Nebraska Redox Biology Center Educational Portal


Thiol oxidoreductases are structurally distinct, but mechanistically similar families of enzymes, which have catalytic cysteine (Cys) in their active sites [ 1, 2 ]. In rare cases, the catalytic Cys could be replaced by selenocysteine (Sec), which is a Cys analog with selenium in place of sulfur. All Cys/Sec replacements have thus far been observed only in active sites of various thiol oxidoreductases [ 1, 2, 3 ]. The reason such a replacement evolved is most likely related to the more pronounced nucleophilic properties of selenium, making Sec a more efficient catalyst compared to Cys [ 2, 4 ]. Selenocysteine is regarded as the major biologically active form of selenium. The synthesis of selenoproteins depends on dietary selenium, making this element essential for mammals and many other organisms including bacteria and archea [ 1, 2, 5 ].

Chemical structure of cysteine and selenocysteine.

There are 18 solved selenoprotein structures in Protein Data Bank at this time (June 2015).

3D structure of Mouse SelW, Mouse Selenoprotein M, Drosophyla Sep15, Mouse Thioredoxin reductase 3 and Mouse Methionine sulfoxide reductase R.

There are more than 50 functionally distinct selenoprotein families and about half of them belongs to thioredoxin forld oxidoreductases [ 1, 2, 3, 6 ]. Considering the large number of known cysteine containing thiol oxidoreductases and comparatively low number of their selenocysteine containing homologs, it will be reasonable to suggest that most of selenoproteins have evolved from the corresponding Cys-containing analogs by mutation of a Cys-codon (either TGT or TGC) to the Sec encoding TGA codon. Such a mutation must occur in a gene encoding a Sec insertion (SECIS) element that occurs in the 3'-untranslated region (3'-UTR) in eukaryotic selenoprotein genes. The SECIS element is required for insertion of Sec into protein during translation of the corresponding mRNA [ 1, 2, 3, 7 ].

Sec is cotranslationally incorporated into nascent polypeptides in response to UGA codons when a specific stem-loop structure - Sec insertion sequence (SECIS) element, is present in the 3' untranslated regions in eukaryotes and in archaea, or immediately downstream of UGA in bacteria. [ 1, 5, 6, 7, 8 ].

Eukaryotic and bacterial selenoprotein genes.

Large group of selenoproteins contain Sec in the C-terminal part and, most probably, evolved by other mechanisms, for example, C-terminal extension of an ancestor protein. Such extension must have occurred after formation of a SECIS-like structure in the 3' UTR of the corresponding gene resulting in recoding of a TGA-stop codon as Sec. As a result, the newly formed protein obtains an additional, redox function, determined by selenocysteine [ 1, 2, 3 ].

Selenocysteine incorporation. Cotranslational incorporation of Sec into proteins is dictated by in-frame UGA codons present in selenoprotein mRNAs. Sec is introduced into selenoproteins by a complex mechanism that requires special trans-acting protein factors, Sec-tRNA and a cis-acting Sec insertion sequence (SECIS) element. When a ribosome encounters the UGA codon, which normally signals translation termination, Sec machinery interacts with the canonical translation machinery to augment the coding potential of UGA codons and prevent premature termination. SECIS elements serve as the factors that dictate recoding of UGA as Sec. In response to the SECIS element in selenoprotein mRNA, Sec-tRNA, which has an anticodon complimentary to the UGA, translates UGA as Sec. At least two trans-acting factors are required for efficient recoding of UGA as Sec in eukaryotes: SECIS binding protein 2 (SBP2) and Sec-specific translation elongation factor (eEFSec) [ 7, 9, 10, 11 ]. SBP2 is stably associated with ribosomes and contains a distinct L7Ae RNA-binding domain that is known to bind SECIS elements with high affinity and specificity. Aside from binding to ribosomes and SECIS elements, SBP2 also interacts with eEFSec, which recruits Sec Sec-tRNA and facilitates incorporation of Sec into the growing polypeptide. Instead of SBP2 and eEFSec, bacteria have a Sec-specific translation elongation factor that directly recognizes SECIS and is required for binding and delivery of SECIS elements to the ribosome [ 7, 12, 13 ].

Mechanism of Sec insertion in eukaryotes. Factors that are required for Sec incorporation into proteins in response to the UGA codon and the factors that may influence the efficiency of the Sec insertion [ 7 ].

Human selenoproteome.

Glutathione peroxidases. Selenocysteine containing glutathione peroxidases (GPx) are widespread in all three domains of life. There are eight GPx paralogs in human, from which five (GPx1, GPx2, GPx3, GPx4, and GPx6) contain a Sec residue in their active site. In the other three GPx homologs (GPx5, GPx7, and GPx8), the active-site Sec is replaced by Cys. GPxs play a wide range of physiological functions in organisms and are involved in hydrogen peroxide signaling, detoxification of hydroperoxides, and maintaining cellular redox homeostasis [ 1, 7, 14 ].

