B.J. Lee, and I.S. Choi). Adaptation of tRNA Population to Codon Usage in Cellular Organelles (L. Marechal-Drouard, A. Dietrich, and J.-H. Weil). Differential tRNA Gene Expression (J.-H. Weil, R. Pirtle, and I. Pirtle). Codon Pair Utilization Bias in Bacteria, Yeast and Mammals (G.W. Hatfield and G.A. Gutman). Variations in Reading the Genetic Code (J. Parker). Selenocysteine: A New Addition to the Universal Genetic Code (I.S. Choi, B.J. lee, J.-E. Jung, and D. Hatfield). tRNA Discrimination in Aminoacylation (L. Pallanck and L. Schulman). The Translational Context Effect (M. Yarus and James Curran). Codon Discrimination in Tr anslation (U. Lagerkvist). Universal Rule of TA/CG Deficiency and TG/CT Excess (S. Ohno). Selective Use of Termination Codons and Variations in Codon Choice (P.M. Sharp, C.J. Burgess, E. Cowe, A.J. Lloyd and K.J. Mitchell).

Selenocysteine tRNA[Ser]Sec Isoacceptors as Central Components in Selenoprotein Biosynthesis in Eukaryotes

In recent years, a variety of experimental results have led to the surprising conclusion that under certain circumstances the UGA termination codon signals the translational insertion of selenocysteine into protein. These studies include the demonstration that (1) a TGA codon (that corresponds to a selenocysteine moiety in the resulting gene products [Cone et al., 1976; Gunzler et al., 1984]) occurs in the open reading frame of genes for formate dehydrogenase in E. coli (Zinoni et al., 1986) and glutathione peroxidase (GPx) in mammals (Chambers et al., 1986; Sukenaga et al., 1987; Mullenbach et al., 1988) and (2) a selenocysteyl-tRNA that decodes UGA occurs in E. coli (Leinfelder et al., 1989) and mammals (Lee et al., 1989b). The genes that utilize UGA for selenocysteine (for review see Stadtman, 1991) and the tRNAs that serve as carrier molecules for the biosynthesis of selenocysteine and donate selenocysteine to protein have been observed in a wide variety of organisms as described below. This phenomenon has evolved in all life kingdoms and thus the universal genetic code has been expanded to include selenocysteine as the 21st encoded amino acid (Hatfield et al., 1992b; Hatfield and Diamond, 1993).

UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins

It is thought that the SelenoCysteine Insertion Sequence (SECIS) element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3′-UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3′-UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3′-UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoprotein-specific manner, and that this process is controlled by the SECIS element in the 3′-UTR.

Selenium metabolism in Drosophila. Characterization of the selenocysteine tRNA population.

The selenocysteine (Sec) tRNA population inDrosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The K m of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nm, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine,N 6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2′-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge,Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date.

Insights into the Chemical Biology of Selenium

The long-sought pathway by which selenocysteyl-tRNA[Ser]Sec is synthesized in eukaryotes has been revealed. Seryl-tRNA[Ser]Sec is O-phosphorylated and SecS, a pyridoxal phosphate-dependent protein, catalyzes the reaction of O-phosphoseryl-tRNA[Ser]Sec with monoselenophosphate to give selenocysteyl-tRNA[Ser]Sec . 1 H- 77 Se HMQC-TOCSY NMR spectroscopy has been developed to detect the selenium-containing amino acids present in selenized yeast after protease XIV digestion. An archived selenized yeast sample is found to contain the novel amino acid S-(methylseleno)cysteine in addition to selenomethionine. Arsenite and selenite react with GSH to form (GS) 2 AsSe−. The structure of this compound has been determined by EXAFS, 77 Se NMR and Raman spectroscopic and chromatographic studies. Its formation under biological conditions has been demonstrated.