Abstract
Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmic-reticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Sec-containing forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli’s native Sec incorporation mechanism.
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References
Liu J, Rozovsky S (2015) Membrane-bound selenoproteins. Antioxid Redox Signal 23(10):795–813. doi:10.1089/ars.2015.6388
Gladyshev VN, Arner ES, Berry MJ, Brigelius-Flohe R, Bruford EA, Burk RF, Carlson BA, Castellano S, Chavatte L, Conrad M, Copeland PR, Diamond AM, Driscoll DM, Ferreiro A, Flohe L, Green FR, Guigo R, Handy DE, Hatfield DL, Hesketh J, Hoffmann PR, Holmgren A, Hondal RJ, Howard MT, Huang K, Kim HY, Kim IY, Kohrle J, Krol A, Kryukov GV, Lee BJ, Lee BC, Lei XG, Liu Q, Lescure A, Lobanov AV, Loscalzo J, Maiorino M, Mariotti M, Sandeep Prabhu K, Rayman MP, Rozovsky S, Salinas G, Schmidt EE, Schomburg L, Schweizer U, Simonovic M, Sunde RA, Tsuji PA, Tweedie S, Ursini F, Whanger PD, Zhang Y (2016) Selenoprotein gene nomenclature. J Biol Chem 291(46):24036–24040. doi:10.1074/jbc.M116.756155
Lilley BN, Ploegh HL (2005) Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane. Proc Natl Acad Sci U S A 102(40):14296–14301. doi:10.1073/pnas.0505014102
Shchedrina VA, Novoselov SV, Malinouski MY, Gladyshev VN (2007) Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif. Proc Natl Acad Sci U S A 104(35):13919–13924. doi:10.1073/pnas.0703448104
Lee JH, Kwon JH, Jeon YH, Ko KY, Lee SR, Kim IY (2014) Pro178 and Pro183 of selenoprotein S are essential residues for interaction with p97(VCP) during endoplasmic reticulum-associated degradation. J Biol Chem 289(20):13758–13768. doi:10.1074/jbc.M113.534529
Lee JH, Park KJ, Jang JK, Jeon YH, Ko KY, Kwon JH, Lee SR, Kim IY (2015) Selenoprotein S-dependent selenoprotein K binding to p97(VCP) protein is essential for endoplasmic reticulum-associated degradation. J Biol Chem 290(50):29941–29952. doi:10.1074/jbc.M115.680215
Ye YH, Shibata Y, Yun C, Ron D, Rapoport TA (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429(6994):841–847. doi:10.1038/nature02656
Lilley BN, Ploegh HL (2004) A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429(6994):834–840. doi:10.1038/nature02592
Turanov AA, Shchedrina VA, Everley RA, Lobanov AV, Yim SH, Marino SM, Gygi SP, Hatfield DL, Gladyshev VN (2014) Selenoprotein S is involved in maintenance and transport of multiprotein complexes. Biochem J 462(3):555–565. doi:10.1042/bj20140076
Liu J, Zhang Z, Rozovsky S (2014) Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential. FEBS Lett 588(18):3311–3321. doi:10.1016/j.febslet.2014.07.037
Fredericks GJ, Hoffmann FW, Rose AH, Osterheld HJ, Hess FM, Mercier F, Hoffmann PR (2014) Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex. Proc Natl Acad Sci U S A 111(46):16478–16483. doi:10.1073/pnas.1417176111
Fredericks GJ, Hoffmann PR (2015) Selenoprotein K and protein palmitoylation. Antioxid Redox Signal 23(10):854–862. doi:10.1089/ars.2015.6375
Hardy D, Bill RM, Jawhari A, Rothnie AJ (2016) Overcoming bottlenecks in the membrane protein structural biology pipeline. Biochem Soc Trans 44(3):838–844. doi:10.1042/bst20160049
Lyons JA, Shahsavar A, Paulsen PA, Pedersen BP, Nissen P (2016) Expression strategies for structural studies of eukaryotic membrane proteins. Curr Opin Struct Biol 38:137–144. doi:10.1016/j.sbi.2016.06.011
