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Selenoprotein reactivity

Selenoprotein reactivity. Elias Arnér , MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden Elias.Arner@ki.se. 21st amino acid 25 human selenoprotein genes Low pKa (≈5.3), nucleophilic and highly reactive

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Selenoprotein reactivity

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  1. Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden Elias.Arner@ki.se Elias Arnér

  2. 21st amino acid • 25 human selenoprotein genes • Low pKa (≈5.3), nucleophilic and highly reactive • Sec incorporated at UGA codons Selenocysteine (Sec, U) + + Elias Arnér

  3. Mammalian TrxR’s are selenoproteins • Human selenoprotein TrxR reported in 1996 • Conserved selenocysteine, essential for catalytic activity • Selenocysteine-containing motif present in other TrxR isoenzymes (mitochondrial: TrxR2, testis: TGR) Tamura & Stadtman (1996) PNAS 93, 1006-1011 Gladyshev et al. (1996) PNAS 93, 6146-6151 Calf: -Arg-Ser-Gly-Gly-Asn-Ile-Leu-Gln-Thr-Gly-Cys-Sec-Gly-COOH Rat: -Arg-Ser-Gly-Gly-Asp-Ile-Leu-Gln-Ser-Gly-Cys-Sec-Gly-COOH Human: -Arg-Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Sec-Gly-COOH Zhong, et al. (1998) J. Biol. Chem., 273, 8581-8591 Lee et al. (1999) J. Biol. Chem. 274, 4722-412; Sun et al. (1999) J. Biol. Chem. 274, 24522-24530; Miranda-Vizuete, A. (1999) Eur. J. Biochem. 261, 405-412 Elias Arnér

  4. Catalytic mechanism of TrxR Arscott, et al. (1997) PNAS, 94, 3621-3626 Zhong, et al. (2000) PNAS, 97, 5854-5859 Lee, et al (2000) PNAS, 97, 2521-2526 Gromer et al (2003) PNAS, 100: 12618-16623 … Cheng et al. (2009) JBC, 284:3998-4008 Rat TrxR1 (GCCG mutant, dimer) Sandalova, et al.(2001) PNAS, 98, 9533-9538 Human TrxR1 (GCGG mutant, dimer) Debreczeni et al.(2006) SGC, unpublished Mouse TrxR2 (GCCG mutant, monomer) Biterova et al (2005) PNAS, 102:15018-23 Johansson et al. (2006) FRBM, 41:649-56. Elias Arnér

  5. Sec-to-Cys in TrxR yields very low activity -Gly-Cys-Sec-Gly-COOH ≈ 1% specific activity -Gly-Cys-Cys-Gly-COOH Zhong and Holmgren (2000) J. Biol. Chem., 275, 18121-18128 Lee, et al (2000) Proc Natl Acad Sci U S A. 97, 2521-2526 Fujiwara et al (2001) J Biochem (Tokyo). 129, 803-812 Bar-Noy et al (2001) Free Radic Biol Med. 30, 51-61 Elias Arnér

  6. -Ser-Cys-Cys-Ser-COOH -Gly-Cys-Sec-Gly-COOH Activity of DmTrxR surprisingly high Kanzok et al (2000) J Biol Chem. 275:40180-6 Elias Arnér

  7. Production in E. coli of recombinant thioredoxin reductase with Sec Arnér, et al. (1999) J. Mol. Biol. 292, 1003-1016 Johansson et al (2004) Nat. Meth. 1, 61-66 Johansson et al (2005) FEBS J. 272, 3449-3460 Cheng et al (2006) Nat. Protocols. 1, 604-613 Elias Arnér

  8. The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR • First set of mutants – Are the flanking Serines important ? • DmTrxR-SCCS (wt) • DmTrxR-SCCG • DmTrxR-GCCS • DmTrxR-GCCG (“Sec-to-Cys” variant of mammalian TrxR) • Second set of mutants – What is the importance of selenium ? • DmTrxR-SCUS (Cys-to-Sec mutant) • DmTrxR-SCUG • DmTrxR-GCUS • DmTrxR-GCUG (“mammalian” motif) Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  9. The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR Km for Dm-Trx was not affected Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  10. The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR Effects on Kcat Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  11. NADP+ Cys489 Ser488 NADPH + H+ Cys489 Ser488 Cys489 Ala487 Ala487 Substrate (ox) Cys489 Ser488 Pro486 Product (red) Ser488 Pro486 Neighboring Ser residues may perhaps activate the Cys residues to reach Sec in reactivity? Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  12. But - the same does not work in mammalian TrxR Rat TrxR1-GCUG 100% Rat TrxR1-SCCS < 0.5%  Main reason for the low activity was avery slow oxidative half-reaction L. Johansson et al. FRBM (2006) 41, 649–656 Elias Arnér

