E. G. Varlamova

Expression of human selenoprotein genes selh, selk, selm, sels, selv, and gpx-6 in various tumor cell lines

The expression level of the genes encoding six selenocysteine-containing human proteins was determined in the brain, cervical, liver, breast, prostate, and human fibrosarcoma cancer cells. It was found that a high level of expression in all studied types genes of tumor cells is characteristic for selh, selk, and selm genes, encoding SelH, SelK, and SelM proteins, respectively, whereas a complete lack of such expression was shown for gpx-6, selv, and sels genes. The results of this work can be regarded as a major prerequisite for further studies on the role of the three selenoproteins SelH, SelK, and SelM in the regulation of carcinogenesis processes associated with these types of cancer.

Cloning, intracellular localization, and expression of the mammalian selenocysteine-containing protein SELENOI (SelI) in tumor cell lines

The intracellular localization of human selenoprotein SelI and the degree of expression of its gene in different human tumor cell lines were determined. It was found that the SelI protein is present in the nucleus, cytoplasm, and endoplasmic reticulum and is absent in the nucleolus. Since the oxidative stress caused by a sharp increase in the content of free radicals in the body is one of the causes of malignant transformation, the study of the role of the trace element selenium and selenocysteine-containing proteins as antioxidants in carcinogenesis is of great scientific interest.

[cDNA cloning, expression and determination of substrate specificity of mice selenocysteine-containing protein SelV (Selenoprotein V)].

To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities.

Determination of mgpx6 and mselv gene mRNA expression during mouse postnatal development

132 To date, 25 seleniummcontaining mammalian proo teins have been identified using bioinformatic approaches [1]. They gained this name due to the presence in their structure of the amino acid selenoo cysteine, which makes these proteins 100 and somee times even 1000 times more active [2]. Seleniummconn taining proteins whose functions are known are oxii doreductases. This group includes deiodinases (DI), glutathione peroxidases (GPx), and thioredoxin reductases (TRx); however, the functions of the majority of selenoproteins are still obscure. In mamm mals, eight glutathione peroxidases were identified, only four of which (GPx1–GPx4) contain selenocyss teine in all mammalian species, whereas GPx6 is repp resented by the selenocysteineecontaining form in only two mammalian species—Sus scrofa and Homo sapiens [1]. At present, information about GPx6 is very scanty. It is known that mRNA of this protein is expressed only in the olfactory epithelium [1]. It has a nuclearr cytoplasmic localization and is absent in mitochonn dria [3]. The presumed function of this protein is the involvement in the metabolism of odorants [4]. Another poorly studied seleniummcontaining mammalian protein is SelV. It was shown that the mRNA of this protein is expressed only in the testes [1]. The protein partners of SelV were identified earlier, and their intracellular localization was established [5]. In this study, we demonstrated the expression of Gpx6 and SelV proteins during postnatal mammalian development at an age of 1 week to 18 months and detected ageerelated fluctuations in the expression of these proteins. The mRNA expression level of mouse genes gpx6 and selv (mgpx6 and mselv, respectively) during the postnatal development of animals was anaa lyzed by realltime PCR. The model organism used in the study was the mouse (Mus musculus) of inbred line C57BL/6. To determine the mRNA expression level of the genes encoding the proteins of interest, male rats aged 1–4 weeks and 2–18 months were used. The total RNA from the olfactory epithelium and testes was isoo lated using the TRIzol ® reagent (Invitrogen, United States) according to the protocol provided by the manufacturer. Then, to produce cDNA, reverse trann scription was performed using a reagent kit containing MMULV reverse transcriptase (Eurogen, Russia). At the next stage, the mRNA expression level of mgpx6 and mselv geones was determined by realltime PCR using the intercalating dye SYBR Green (Eurogen, Russia). Simultaneous amplification performed with the primers specific for the mouse βactin gene served as a control to …

Effect of Sodium Selenite on Gene Expression of SELF, SELW, and TGR Selenoproteins in Adenocarcinoma Cells of the Human Prostate

Selenium is an essential trace element, the deficiency of which leads to the development of several serious diseases, including male infertility, prostate cancer, etc. It has been shown that oxidative stress contributes to the progression of prostate cancer, and antioxidants such as selenium and vitamin E can significantly reduce the risk of this disease. Sodium selenite, one of the selenium compounds that induce the formation of reactive oxygen species, is considered as a potential anticancer agent. The SS concentrations that lead to a decrease in the viability of human prostate adenocarcinoma cells (line Du-145) have been selected, and the effect of sodium selenite on the expression of mRNA of the SELV, SELW, and TGR selenocysteine proteins in these cells has been analyzed.

