Chryssostomos Chatgilialoglu

Transcription

1 Chryssostomos Chatgilialoglu 2011

2 RS/RS in physiological concentrations play a role in regulating the activation of transcription factors, cell proliferation and apoptosis. RS/RS play a critical role in eliminating viral and microbial infections and thus function as an efficient cellular defense mechanism. The overproduction of RS/RS has been linked with the etiology of various diseases and the inflammatory response.

3 2 3 Physiological [ ] ~ 10 nm Inflammed tissues up to ~ 1 µm arginine citrulline S 2 2 H s 1 pk a = x 10 9 M 1 s 1 2 ADPH oxidase Xanthine oxidase mitochondria Basal level of 0.1 nm ~ 10 nm 2 C 2 C x 10 4 M 1 s 1 <100 ns C 3

4 H Fe(III) Fe(II) 2 MP 2 H + Lipids Proteins DA HCl Cl, MP x Cl, 2 SC or Br MP H 2 2 SD 2 C 2 H HSC or HBr C 2 C 3

5 -Cell radiation source from 60 Co Dose rate 5 Gy/min 20 min irradiation 100 Gy dose ca µm of reactive species 45% 45% 10% H 2 e H (0.28), H H + aq (0.27), (0.06), (0.27), H 2 2 (0.07) (radiation chemical yields, µmol/j)

6 H 2 e aq (0.27), H (0.28), H (0.06) (radiation chemical yields, µmol/j) 2 -saturated aqueous solution [ 2 ]= M 2 / 2 (90:10) aqueous solution [ 2 ]=1.34 x 10 4 M 90% H + 10% H H e aq H x 10 9 M 1 s 1 75% H + 25% 2 H + 2 H e aq x M 1 s x M 1 s 1

7 LIPIDS Trans Lipids (geometrical) Lipidomics of cell membranes utrilipidomics Defences PRTEIS S-containing residues Methionine Cysteine/Cystine Reductive stress xidative stress UCLEIC ACIDS DA damage Sugar radicals Guanyl radicals xidative stress & lesion detection Excess Electron Transfer G-quadruplex Molecular libraries and reaction mechanisms

8 Double bonds of unsaturated fatty acids are cis Desaturase enzymes provide only cis geometry during lipid biosynthesis The cis geometry regulates membrane properties The cis geometry is requested for biological activity

9 CIS TRAS Increases of trans fatty acids in membrane lipids Decrease Fluidity and Permeability Signal transduction Protein access Membrane receptor functions Metabolite transport

10 1) Microbial biohydrogenation 2) Industrial processes of vegetable and fish oils 3) Deep frying of edible oils 4) Enzymatic pathway in bacterial 5) By radical stress in mammals

11 (The capacity of bacteria to alter their membrane composition) cis trans isomerase 9 9t C16:1 9 9c C16:1 Synthesis of cyclopropane 9

12 Bacteria like Pseudomonas and Vibrio use enzymatic cis trans isomerization of membrane lipids as a short-term adaptation response (e.g. temperature increase, environmental toxicity). H 3 P R 2 R 1 sn-2 sn-1 Phosphatidyl ethanolamine 9 9c C16:1 9 isomerase 9 9t C16:1 Mammals do not have any enzymatic pathway to form trans-fa.

13 Living cells top-down approach Cell cultures Analyses of tissues (with pathologies) Analyses of red blood cell membrane Trans Phospholipids bottom-up approach Model studies in solution and vesicles Cis Phospholipids

14 kcal/mol SR R 2 R 1 RS + R 1 R 2 k cis a k cis f k cis a = 1.6 x 10 5 M 1 s 1 k trans a = 2.9 x 10 5 M 1 s RS H H R 1 R k trans a k trans f RS + k f cis = 1.7 x 10 7 s 1 k f trans = 1.6 x 10 8 s 1 R 1 R 2 J. Am. Chem. Soc. 2002, 124, ChemPhysChem 2005, 6, 286.

