Distribution and functional consequences of nucleotide polymorphisms in the 3'-untranslated region of the human Sep15 gene.

Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3'-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3'-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3'-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both.

Association between the 15-kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells.

Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding.

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.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.

Selenocysteine-containing thioredoxin reductase in C. elegans.

Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases. Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family. The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase. The other gene contains TGA and its product is designated TR-Se. This Sec-containing thioredoxin reductase lacks a canonical Sec insertion sequence element in the 3'-untranslated area of the gene. TR-Se shows greater sequence similarity to mammalian thioredoxin reductase isozymes TR1 and TR2, whereas TR-S is more similar to TR3. TR-Se was identified as a thioredoxin reductase selenoprotein by labeling C. elegans with 75Se and characterizing the resulting 75Se-labeled protein by affinity and other column chromatography and gel-electrophoresis. TR-Se was expressed in Escherichia coli as a selenoprotein when a bacterial SECIS element was introduced downstream of the Sec TGA codon. The data show that TR-Se is the major naturally occurring selenoprotein in C. elegans, and suggest an important role for selenium and the thioredoxin system in this organism.

[Characteristics of N-terminal 60-kDa fragment of elongation factor 2]. / Svoistva N-kontsevogo 60-kDa fragmenta faktora élongatsii 2.

The N-terminal 60-kDa-fragment of elongation factor 2 from rat liver (EF-2) was obtained by the limited proteolysis of native EF-2 with elastase. This fragment consists of 506 N-terminal amino acid residues of EF-2. The conformational properties of both this fragment and EF-2 in solution were studied by circular dichroism and fluorescent spectroscopy. The contents of secondary structure components in the fragment and in the factor that were deduced from CD measurements agreed well with values predicted from their primary structures. Both proteins were resistant to denaturation with < or = 3 M urea and exhibited cooperative denaturation transitions. Temperature melting also proceeded cooperatively for the fragment and EF-2. Structural properties of the N-terminal 60-kDa-fragment are discussed in comparison with the biochemical characteristics and 3D structure of prokaryotic elongation factor EF-G.

[Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution]. / Biosintez i konformationnoe sostoianie v rastvore 17-kDa- i 27-KDa- N-kontsevykh fragmentov faktora élongatsii EF-2.

N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E. coli cells and in a wheat germ cell-free translation system, and their conformations were studied. Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates). The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone. Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules). A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure. Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein.

[Structure-activity domain of elongation factor EF-2. Analysis of fragments of limited EF-2 hydrolysis, obtained using trypsin and elastase]. / Strukturno-funktsional'nye domeny faktora élongatsii EF-2. Analiz fragmentov ogranichennogo gidroliza EF-2, poluchennykh s pomoshch'iu tripsina i élastazy.

Limited hydrolysis of EF-2 with trypsin in mild conditions leads to cleavage at the N-terminal part of the protein, at the region of phosphorylation, at the Arg54 and Arg65 residues. The trypsinolysis product, fragment T1', containing Thr56 and Thr58, which are phosphorylated in EF-2, is also phosphorylated by EF-2-kinase at the same residues. In the phosphorylated EF-2, digestion by trypsin takes place only at Arg65, resulting in a reduction of the rate of hydrolysis in comparison with the native EF-2. Digestion of EF-2 with elastase results in the formation of two fragments E1 and E2 (60 and 40 kDa, respectively). Fragment E1 represents the N-terminal part of EF-2. It is resistant to the further action of elastase, is not cleaved by trypsin, and loses its capability for phosphorylation. Fragment E2, the C-end part of the molecule, is not resistant to the further action of elastase and retains its capability for ADP-ribosylation with the A fragment of diptheria toxin and NAD+. Electrophoretic analysis of EF-2 and its proteolytic fragments according to O'Farrell showed that the modification, resulting in the presence of two initial forms of EF-2, is located between the amino acid residues 66 and 506 of the polypeptide chain. In conclusion a possibility of studying the formation of partial functional activities within the framework of individual structure-functional domains using a set of N-terminal fragments of various length is discussed.