Influence of Sodium Selenite on the mRNA Expression of the Mammalian Selenocysteine-Containing Protein Genes in Testicle and Prostate Cancer Cells

The sodium selenite concentration that reduces the viability of Du-145 human prostate adenocarcinoma cells and F-9 mouse testicular teratocarcinoma cells was determined. We investigated the effect of sodium selenite on the mRNA expression level of the genes encoding mammalian selenocysteine-containing glutathione peroxidases and thioredoxin reductases (key antioxidant enzymes involved in the regulation of intracellular thiol redox balance), endoplasmic reticulum selenoproteins, and selenoproteins located in the testes and prostate.

[Methods to Biosynthesize Mammalian Selenocysteine-Containing Proteins in vitro].

The main problem in studying mammalian selenocysteine-containing proteins is that the proteins are difficult to obtain in a recombinant form because the amino acid selenocysteine (Sec), which is their component, is encoded by TGA, which is one of the stop codons. When only the open reading frame of a target protein is cloned in a plasmid, translation is prematurely terminated at the TGA codon. An intricate natural mechanism allows the codon to be recognized as a selenocysteine codon and involves various cis- and trans-acting factors, such as the selenocysteine insertion sequence (SECIS), mRNA secondary structure, selenocysteine tRNA Sec-tRNA[Ser]Sec, SECIS-binding protein 2 (SBP2), selenocysteine-specific elongation factor EFsec, and others. Generation of recombinant selenoproteins in preparative amounts directly depends on the expression levels of the cis- and trans-acting transcription and translation factors to further complicate the problem, and cysteine homologs of selenoproteins are consequently used in many studies. Several methods designed to express mammalian selenoproteins in vitro are considered in the review.

Characterization of several members of the thiol oxidoreductase family

There is no doubt as to the important role that free radicals and reactive oxygen species play in the cell. Disturbances in intracellular redox proteins are often accompanied by common pathologies, including diabetes, myocardial infarction, neurodegeneration, bronchopulmonary diseases, cancer, etc. Numerous antioxidant enzymes are related to various redox biology systems, the thiol oxidoreductase superfamily playing a key role. The superfamily includes thioredoxin, glutaredoxin, peroxiredoxin, protein disulfide isomerase, and glutathione peroxidase families and a number of other proteins. Apart from their antioxidant function, thiol oxidoreductases are capable of recycling hydroperoxyde to produce specific disulfide bonds within and between proteins, which significantly expands their functional range. In view of this, it is a topical problem of redox biology to characterize the superfamily members biochemically and to study their functional mechanisms.

Selenocysteine biosynthesis and mechanism of incorporation into growing proteins

The universal genetic code codes for the 20 canonical amino acids, while selenocysteine (Sec) is encoded by UGA, one of the three well-known stop codons. Selenocysteine is of particular interest of molecular biology, principally differing in the mechanism of incorporation into growing polypeptide chains from the other 20 amino acids. The process involves certain cis- and trans-active factors, such as the Sec insertion sequence (SECIS). The SECIS is in the 3′-untranslated mRNA region in eukaryotes and within the open reading frame located immediately downstream of the Sec UGA codon in bacteria, the difference leading to differences in the mechanism of Sec incorporation between the two domains of life. The trans-active factors include Sec-tRNA[Ser]Sec, which is synthesized by a unique system; the Sec-specific elongation factor EFsec; and a SECIS-binding protein (SBP2). Thus, many additional molecules are to be synthesized in the cell to allow Sec incorporation during translation. The fact makes Sec-containing proteins rather “expensive” and emphasizes their crucial role in metabolism.