  13. 140 Percentage of activity determined at pH 7.4 120 100 80 Percentage of activity determined at pH 7.4 60 GCCG 40 SCCG 140 GCCS 20 120 SCCS 0 100 6.0 6.5 7.0 7.5 8.0 8.5 9.0 80 60 U GC G 40 SC U G U GC S 20 U SC S 0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Why selenoprotein? — Could another pH-optimum profile be the answer? Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  14. Why selenoprotein? — Could reactions with electrophilic agents be the answer? Gromer et al (2003) PNAS; 100:12618-12623 Elias Arnér

  15. SH SH NADPH + H+ NADP+ S Se Se- Se Electrophilic compounds easily target the selenocysteine residue in TrxR Electrophilic drugs inactivating TrxR1: • DNCB, DNFB, ... • iodoacetic acid, iodoacetamide, 4-vinylpyridine, … • Aurothioglucose, auranofin, … • Nitrosoureas, other alkylating anticancer agents • quinone derivatives • electrophilic prostaglandin derivatives • platinum compounds (cisplatin, oxaliplatin) • …and many more…. Elias Arnér

  16. Cell growth Redox regulation Antioxidant defense p53 function Increased cell proliferation, modulation of cell function, or induction of apoptosis, depending upon additional stimuli and growth conditions Intact TrxR/Trx system (Induction of TrxR1 through an Nrf2 response) Electrophilic compounds targeting TrxR FAD FAD S S ROS production S S S S Se Se Cell death, cell cycle arrest or other cellular effects due to an impaired thioredoxin system Effects of targeting Sec in TrxR1? SecTRAPs • Rapid process • Caspase involvement • Membrane integrity lost • Not requiring protein synthesis Complete active site inhibited - similar effect as TrxR knockdown Only Sec residue targeted Apoptosis and/or necrosis due to lethal prooxidant gain of function in selenium compromised forms of TrxR Anestål et al (2008) PLoS ONE, e1846; Arnér (2009) BBA, In press Elias Arnér

  17. How about Sec properties compared to Cys if introduced into any type of protein? Elias Arnér

  18. Engineered SECIS Engineered SECIS TrxR ORF peptide/protein ORF rTrxR (...-Gly-Cys-Sec-Gly-COOH) Sel-tagged peptide or protein (...-Gly-Cys-Sec-Gly-COOH) The concept of a Sel-tag Elias Arnér

  19. Sel-tag Properties of a Sel-tag • is a small modification of parent peptide • is redox active • has unique biochemical characteristics due to the Sec residue and selenolthiol motif Johansson et al (2004) Nat. Meth. 1, 61-66 Johansson et al (2005) FEBS J. 272, 3449-3460 Cheng et al (2006) Nat. Protocols. 1, 604-613 Elias Arnér

  20. COOH Sepharose matrix Gly Sec Se As N S Cys Gly Protein Using the Sel-tag for purification • One-step purification over PAO Sepharose Cheng et al (2006) Nat. Protocols. 1, 604-613 Elias Arnér

  21. Which “Sel-tag”? • Mammalian TrxR: -Gly-Cys-Sec-Gly-COOH • Drosophila TrxR: -Ser-Cys-Cys-Ser-COOH Elias Arnér

  22. -Gly-Cys-Sec-Gly (GCUG) • -Ser-Cys-Sec-Ser (SCUS) • -Gly-Cys-Cys-Gly (GCCG) • -Ser-Cys-Cys-Ser (SCCS) Selenolthiol variants Fel d 1 Dithiol variants Which “Sel-tag”? Fel d 1, one of the allergens from the domestic cat (Felis domesticus). Elias Arnér

  23. Protein Purification w/ PAO sepharose Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981 Elias Arnér

  24. Protein labeling – Fluorescent labeling • Pre-reduced with 2 mM DTT • React with 5-IAF, 5 min • Addition of ß-mercaptoethanol • SDS-PAGE • Fluorescence documentation under UV light • Coomassie staining Non-specific labeling through six structural cysteines • Short labeling time • Lower reaction pH Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981 Elias Arnér

  25. S | Se [11C]CH3I DTT SH Se- SH Se-[11C]CH3 + I- Selenolate-targeted radiolabeling with a positron emitter Elias Arnér

  26. Selenolate-targeted radiolabeling with a positron emitter Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981 Elias Arnér

  27. Before NAP-5 (~30%) After NAP-5 (>99%) Carbon-11 labeling and MicroPET imaging of Annexin A5 Elias Arnér

  28. Carbon-11 labeling and MicroPET imaging Elias Arnér

  29. PET imaging of apoptosis ─ Tumor model • Mice with tumor xenograft (multiple myeloma) • i.v. injection of drug • 19 hours later, i.v. injection of 11C-Annexin V • One hour MicroPET scanning Elias Arnér

  30. Oxidoreductases may evolve to use either Cys or Sec • Sec may substitute Cys in DmTrxR, but Cys can not substitute Sec in rat TrxR even if “activated” by neighboring Ser residues - importance of the active site microenvironment • Sec is clearly more reactive with electrophilic compounds than “activated Cys”, and Sec furthermore gives a broader pH-range of reactivity • The Sec reactivity can be used for some biotech applications where Cys is not useful Summary of Sec reactivity – How about the differences between Sec and Cys in Nature….? Elias Arnér

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