Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Selenoprotein reactivity Elias Arnér, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden [email protected] Elias Arnér June 17, 2010 Berzelius and selenium Jöns Jacob Berzelius 17791848 Elias Arnér Berzeli park, Stockholm June 17, 2010 Berzelius and selenium Gripsholm chemical factory Where Berzelius discovered selenium in 1817-1818 Elias Arnér June 17, 2010 Berzelius and selenium “[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.” Elias Arnér From pp. 49-50: June 17, 2010 Selenium is closely related to oxygen and sulfur Elias Arnér June 17, 2010 Selenocysteine (Sec, U) H | +H3N—C—COO| CH2 | SeSelenocysteine Elias Arnér H | +H3N—C—COO| CH2 | SH Cysteine 21st amino acid 25 human selenoprotein genes Low pKa (≈5.3), nucleophilic and highly reactive Sec incorporated at UGA codons June 17, 2010 Chemical properties of Sec vs. Cys Elias Arnér June 17, 2010 Chemical properties of Sec vs. Cys High Sec reactivity at pH ≈ 2.0 (!) with IAA or IAM, much more reactive than Cys Sec pKa ≈ 5.2 Elias Arnér June 17, 2010 Nucleophilicity is more than what is solely reflected by pKa values Nucleophilicity is governed by a number of factors, including: • • • • • • • • pKa 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 Elias Arnér June 17, 2010 Nucleophilicity is more than what is solely reflected by pKa values Nulecophile-assisted catalysis: u n c a t a l y s e d + Y RY R X -X + N u + Y RNu RY n u c l e o p h i l i c c a t a l y s e d R X -X - Nu Nucleophile has to react faster with R-X than Y(facilitated by Nu- having high nucleophilicity) Elias Arnér Nucleophile has to allow rapid reaction of R-Nu with Y(facilitated by Nu- being a good leaving group) June 17, 2010 So what selenoproteins are there - and what functions do they catalyze…? Elias Arnér June 17, 2010 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 Elias Arnér June 17, 2010 Glutathione peroxidases These enzymes detoxify peroxides (ROOH) by a seleniumdependent redox cycling mechanism, involving Sec in the active site and consuming glutathione: Elias Arnér June 17, 2010 Elias Arnér June 17, 2010 Effects of selenium on cancer are dependent upon context, type of selenium compound, duration and concentration Se compounds and selenoproteins SeO32- Elias Arnér June 17, 2010 Expanding the genetic code Elias Arnér June 17, 2010 Expanding the genetic code Sec Elias Arnér June 17, 2010 Mechanism of selenocysteine insertion in eukaryotes Allmang, C. et al (2009). BBA 1790, 1415-1423. Elias Arnér June 17, 2010 Mechanism of selenocysteine insertion in bacteria (E. coli) Yoshizawa & Böck (2009). BBA 1790, 1404-1414. Elias Arnér June 17, 2010 Requirements for co-translational selenoprotein synthesis tRNASec 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 tRNASec and the SECIS element(directly or indirectly) Accessory factors to produce the Sec-tRNASec and to facilitate the Sec insertion at the UGA codon Elias Arnér June 17, 2010 Differences between eukaryotic and bacterial SECIS elements Eukaryotic * In 3’-UTR Elias Arnér Bacterial * Within ORF June 17, 2010 Can mammalian selenoproteins be expressed in E. coli? Bacterial selenoprotein mRNA: 5’ bacterial-type SECIS