1. June 17, 2010Elias Arnér Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden Elias.Arner@ki.se

2. Berzelius and selenium Berzeli park, Stockholm June 17, 2010Elias Arnér Jöns Jacob Berzelius 1779- 1848

3. Berzelius and selenium June 17, 2010Elias Arnér Gripsholm chemical factory  Where Berzelius discovered selenium in 1817-1818

4. Berzelius and selenium From pp. 49-50: June 17, 2010Elias Arnér “[the element]…which I, to mark its akin properties with tellurium, have named Selenium, from Σελήνη, moon (goddess). What is more, it is in this regard, midway between sulfur and tellurium, and has almost more characters of sulfur than of tellurium.”

5. June 17, 2010Elias Arnér Selenium is closely related to oxygen and sulfur

6. June 17, 2010Elias Arnér Selenocysteine (Sec, U) 21st amino acid 25 human selenoprotein genes Low pKa (≈5.3), nucleophilic and highly reactive Sec incorporated at UGA codons ++

7. Chemical properties of Sec vs. Cys Elias ArnérJune 17, 2010

8. Chemical properties of Sec vs. Cys Elias ArnérJune 17, 2010 Sec pKa ≈ 5.2 High Sec reactivity at pH ≈ 2.0 (!) with IAA or IAM, much more reactive than Cys

9. Nucleophilicity is more than what is solely reflected by pKa values Elias ArnérJune 17, 2010  Nucleophilicity is governed by a number of factors, including: pK a basicity vs. acidity (Lewis or Brønstedt theories) polarizability electronegativity atomic radius solvation energy (solvent) chemical bond strength between reacting atoms …  Sec is clearly much more nucleophilic than Cys Arnér ES. Exp Cell Res. 2010, 316:1296-303

10. Nucleophilicity is more than what is solely reflected by pKa values Elias ArnérJune 17, 2010 Nulecophile-assisted catalysis: Nucleophile has to react faster with R-X than Y - (facilitated by Nu - having high nucleophilicity) Nucleophile has to allow rapid reaction of R-Nu with Y - (facilitated by Nu - being a good leaving group)

11. So what selenoproteins are there - and what functions do they catalyze…? Elias ArnérJune 17, 2010

12. Elias Arnér The Human Selenoproteome (Vadim Gladyshev and collaborators) Kryukov et al, Science. 2003;300:1439-1443 Known antioxidant enzymes  Many more and other selenoproteins in other organisms!  Plants and yeast are examples of organisms not having selenoproteins

13. June 17, 2010Elias Arnér These enzymes detoxify peroxides (ROOH) by a selenium- dependent redox cycling mechanism, involving Sec in the active site and consuming glutathione: Glutathione peroxidases

14. June 17, 2010Elias Arnér

15. June 17, 2010Elias Arnér Effects of selenium on cancer are dependent upon context, type of selenium compound, duration and concentration Se compounds and selenoproteins SeO 3 2-

16. Elias Arnér Expanding the genetic code June 17, 2010

17. Elias Arnér Sec Expanding the genetic code June 17, 2010

18. Elias Arnér Mechanism of selenocysteine insertion in eukaryotes Allmang, C. et al (2009). BBA 1790, 1415-1423. June 17, 2010

19. Elias Arnér Mechanism of selenocysteine insertion in bacteria (E. coli) June 17, 2010 Yoshizawa & Böck (2009). BBA 1790, 1404-1414.

20. June 17, 2010Elias Arnér Requirements for co-translational selenoprotein synthesis tRNA Sec that is charged with Sec An in-frame UGA codon A secondary structure in the selenoprotein mRNA, called a Sec Insertion Sequence (SECIS) element A dedicated elongation factor for co-translational Sec insertion, interacting with both the tRNA Sec and the SECIS element(directly or indirectly) Accessory factors to produce the Sec-tRNA Sec and to facilitate the Sec insertion at the UGA codon

21. June 17, 2010Elias Arnér Bacterial * Within ORF Eukaryotic * In 3’-UTR Differences between eukaryotic and bacterial SECIS elements

22. June 17, 2010Elias Arnér Can mammalian selenoproteins be expressed in E. coli?

23. June 17, 2010Elias Arnér Production in E. coli of recombinant thioredoxin reductase with Sec at the C-terminus C G U G U C U G A C A C G G C C C A U C G G U U G C A G GU C U G C A C C A A U C G G U C G G U A A U G G C G C A UGA codon for selenocysteine insertion { SelB binding  FDH-H: 11 nt G G A U G C U G A G G C U A A U A A U C G G U U G C A G GU C U G C A C C A A U C G U U A G C C U A U G C G G C C New SECIS:...- Gly - Cys - Sec - Gly -COOH Arnér, et al. (1999) J. Mol. Biol. 292, 1003-1016

24. June 17, 2010Elias Arnér The thioredoxin glutathione reductase of Schistoma mansoni A collaboration with David L. Williams, Illinois State University, USA

25. June 17, 2010Elias Arnér Schistosoma mansoni  An infectious worm  May cause schistosomiasis (bilharzia), with fever, portal hypertension, abdominal symptoms, death due to fatigue, diarrhea or CNS symptoms  About 200 million people infected, more than 200,000 deaths annually

