Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells

Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA, USA.
Molecular and Cellular Biochemistry (Impact Factor: 2.39). 03/2009; 327(1-2):111-26. DOI: 10.1007/s11010-009-0049-x
Source: PubMed

ABSTRACT Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3'-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3'-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3'-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.

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    • "For some selenoprotein mRNAs, additional sequences flanking the SECIS may be required for efficient selenocysteine incorporation. Indeed, a very recent study analyzing the expression of human Gpx3 in transfected cells reported that selenocysteine incorporation was supported by a 3′-UTR of 500 nt, but not by a 100 nt 3′-UTR that encompassed the SECIS (48). While the underlying basis for this difference was not investigated, we speculate the additional sequences may be required to stabilize the stem-loop structure of the SECIS or to recruit other factors that may enhance the efficiency of UGA recoding. "
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    ABSTRACT: The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3'-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5'-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.
    Nucleic Acids Research 09/2009; 37(17):5868-80. DOI:10.1093/nar/gkp635 · 9.11 Impact Factor
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    ABSTRACT: Selenium (Se) is a nutritional trace mineral essential for various aspects of human health that exerts its effects mainly through its incorporation into selenoproteins as the amino acid, selenocysteine. Twenty-five selenoprotein genes have been identified in humans and several selenoproteins are broadly classified as antioxidant enzymes. As progress is made on characterizing the individual members of this protein family, however, it is becoming clear that their properties and functions are quite diverse. This review summarizes recent insights into properties of individual selenoproteins such as tissue distribution, subcellular localization, and regulation of expression. Also discussed are potential roles the different selenoproteins play in human health and disease.
    Cellular and Molecular Life Sciences CMLS 05/2009; 66(15):2457-78. DOI:10.1007/s00018-009-0032-4 · 5.81 Impact Factor
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    ABSTRACT: The co-translational incorporation of selenocysteine (Sec) requires that UGA be recognized as a sense rather than a nonsense codon. This is accomplished by the concerted action of a Sec insertion sequence (SECIS) element, SECIS binding protein 2, and a ternary complex of the Sec specific elongation factor, Sec-tRNA(Sec), and GTP. The mechanism by which they alter the canonical protein synthesis reaction has been elusive. Here we present an overview of the mechanistic perspective on Sec incorporation, highlighting recent advances in the field.
    Antioxidants & Redox Signaling 09/2009; 12(7):881-92. DOI:10.1089/ars.2009.2878 · 7.41 Impact Factor
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