Bacterial selenoprotein ORF 3’ UGA Mammalian selenoprotein mRNA: 5’ mammalian-type SECIS Mammalian selenoprotein ORF 3’ UGA Elias Arnér June 17, 2010 Production in E. coli of recombinant thioredoxin reductase with Sec at the C-terminus FDH-H: 11 nt UGA codon for s el enocy s t ei ne i ns ert i on { GU G C A U C G G C U U A G C G C C A U A A U C C C G C G G U G C C G A C G A UA G A U U C G U G G C U G G C C A } New SECIS: S el B bi ndi ng G G A C G UU G G C U A A U A A U C G G A G U C G U A G G U 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 Arnér, et al. (1999) J. Mol. Biol. 292, 1003-1016 Elias Arnér June 17, 2010 The thioredoxin glutathione reductase of Schistoma mansoni A collaboration with David L. Williams, Illinois State University, USA Elias Arnér June 17, 2010 Schistosoma mansoni Elias Arnér 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 June 17, 2010 The “TGR” of S. mansoni Elias Arnér June 17, 2010 The “TGR” of S. mansoni Elias Arnér June 17, 2010 Substrate spectrum of pure recombinant SmTGR K m (µM) Vmax (µM/min) K ca t (s - 1) Kca t /Km (M - 1s- 1) D isul fi de proteins SmTrx1 SmPrx 6.37 ± 0.9 NA 36 ± 1.7 NA 30 NA 4.7 x 10 NA 6 Low M W di sulfides D TN B 114 ± 9.0 19.2 ± 1.6 16 1.4 x 10 5 GSSG 71.5 ± 11.8 26 ± 3.5 21.7 3.0 x 10 5 H ED 1867 ± 92 21.4 ± 0.33 17.8 9.6 x 10 3 GSH 248.6 ± 7.8 24.1 ± 3.9 20.1 8.1 x 10 4 Se substrates N a2SeO 3 66.7 ± 6.9 2.54 ± 0.046 2.1 3.1 x 10 4 Peroxi des H 2O 2 68,000 ± 5000 11.3 ± 0.46 9.4 1.4 x 10 2 t-Butyl -OOH 48,550 ± 2000 12.8 ± 0.33 10.7 2.2 x 10 2 al loxan 1140 ± 220 29.5 ± 4.5 24.6 2.2 x 10 4 l ipoi c aci d 2610 ± 300 2.03 ± 0.135 1.7 6.5 x 10 2 l ipoamide 1342 ± 11.4 14.2 ± 0.46 11.8 8.8 x 10 3 Coupled Grx assay Other subsrates D HAA ubi qui none NA NA NA NA NA NA NA NA SmTGR, in essence, combines the activities of mammalian TrxR’s, GR’s and Grx’s Kuntz et al. (2007) PLoS Med 4(6): e206 Elias Arnér June 17, 2010 Finding inhibitors of SmTGR? gold comp le xes 14-15 cis -platinum complexes 16 Mannich ba ses17 1,4-naph thoquinones 18-20 auranofin aurothioglucose aurothiomalate RMA19 RMA35 3-DAP CDE4 CDE16-2 menadione plumbagin naphthazarin JD155 JD159 M5 LS766 LS852 LS1121 LS908 LS1108 LS1114 LS1105 LS808 LS826 100 100 100 80 98 100 99 98 82 85 100 70 100 63 24 26 29 30 41 44 63 74 95 (Incubation with 50 µM inhibitor 15 min in presence of NADPH, then DTNB assay) Kuntz et al. (2007) PLoS Med 4(6): e206 Elias Arnér June 17, 2010 Auranofin inhibits SmTGR in larvae GR activity 25 control 20 15 10 5 + 10 µM auranofin 0 0 2 4 6 0 Time (hr) TrxR activity 60 control Activity (nmol/min/mg) 60 50 40 30 20 10 + 10 µM auranofin 0 0 2 Time (hr) 6 4 2 Time (hr) 70 Activity (nmol/min/mg) LDH activity 160 140 120 100 80 60 40 20 0 Activity (nmol/min/mg) Activity (nmol/min/mg) 30 4 6 GPx activity 50 40 30 20 10 0 0 2 4 6 Time (hr) SmTGR was specifically inhibited in cultured Schistosoma mansoni by treatment with auranofin Kuntz et al. (2007) PLoS Med 4(6): e206 Elias Arnér June 17, 2010 GR activity 70 TrxR activity 60 25 Activity (nmol/min/mg) Activity (nmol/min/mg) 30 20 15 10 5 50 40 30 20 10 0 0 0 2 4 6 0 Time (hr) 2 Time (hr) 4 6 GSH:GSSG ratio 25 20 15 10 5 0 0 2 4 6 Time (hr) 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 