15 L- -Phosphatidyl Choline P sn-2 sn-1 R 2 R 1 polar head hydrophobic chains H 2 alcol MLV LUVET

16 LUVET Hydrophilic substrate Amphiphilic substrate

17 Phospholipid Hydrophobic substrate Water or buffer Magnetic stirring Suspension of unilamellar vesicles incorporating the hydrophobic substrate

18 H 2 e aq (45%), H (45%), H (10%) acqua e H aq RSH + H /H 2 + H + RS aqueous interior R 1 R 2 RS RS H H R 1 R 2 -RS acqua R 1 R 2

19 9c,12c C18:2 Exogenous (diet) desaturase 6 6c,9c,12c C18:3 elongase 8c,11c,14c C20:3 desaturase 5 5c,8c,11c,14c C20:4 5trans- and 8trans-isomers are markers of endogenous formation 5 8 Endogenous (enzyme) Free Radical Biol. Med. 2002, 33, 1516 J. Am. Chem. Soc. 2004, 126, 1063

20 Cultured in a trans-free environment Human monocytic leukemia cell (THP-1) incubated with 10 mm of a thiol under radical stress Total trans lipids % Total trans lipids % Total trans lipids up to 20% Free Radical Biol. Med. 2005, 38, 1180

21 Liver Heart Total trans-fa (%) Total trans-fa (%) Kidney homogenates Kidney Adipose tissue Erythrocytes ~50% arachidonic acid trans isomers derived from an endogenous proccess Total trans-fa (%) control Plasma lipids CCl 4 treated rats Free Radical Biol. Med. 2006, 40, 1549

22 Trans Lipids: The Free Radical Path C. Chatgilialoglu, C. Ferreri Acc. Chem. Res. 2005, 38, (DI: /S X) (DI: /tx )

23 500 µm LH 400 Gy 20 µm L chain propagation length: µm trans LH catalytic cycle: 370 rg. Biomol. Chem. 2011, 9, 3541.

24 J. Am. Chem. Soc. 2011, 133,

25 15 samples % mono-trans J. Am. Chem. Soc. 2011, 133,

26 Radical Stress Sulfur-containing Protein/peptide Diffusible thiyl radicals (e.g. HS or CH 3 S ) Protein/peptide modification Lipid modification Biomarkers identification Molecular libraries

27 H 2 S pk a =6.9 HS + H H 2 S + h 75 HS + h HS + H ph =3, 5, 7 HS + e aq H 2 S/HS S + HS -irr. or h HS /S HSS 2 S S + R 1 R 2 H R1 % Trans isomer H R 2 50 e aq + H 2 S 25 H + H 2 S/HS 0 H + H 2 S/HS [H 2 S] = 0.2 mm HS Time / min pk a S4 + HS + H R 1 ph =9, H 2 + HS H 2 + HS S + H + R 2 Angew. Chem. Int. Ed. 2007, 46, 1914

28 protein Met S CH 3 aqueous Cys S S HS -irr. protein CH 3 S aqueous interior S -irr. Aba Ala

29 Cys110, Cys58, Cys72 Ala -irr. S S CH 3 S CH 3 S Met79 Aba LUVET ChemBioChem 2006, 7, 1738 J. Proteome Res. 2008, 7, 2007

30 Cys34 Cys514 Cys567 Ala S S -irr. Met123 Met298 Met548 Aba CH 3 S CH 3 S LUVET

31 protein Met -irr. protein Aba H CH 3 S protein + CH 3 SH 2 protein Chem. Res. Toxicol. 2010, 23, 254 protein Hser

32 disulfide radical anion Rase A, HSA e aq S CH 2 H 2 C S H + H 2 C S S CH 2 S H H S CH 2 H 2 C S sulfuranyl radical H H 3 C H 2 C S Cysteine residue S H 3 C H 2 C S Alanine residue

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35 5,8-Cyclo-2 -deoxyadenosine H 5' 8 H 2 H 5' 8 H 2 (5 R) (5 S) 5,8-Cyclo-2 -deoxyguanosine H 5' 8 H 8 H H 5' H 2 H 2 (5 R) (5 S)

36 Identified in mammalian cellular DA in vivo (5 S)-cyclo-dA levels similar to 8-oxoG Their levels are enhanced by oxidative stress conditions Jaruga et al. ucleic Acids Res. 2004, 32, e87; D Errico et al. EMB J. 2006, 25, 4305; Rodriguez et al. Biochemistry 2007, 46, 2488; Jaruga & Dizdaroglu, DA Repair 2008, 7, Associated with neurological diseases in xeroderma pigmentosum patients P. J. Brooks, euroscience 2007, 145, P. J. Brooks, DA Repair 2008,