Thioredoxin reductases. Thioredoxin reductases (TRs) are oxidoreductases that comprise the major disulfide reduction system of the cell by reducing of thioredoxins and another substrates. In mammalian cells, there are three TR isozymes, all of which are Sec-containing proteins. These proteins contain a Sec residue in the C-terminal part of protein. Thioredoxin reductase 1 localized in the cytosol and nucleus. Thioredoxin reductase 3 is localized in the mitochondria, where it is involved in reduction of mitochondrial thioredoxin (Trx2) and glutaredoxin 2 (Grx2). TGR or thioredoxin reductase 2 contains an additional glutaredoxin (Grx) domain, which is located in the N-terminal part of the protein [ 1, 7, 15 ].

Thyroid hormone deodinases. The human genome encodes three deiodinases D1, D2 and D3, which are thioredoxin-like fold selenoproteins sharing a SxxU redox motif (DI1, DI2, and DI3), and are involved in regulation of thyroid hormone activity by reductive deodination. These proteins have distinct subcellular localizations and tissueexpression. DI1 and DI3 are located on the plasma membrane, but DI2 is localized to the endoplasmic reticulum [ 1, 7, 16, 17 ].

Selenoproteins K and S. SelK and SelS are endoplasmic reticulum resident selenoproteins with unknown funtion. These proteins contain Sec in the C-terminal part and, most probably, evolved by other mechanisms, for example, C-terminal extension of an ancestor protein. They are involved in redox control of protein translocation from ER to cytosol as a component of the ER-Associated Protein Degradation (ERAD) system. ERAD is a pathway which protects cells from accumulation of misfolded proteins by transferring these proteins from the ER to cytosol for subsequent ubiquitination and proteasomal degradation [ 1, 7, 18, 19, 20 ].

Selenoproteins M and Sep15. These thioredoxin fold selenoproteins with unknown function are involved in redox regulation of N-linked glycoprotein folding quality control pathway. Sep15 contain N-terminal domain responsible for its interaction with UGTR, an essential glycoprotein that plays a role as a folding quality control sensor. Being similar to each other, Sep15 or SelM show very weak similarity to other thioredoxin fold proteins [ 1, 7, 21, 2223 ].

Selenoprotein T and W, V and H. These thioredoxin fold selenoproteins with unknown function belongs to RDX superfamily. SelT is localized in endoplasmic reticulum, SelW and SelV in cytosol and SelH in nucleus [ 1, 7, 24, 25, 26 ].

Selenoprotein P. SelP is an abundantly expressed secreted selenoprotein that accounts for almost 50% of the total Se in plasma. The unique feature of SelP is the presence of multiple Sec residues. The fact that SelP is secreted into the plasma and the presence of multiple Sec residues in its sequence suggest that this selenoprotein might function as a Se supplier in comlpex organisms [ 1, 7, 27, 28, 29 ].

Selenoprotein R (MsrB1). This selenoprotein is a stereospecific methionine-R-sulfoxide reductase, which catalyzes repair of the R enantiomer of oxidized methionine residues in proteins. The Sec-containing MsrB1 protein is the major MsrB in mammals, which is primarily localized in the cytosol and nucleus [ 1, 7, 30, 31, 32, 33 ].

Human selenoproteome. Thioredoxin fold selenoproteins are shown on blue background. Selenoproteins evolved by C-terminal extention mechanism are shown on orange background. Secondary structure end Sec insertion sites are shown at the right part of the table. β-sheets are shown in blue and α-helices in orange 1, 2 ].

Selenocysteine biosyntesis. Selenocysteine is unique among other amino acids in that it is the only known amino acid in eukaryotes whose biosynthesis occurs on its own tRNA, designated Sec tRNA[Ser]Sec. tRNA[Ser]Sec is initially aminoacylated with serine in a reaction catalyzed by seryl-tRNA synthetase (SerRS) to form seryl-tRNA[Ser]Sec, which provides the backbone for Sec biosynthesis [ 7, 34, 35, 36 ].

Mechanism of Sec biosynthesis in eukaryotes and the Sec machinery-based pathway for synthesis of Cys. The phosphoseryl-tRNA kinase (PSTK) provides the phosphorylated intermediate PSer-tRNA[Ser]Sec serving as a substrate for SecS. Selenophosphate (H2SePO3-) generated by SPS2 from selenite and ATP is used as a donor of active Se for SecS. The de novo synthesis of Cys using the Sec biosynthetic machinery is shown on the bottom right [ 7 ].

In silico selenoprotein identification. Several independent approaches such as thermodynamics based tool for SECIS element prediction in mRNA structure and identification of cysteine/selenocysteine pairs in homologous sequences were developed recently and applied for genomic data bases analysis. Application of these tools to various completely sequenced genomes resulted in prediction of full sets of selenoproteins (selenoproteomes). As result, wide distribution of selenoproteins was demonstrated for all three domains of life. 25 human and 24 mouse selenoproteins were predicted and experimentally confirmed. Eucariotins SECIS element prediction tool and bacterial SECIS element prediction tool are based on thermodynamics algorithm of SECIS-like structured identification [ 1, 2, 3, 37 ].

Computational search for mammalian selenoprotein genes.

High-throughput identification of catalytic redox-active Cys and selenocysteines in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases is independent of protein family and structure. This method allows selectively detect the majority of known proteins with redox-active Cys and Sec [ 1, 2, 3, 6 ].

High-throughout prediction of redox-active cysteine and selenocysteine residues. The method used for prediction of redox-active Cys residues by homology to sporadic selenoproteins.

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