Christianson JC, Olzmann JA, Shaler TA, Sowa ME, Bennett EJ, Richter CM, Tyler RE, Greenblatt EJ, Harper JW, Kopito RR (2011) Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol 14(1):93–105. doi:10.1038/ncb2383
Liu J, Srinivasan P, Pham DN, Rozovsky S (2012) Expression and purification of the membrane enzyme selenoprotein K. Protein Expr Purif 86(1):27–34. doi:10.1016/j.pep.2012.08.014
Liu J, Li F, Rozovsky S (2013) The intrinsically disordered membrane protein selenoprotein S is a reductase in vitro. Biochemistry 52(18):3051–3061. doi:10.1021/bi4001358
Liu J, Rozovsky S (2013) Contribution of selenocysteine to the peroxidase activity of selenoprotein S. Biochemistry 52(33):5514–5516. doi:10.1021/bi400741c
Muller S, Senn H, Gsell B, Vetter W, Baron C, Bock A (1994) The formation of diselenide bridges in proteins by incorporation of selenocysteine residues - biosynthesis and characterization of (Se)(2)-thioredoxin. Biochemistry 33(11):3404–3412. doi:10.1021/bi00177a034
Strub MP, Hoh F, Sanchez JF, Strub JM, Bock A, Aumelas A, Dumas C (2003) Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing. Structure 11(11):1359–1367. doi:10.1016/j.str.2003.09.014
Yoshizawa S, Böck A (2009) The many levels of control on bacterial selenoprotein synthesis. Biochim Biophys Acta 1790(11):1404–1414. doi:10.1016/j.bbagen.2009.03.010
Arnér ESJ, Sarioglu H, Lottspeich F, Holmgren A, Bock A (1999) High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes. J Mol Biol 292(5):1003–1016. doi:10.1006/jmbi.1999.3085
Rengby O, Johansson L, Carlson LA, Serini E, Vlamis-Gardikas A, Karsnas P, Arner ESJ (2004) Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis. Appl Environ Microbiol 70(9):5159–5167. doi:10.1128/AEM.70.9.5159-5167.2004
Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41(1):207–234. doi:10.1016/j.pep.2005.01.016
Cheng Q, Johansson L, Thorell JO, Fredriksson A, Samen E, Stone-Elander S, Arner ESJ (2006) Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli. ChemBioChem 7(12):1976–1981. doi:10.1002/cbic.200600326
Blommel PG, Fox BG (2007) A combined approach to improving large-scale production of tobacco etch virus protease. Protein Expr Purif 55(1):53–68. doi:10.1016/j.pep.2007.04.013
Cheng Q, Stone-Elander S, Arner ESJ (2006) Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with C-11. Nat Protoc 1(2):604–613. doi:10.1038/nprot.2006.87
Acknowledgments
We thank Prof. E. S. J. Arner from the Karolinska Institutet for the generous donation of the pSUABC plasmid and Dr. Marie-Paule Strub at the NIH laboratory of molecular biophysics for the donation of BL21(DE3)cys cells. The Delaware COBRE program supported this research with grants from the National Institute of General Medical Sciences under awards P30 GM110758-02 and P20 GM104316. We acknowledge the use of instrumentation made accessible through the National Science Foundation under Grant No. CHE-1337449. This work was primarily supported by the National Science Foundation under Grant No. MCB-1054447 “CAREER: Reactivity of Selenoproteins” and Grant No. MCB-1616178.
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Zhang, Z., Liu, J., Rozovsky, S. (2018). Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS. In: Chavatte, L. (eds) Selenoproteins. Methods in Molecular Biology, vol 1661. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7258-6_18
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DOI: https://doi.org/10.1007/978-1-4939-7258-6_18
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