1. May 20, 2009Elias Arnér Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden Elias.Arner@ki.se

2. May 20, 2009Elias Arnér Selenocysteine (Sec, U) 21st amino acid 25 human selenoprotein genes Low pKa (≈5.3), nucleophilic and highly reactive Sec incorporated at UGA codons ++

3. May 20, 2009Elias Arnér 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 Tamura & Stadtman (1996) PNAS 93, 1006-1011 Gladyshev et al. (1996) PNAS 93, 6146-6151 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 Zhong, et al. (1998) J. Biol. Chem., 273, 8581-8591 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)

4. May 20, 2009Elias Arnér Johansson et al. (2006) FRBM, 41:649-56. 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 … Rat TrxR1 (GCCG mutant, dimer) Sandalova, et al.(2001) PNAS, 98, 9533-9538 Mouse TrxR2 (GCCG mutant, monomer) Biterova et al (2005) PNAS, 102:15018-23 Human TrxR1 (GCGG mutant, dimer) Debreczeni et al.(2006) SGC, unpublished Catalytic mechanism of TrxR Cheng et al. (2009) JBC, 284:3998-4008

5. May 20, 2009Elias Arnér -Gly-Cys-Sec-Gly-COOH -Gly-Cys-Cys-Gly-COOH ≈ 1% specific activity 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 Sec-to-Cys in TrxR yields very low activity

6. May 20, 2009Elias Arnér Activity of DmTrxR surprisingly high Kanzok et al (2000) J Biol Chem. 275:40180-6 -Gly-Cys-Sec-Gly-COOH -Ser-Cys-Cys-Ser-COOH

7. May 20, 2009Elias Arnér 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

8. May 20, 2009Elias Arnér 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

9. May 20, 2009Elias Arnér 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

10. May 20, 2009Elias Arnér The role of the C-terminal motif in TrxR assessed using recombinant variants of DmTrxR Gromer et al (2003) PNAS; 100:12618-12623 Effects on Kcat

11. May 20, 2009Elias Arnér Neighboring Ser residues may perhaps activate the Cys residues to reach Sec in reactivity? Ser 488 Cys 489 Ala 487 Pro 486 Ser 488 Cys 489 Ala 487 Pro 486 Cys 489 Gromer et al (2003) PNAS; 100:12618-12623 Substrate (ox) Product (red) NADPH + H + NADP +

12. May 20, 2009Elias Arnér But - the same does not work in mammalian TrxR Rat TrxR1-GCUG100% Rat TrxR1-SCCS < 0.5%  Main reason for the low activity was a very slow oxidative half-reaction L. Johansson et al. FRBM (2006) 41, 649–656

13. May 20, 2009Elias Arnér Gromer et al (2003) PNAS; 100:12618-12623 — Could another pH-optimum profile be the answer? Why selenoprotein? 6.06.57.07.58.08.59.0 Percentage of activity determined at pH 7.4 0 20 40 60 80 100 120 140 GCCG SCCG GCCS SCCS 6.06.57.07.58.08.59.0 0 20 40 60 80 100 120 140 GC U G SCUG GC U S SC U S Percentage of activity determined at pH 7.4

14. May 20, 2009Elias Arnér Gromer et al (2003) PNAS; 100:12618-12623 — Could reactions with electrophilic agents be the answer? Why selenoprotein?

15. May 20, 2009Elias Arnér NADPH + H + NADP + Electrophilic drugs inactivating TrxR1: Electrophilic compounds easily target the selenocysteine residue in TrxR SSe SHSe - SH Se 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….

16. May 20, 2009Elias Arnér Effects of targeting Sec in TrxR1? Anestål et al (2008) PLoS ONE, e1846; Arn é r (2009) BBA, In press 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 ROS production Rapid process Caspase involvement Membrane integrity lost Not requiring protein synthesis S S S Se SecTRAPs Apoptosis and/or necrosis due to lethal prooxidant gain of function in selenium compromised forms of TrxR S S Se S Cell death, cell cycle arrest or other cellular effects due to an impaired thioredoxin system Complete active site inhibited - similar effect as TrxR knockdown Only Sec residue targeted (Induction of TrxR1 through an Nrf2 response) Electrophilic compounds targeting TrxR Intact TrxR/Trx system

17. May 20, 2009Elias Arnér How about Sec properties compared to Cys if introduced into any type of protein?

18. May 20, 2009Elias Arnér The concept of a Sel-tag TrxR ORF Engineered SECIS rTrxR (...-Gly-Cys-Sec-Gly-COOH) peptide/protein ORF Engineered SECIS Sel-tagged peptide or protein (...-Gly-Cys-Sec-Gly-COOH)

19. May 20, 2009Elias Arnér 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 Sel-tag

20. May 20, 2009Elias Arnér Using the Sel-tag for purification  One-step purification over PAO Sepharose N As Sepharose matrix Se S Sec Cys Gly COOH Protein Cheng et al (2006) Nat. Protocols. 1, 604-613

21. May 20, 2009Elias Arnér Mammalian TrxR: -Gly-Cys-Sec-Gly-COOH Drosophila TrxR : -Ser-Cys-Cys-Ser-COOH Which “Sel-tag”?

22. May 20, 2009Elias Arnér  -Gly-Cys-Sec-Gly (GCUG)  -Ser-Cys-Sec-Ser (SCUS)  -Gly-Cys-Cys-Gly (GCCG)  -Ser-Cys-Cys-Ser (SCCS) Dithiol variants Selenolthiol variants Fel d 1 Fel d 1, one of the allergens from the domestic cat (Felis domesticus). Which “Sel-tag”?

23. May 20, 2009Elias Arnér Protein Purification w/ PAO sepharose Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981

24. May 20, 2009Elias Arnér  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 Protein labeling – Fluorescent labeling Non-specific labeling through six structural cysteines Short labeling time Lower reaction pH Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981

25. May 20, 2009Elias Arnér Selenolate-targeted radiolabeling with a positron emitter S | Se SH Se - DTT SH Se-[ 11 C]CH 3 + I - [ 11 C]CH 3 I

26. May 20, 2009Elias Arnér Selenolate-targeted radiolabeling with a positron emitter Cheng, Q., et al (2006) ChemBioChem 7, 1976-1981

27. May 20, 2009Elias Arnér Carbon-11 labeling and MicroPET imaging of Annexin A5 Before NAP-5 (~30%) After NAP-5 (>99%)

28. May 20, 2009Elias Arnér Carbon-11 labeling and MicroPET imaging

29. May 20, 2009Elias Arnér PET imaging of apoptosis ─ Tumor model  Mice with tumor xenograft (multiple myeloma)  i.v. injection of drug  19 hours later, i.v. injection of 11 C-Annexin V  One hour MicroPET scanning

30. May 20, 2009Elias Arnér Summary of Sec reactivity 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 – How about the differences between Sec and Cys in Nature….?