37 major groove H H H H base pair H H H minor groove rder of reactivity of H radical: H5 >H4 >H3 H2 H1

38 H 2 H 2 H xid. Base 2 Base Base

39 H 2 H 2 H H 10-11% H H / % 2 Adenine H 2 H 2 H H (5 R):(5 S) = 6:1

40 H H H H 2 H 8 10% H H 2 6 8% H / 2 Guanine 2 H H H H 2 H H 2 (5 R):(5 S) = 8:1

41 ES-MS: m/z 250 (MH+). MS 2 (250):164 H 2 H 2 m/z 164 m/z 164 H H J 4'5' 1 Hz 1 H MR J 4'5' 6 Hz m/z 180 H m/z 180 H H H 2 H H 2 ES-MS: m/z 266 (MH+). MS 2 (266):180

42 Pulse radiolysis and -radiolysis e aq H 2 H 2 H 2 H Br H H

43 720 nm 360 nm H 2 H 2 Br H e aq M 1 s 1 H H 2 H H 2 H s s 1

44 H 2 H H 1.6 x 10 5 s -1 H H 2 H GSH 5 x 10 7 M 1 s 1 A 2 Fe(C) x 10 9 M 1 s 1 k obs = k c + k[gsh] 2 x 10 9 M 1 s 1 H A H A

45 H 2 H 2 H H k c = 1.6 x 10 5 s -1 H H H H H H 2 k c = 6.9 x 10 5 s 1 H H 2

46 H 2 e aq (0.27), H (0.28), H (0.062) (radiation chemical yields (G) in µmol/j) 45% e aq + 45% H + 10% H H e aq H + H + 2 k(e aq ) = M -1 s -1 90% H + 10% H

47 1) Irradiated sample of DA ( 2 - or 2 -saturated and up to 60 Gy of dose) 2) DA enzymatic digestion (uclease P, DAse II, phosphodiesterase I and II, and alkaline phosphatase) 3) LC-MS/MS analysis Lesions / 10 6 nucleosides / Gy Lesions calf thymus DA 2 2 5,8-cyclo-dGuo ,8-cyclo-dAdo

48 H 2 e aq (0.27), H (0.28), H (0.062) (radiation chemical yields (G) in µmol/j) e aq + 2 H H + k(e aq ) = M -1 s -1 k(h ) = M -1 s -1

49 (5 R):(5 S) = 4:1 (5 R):(5 S) = 3:1

50 Modification* 2 2 / 2 (9:1) 8-xo-dGuo xo-dAdo ,8-cyclo-dGuo ,8-cyclo-dAdo *Expressed in number of modified nucleosides per 10 6 nucleosides and per Gy

51 H 2 H H 2 H H 8-oxo-dGuo 8-oxo-dAdo

52 Biomimetic Chemistry Reaction mechanisms Radiation chemistry Biomarkers Identification Biomarkers Validation Libraries of Modifications Clinical Research Genomics Epigenomics Proteomics Metabolomics Lipidomics rganic synthesis Analytical protocols

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56 CTRL Re-equilibrium Unbalance Ad hoc nutrition & nutrilipidomics Familiarity Metabolism Diet Radical stress

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58 Acknowledgements Carla Ferreri Armida Torreggiani M. Guerra Q.G. Mulazzani M. Tamba Action CM0603 Free Radicals in Chemical Biology 15/06/ /06/2011 K. Bobrowski (Warsaw) J. Cadet (Grenoble) B. Mihaljevic (Zagreb) A. Scaloni (apoli) Abdelouahid Samadi Antonio Manetto Clara Caminal Liliana B. Jimenez Marialuisa Russo Katalin Dajka Ioannis Lykakis Tamara Perchyonok Maria Panagiotaki Jordi Domenech Casal livier Mozziconacci Panagiotis Kaloudis Fabien Boussicault Sebastian Barata Vallejo Marie Spadafora Fernando Moyano Zuzana Jurasekova Michail A. Terzidis Michele Melchiorre Kerry Gilmore (2002/03) (2003/04) (2003/05) (2004/06) (2004/06) (2004/06) (2005/06) (2005/06) (2006/07) (2006/07) (2006/07) (2006/08) (2006/07) (2008/10) (2009) (2009/10) (2010) (2010/11) (2010/11) (2010/11)