Elias Arnér June 17, 2010 Is SmTGR an essential protein? TGR C GAPDH C RNAi RNAi 100 % Survival 80 60 40 20 0 1 2 Days 3 4 SmTGR is essential for Schistosoma mansoni survival in culture, under both aerobic and anaerobic conditions Kuntz et al. (2007) PLoS Med 4(6): e206 Elias Arnér June 17, 2010 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 Worm counts • • 50 40 30 20 10 0 ** ** Control AF Treatment Treatment of infected mice with auranofin gave significantly lower worm burden (p<0.015, n=7) Kuntz et al. (2007) PLoS Med 4(6): e206 Elias Arnér June 17, 2010 Mammalian thioredoxin reductase - kinetic properties - importance for cancer therapy Elias Arnér June 17, 2010 The mammalian thioredoxin system ASK-1 Apoptosis induction Dehydroscorbic acid Trx-S2 NADPH + H+ Ascorbic acid Lipid hydroperoxides Alcohols Secreted Trx as cocytokine Converted to Trx80 as secreted chemokine TrxR Ribonucleotide reductase ASK-1 Apoptosis repression GSSG α-Lipoic Acid DHLA 2 GSH NADP+ P53 maturation and function Trx-(SH)2 H2O2 H2O+O2 GPxox AP-1 activation NF-κB repression (in cytosol) Prxox GPxred Prxred Nordberg J. & Arnér E.S.J. (2001). Free Rad. Biol. Med.31,1287-1312. Elias Arnér June 17, 2010 Catalytic mechanism of TrxR 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 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 … Elias Arnér June 17, 2010 Catalytic mechanism of TrxR Arnér, E.S.J. (2009) BBA 1790, 495-526 Elias Arnér June 17, 2010 The selenenylsulfide of TrxR Cheng et al. (2009) JBC, 284:3998-4008 Elias Arnér June 17, 2010 Catalytic cycle(s) of TrxR1 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 Elias Arnér June 17, 2010 Electrophilic compounds easily target the selenocysteine residue in TrxR NADPH + H+ S NADP+ SH Se Se- 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 SH Se June 17, 2010 A549 cells Controls/MOCK V1/V2 siRNA 75Se: TrxR1 Protein: Eriksson et al (2009) FRBM, 47:1661-1671 Elias Arnér June 17, 2010 Effects of targeting TrxR1? TrxR1 siRNA MOCK Knockdown of TrxR1 to ≈10% sensitizes cells towards DNCB and menadoione No difference in sensitivity towards auranofin Eriksson et al (2009) FRBM, 47:1661-1671 Elias Arnér June 17, 2010 Effects of targeting TrxR1? TrxR1 siRNA MOCK Knockdown of TrxR1 to ≈10% makes cells more resistant to cisplatin No difference in sensitivity towards oxaliplatin or juglone Eriksson et al (2009) FRBM, 47:1661-1671 Elias Arnér June 17, 2010 Effects of targeting Sec in TrxR1? 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 S S S Se S S ROS production SecTRAPs S Se • Rapid process Complete active site inhibited - similar effect as TrxR knockdown Only Sec residue targeted • Caspase involvement • Membrane integrity lost • Not requiring protein synthesis Cell death, cell cycle arrest or other cellular effects due to an impaired thioredoxin system 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 June 17, 2010 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 Elias Arnér June 17, 2010 Elias Arnér June 17, 2010 Acknowledgements Hanna-Stina Ahlzén Marcus Cebula Qing Cheng Victor Croitoru Pascal Dammeyer Sofi Eriksson Stefanie Prast-Nielsen Jianqiang Xu Elias Arnér 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