26. June 17, 2010Elias Arnér The “TGR” of S. mansoni

27. June 17, 2010Elias Arnér The “TGR” of S. mansoni

28. June 17, 2010Elias Arnér Substrate spectrum of pure recombinant SmTGR Coupled Grx assay  SmTGR, in essence, combines the activities of mammalian TrxR’s, GR’s and Grx’s Kuntz et al. (2007) PLoS Med 4(6): e206

29. June 17, 2010Elias Arnér Finding inhibitors of SmTGR? (Incubation with 50 µM inhibitor 15 min in presence of NADPH, then DTNB assay) Kuntz et al. (2007) PLoS Med 4(6): e206

30. June 17, 2010Elias Arnér control + 10 µM auranofin control + 10 µM auranofin  SmTGR was specifically inhibited in cultured Schistosoma mansoni by treatment with auranofin Auranofin inhibits SmTGR in larvae Kuntz et al. (2007) PLoS Med 4(6): e206

31. June 17, 2010Elias Arnér  Inhibition of SmTGR coincides with oxidation of GSH  83% of the treated worms were dead after 6 hrs Kuntz et al. (2007) PLoS Med 4(6): e206

32. June 17, 2010Elias Arnér Is SmTGR an essential protein? TGR GAPDH C RNAi C RNAi  SmTGR is essential for Schistosoma mansoni survival in culture, under both aerobic and anaerobic conditions Kuntz et al. (2007) PLoS Med 4(6): e206

33. June 17, 2010Elias Arnér  Treatment of infected mice with auranofin gave significantly lower worm burden (p<0.015, n=7) Can inhibition of SmTGR cure schistosomiasis? Mice were infected with 100 S. mansoni cercariae Injected i.p. with 6 mg auranofin/kg body weight (safe dose for healthy mice), twice daily for nine days, beginning 7 days post infection One week after final dose, the mice were perfused and worms counted ** Kuntz et al. (2007) PLoS Med 4(6): e206

34. June 17, 2010Elias Arnér Mammalian thioredoxin reductase - kinetic properties - importance for cancer therapy

35. June 17, 2010Elias Arnér The mammalian thioredoxin system TrxR NADPH + H + NADP + Trx-S 2 Trx-(SH) 2 Dehydroscorbic acid Ascorbic acid Lipid hydroperoxides Alcohols α-Lipoic Acid DHLA GSSG 2 GSH H2O2H2O2 H 2 O+O 2 GPx ox GPx red Prx ox Prx red NF-κB repression (in cytosol) AP-1 activation ASK-1  ASK-1  Apoptosis induction Apoptosis repression P53 maturation and function Secreted Trx as co- cytokine Converted to Trx80 as secreted chemokine Ribonucleotide reductase Selenium dependence Nordberg J. & Arnér E.S.J. (2001). Free Rad. Biol. Med.31,1287-1312.

36. June 17, 2010Elias Arnér 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

37. June 17, 2010Elias Arnér Catalytic mechanism of TrxR Arnér, E.S.J. (2009) BBA 1790, 495-526

38. June 17, 2010Elias Arnér Cheng et al. (2009) JBC, 284:3998-4008 The selenenylsulfide of TrxR

39. Catalytic cycle(s) of TrxR1 June 17, 2010Elias Arnér Both subunits required for normal Sec-dependent Trx reducing activity One single subunit half-active site is still redox active, and may redox cycle with certain substrates if the Sec residue becomes destroyed

40. June 17, 2010Elias 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….

41. June 17, 2010Elias Arnér TrxR1 Controls/MOCKV1/V2 siRNA A549 cells 75 Se: Protein: Eriksson et al (2009) FRBM, 47:1661-1671

42. June 17, 2010Elias Arnér TrxR1 siRNA MOCK Knockdown of TrxR1 to ≈10% sensitizes cells towards DNCB and menadoione No difference in sensitivity towards auranofin Effects of targeting TrxR1? Eriksson et al (2009) FRBM, 47:1661-1671

43. June 17, 2010Elias Arnér TrxR1 siRNA MOCK Knockdown of TrxR1 to ≈10% makes cells more resistant to cisplatin No difference in sensitivity towards oxaliplatin or juglone Effects of targeting TrxR1? Eriksson et al (2009) FRBM, 47:1661-1671

44. June 17, 2010Elias Arnér Effects of targeting Sec in TrxR1? Anestål et al (2008) PLoS ONE, e1846; Arnér (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

45. June 17, 2010Elias Arnér Summary of selenoprotein reactivity Oxidoreductases may evolve to use either Cys or Sec Cys-containing orthologues of selenoproteins likely to depend upon either i) “activated Cys” residues, and ii) higher expression levels Sec is clearly more reactive with electrophilic compounds than “activated Cys” residues The Sec reactivity in TrxR may play a role in chemotherapy - either against parasites such as Schistosoma, or in human cancer treatment The Sec reactivity can be used for a number of biotech applications, where Cys is not as useful

46. Elias ArnérJune 17, 2010

47. Elias Arnér Acknowledgements Hanna-Stina Ahlzén Marcus Cebula Qing Cheng Victor Croitoru Pascal Dammeyer Sofi Eriksson Stefanie Prast-Nielsen Jianqiang Xu Sharon Stone-Elander Jan-Olov Thorell Erik Samén Li Lu Stig Linder Lars Holmgren Stefan Ståhl Lars Abrahmsén Ylva Lindqvist Tanja Sandalova Charles Williams, Jr. Narimantas Cenas Stephan Gromer June 17, 2010