Selenoprotein P Expression, Purification, and Immunochemical Characterization

Tujebajeva, Rosa M.; Harney, John W.; Berry, Marla J.

doi:10.1074/jbc.275.9.6288

Cited by 52 publications

(33 citation statements)

References 36 publications

(37 reference statements)

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“…75 Se]selenite labeling of endogenous SEPP1 in HepG2 cells (48). We can therefore conclude that our results are concordant with the nature of SEPP1 production in vivo, with full-length or nearly full-length protein being predominant albeit with some level of termination occurring at the second UGA codon.…”

Section: Discussionsupporting

confidence: 77%

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Shetty

Shah

Copeland

2014

Journal of Biological Chemistry

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Background: Selenocysteine incorporation into selenoprotein P (SEPP1), which contains 10 Sec residues, is unique. Results: Processive Sec incorporation can be reconstituted in vitro, independently of the conserved non-SECIS portions of the 3Ј-UTR. Conclusion: Processivity is intrinsic, but efficiency is governed by selenium levels. Significance: SEPP1 synthesis is essential for male fertility and proper neurologic function.

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Section: Discussionsupporting

confidence: 77%

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Shetty

Shah

Copeland

2014

Journal of Biological Chemistry

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“…1A), for these studies. Previous studies have shown that premature termination of translation occurs at several of the UGA codons in selenoprotein P in the intact rat (see introduction) as well as in both rat and zebra fish selenoprotein P expressed by transient transfection of mammalian cells (27,28). However, because 75 Se labeling was used in these prior studies, the degree of termination at the first UGA codon, which would result in a product containing no selenium (Fig.…”

Section: Resultsmentioning

confidence: 70%

“…75 Se labeling was carried out as previously described (27). On day 2, media were exchanged for media without serum, with no added unlabeled selenium, at which time 75 Se-sodium selenite (1,000 Ci/g, 3 to 6 Ci/ml) was added.…”

Section: Methodsmentioning

confidence: 99%

Efficient Incorporation of Multiple Selenocysteines Involves an Inefficient Decoding Step Serving as a Potential Translational Checkpoint and RibosomeBottleneck

Stoytcheva

Tujebajeva

Harney

et al. 2006

Molecular and Cellular Biology

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Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3 untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.Selenoprotein P is an enigma of genetic recoding. It is the only known protein in which multiple potential stop codons are recoded to function as sense codons. In selenoprotein mRNAs, UGA codons, which would normally signal the termination of protein synthesis, are decoded as the amino acid selenocysteine. This process involves a unique tRNA with an anticodon complementary to UGA and specialized secondary structures located in the 3Ј untranslated regions (UTRs) of selenoprotein mRNAs, termed selenocysteine incorporation sequence (SECIS) elements (3). For selenocysteine incorporation to occur, the SECIS element must recruit a SECIS binding protein, SBP2 (6). SBP2, in turn, recruits a dedicated elongation factor, EFsec (7, 26), complexed with selenocysteyl-tRNA (2, 31). Most selenoprotein mRNAs contain a single UGA codon and a single SECIS element. Selenoprotein P mRNAs contain 10 to 18 UGA codons, depending on the species, and two SECIS elements differing in secondary structure. The majority of studies to date on the mechanism of selenoprotein synthesis have focused on selenoprotein mRNAs containing a single UGA codon and a single SECIS element. Thus, little is known about the mechanism for incorporating multiple selenocysteines.The incorporation of selenium into selenoprotein P serves several important biological functions: it allows the accumulation of this essential but potentially toxic trace element in a biologically stable and nontoxic form, it plays a critical role in the transport of selenium from the liver via the circulation to target organs (13,25), it is thought to function in sequestering and thereby detoxifying heavy metals in plasma...

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“…Accordingly, to express a full-size polypeptide in the absence of the Sec insertion sequence element, TGA that encodes Sec at position 93 in Sep15 was replaced in these constructs with TGC that encodes cysteine. These mutations were made to increase translation efficiency because efficiency of selenoprotein synthesis from transfected constructs in mammalian cells is low (19). The constructs were transiently transfected into monkey CV-1 cells, and confocal microscopy was used to localize fusion proteins by detecting the GFP green fluorescence (Fig.…”

Section: Page (mentioning

confidence: 99%

Association between the 15-kDa Selenoprotein and UDP-glucose:Glycoprotein Glucosyltransferase in the Endoplasmic Reticulum of Mammalian Cells

Korotkov¹,

Kumaraswamy

Zhou

et al. 2001

Journal of Biological Chemistry

148

109

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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.

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Selenoprotein P Expression, Purification, and Immunochemical Characterization

Cited by 52 publications

References 36 publications

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Efficient Incorporation of Multiple Selenocysteines Involves an Inefficient Decoding Step Serving as a Potential Translational Checkpoint and RibosomeBottleneck

Association between the 15-kDa Selenoprotein and UDP-glucose:Glycoprotein Glucosyltransferase in the Endoplasmic Reticulum of Mammalian Cells

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