1 / 40

Thiol Proteins Thioredoxin, glutaredoxin, protein disulfide isomerase,

Thiol Proteins Thioredoxin, glutaredoxin, protein disulfide isomerase, peroxidases, methionine sulfoxide reductase Thiol Protein – at least one very reactive cysteine. Thiols in Biology. COO - COO -

hubert
Télécharger la présentation

Thiol Proteins Thioredoxin, glutaredoxin, protein disulfide isomerase,

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Thiol Proteins Thioredoxin, glutaredoxin, protein disulfide isomerase, peroxidases, methionine sulfoxide reductase Thiol Protein – at least one very reactive cysteine

  2. Thiols in Biology COO - COO - H-C-CH2-SH H-C-CH2-S- + H+ pKa = 8,37 NH3+ NH3+ cysteine pH = 8,37: [RSH] = [RS-] thiol thiolate pH < 8,37 [RSH] > [RS-] pH > 8,37 [RSH] < [RS-]

  3. N H O H O 3 O O C C H C H C H C N H C C N H C H C 2 2 2 O O C H 2 S H c y s t e i n e g l y c i n e - g l u t a m a t e l Glutathione (GSH) pKa = 9,2 Low toxicity High intracellular concentrations (1-10mM)

  4. Thiolate stabilization in proteins

  5. Thiol/disulfide exchange reactions R1SH + R2SS R2 R1SS R2 + R2SH • central theme in biology • structure of proteins • Regulation of protein activity • (enzyme, transcription factors...) • -cellular redox homeostasis etc...

  6. Rlg Rn Rn-S- + S-S S-S + Rlg-S- R R k nucleophilic agent leaving group Mechanism of thiol/disulfide exchange reactions nucleophilic substitution V = k[Rn-S- ] [ RSSRlg]

  7. Thioredoxin (Trx) Dissulfide reductase - 12-13kDa - plants, animals, yeast , bacteria target trx trx H H target target trx trx Cys35-SH Cys32-S - Cys35-SH Cys32-S - S S S S Cys35-S Cys32-S - -S S -S S Cys35-S Cys32-S Cys35-S Cys32-S HS HS HS HS thioredoxin reductase NADPH + trx (-SS-) NADP + trx (-2SH) glutathione reductase NADPH + GSSG NADP + 2 GSH

  8. Thioredoxin fold thioredoxin

  9. Ribonucleotide Reductase, other target oxidized Grx GSSG (2-RSH) (RSSR) GSSG reductase NADPH,H+ Ribonucleotide Reductase, other target reduced Grx 2 GSH ) Glutaredoxin (Grx) 12-13kDa CXXC motifi (CPYC)

  10. Trx and Grx targets yeast PAPS Tioredoxin reductase peroxidase Ribonucleotide Reductase Methionine ? ? sulphoxide ? reductase Oxidized Grx1 GSSG Trx1 Target Grx2 Trx2 (2 - RSH) (2 - RSH) Glr1, NADPH, H Trr1, NADPH, H + + Reduced Grx1 Trx1 Target Grx2 GSH Trx2 ( - SS - ) ( - SS - )

  11. Methionine sulfoxide reductase Reviewed by Weissbach et al. (2002) Arch. Biochem. Biophys., 397:172.

  12. Deglutathionylation by Grx monothiol mechanism Grx-S- + protein –SS G Grx – SS G + protein-SH Grx – SS G + GSH GSSG + Grx-S-

  13. Protein Motif in active site Redox Potential (mV) TrxCys-Gly-Pro-Cys -270 GrxCys-Pro-Tyr-Cys -200 to –235 TryparedoxinCys-Por-Pro-Cys -249 Protein disulfide isomerase Cys-Gly-His-Cys -127 (PDI) DsbA Cys-Pro-His-Cys -125 Thiol/disulfide oxido-reductases

  14. PDI and DsbA generate disulfide bonds in proteins PDI is in the endoplasmatic reticulum (ER) DsbA in periplasm (bacteria) GSH:GSSG in ER 1:1 to 3:1 (100:1 to 30:1 in cytoplasm)

  15. Protein Oxidation States TrxCys-Gly-Pro-Cys-2 (thiol), -1 (disulfide) GrxCys-Pro-Tyr-Cys-2 (thiol), -1 (disulfide) TryparedoxinCys-Por-Pro-Cys-2 (thiol), -1 (disulfide) Protein disulfide isomerase Cys-Gly-His-Cys-2 (thiol), -1 (disulfide) (PDI) DsbA Cys-Pro-His-Cys -2 (thiol), -1 (disulfide) Glutathione Peroxidase Asn-Val-Ala-Ser-Lys-Cys -Gli -2 (thiol), 0 (sulfenic acid) non-selenium (GPx) Thiol proteins oxidation states

  16. Prx = peroxiredoxin 2 RSH + H2O2 RSSR + 2 H2O 2 RSH + ROOH RSSR + ROH + H2O 197 residues - 25kDa Active site: cysteine 47 cysteine 170 – catalysis Netto et al.(1996) J. Biol. Chem., 271, 15315-15321.

  17. Trivelli, X., Krimm, I., Ebel, C., Verdoucq, L., Prouzet-Mauléon, V., Chartier, Y., Tsan, P., Lauquin, G., Meyer, Y., Lancelin, J. (2003) Characterization of the yeast peroxiredoxin Ahp1 in its reduced and overoxidized inactive forms using NMR. Biochemistry42: 14139-49. Type A YPxDF[T/S]FVCPP[T/S]E[I/L/V] .....C-terminal VCrP Type B HPxDFTPVCPTTE Type C YPx[A/D]xTP[G/V] CPTx[Q/E]xCrx[F/L] Type D xP[G/A]A[F/Y][T/S][P/G]xCP[S/T]xxHxP Type E xP[D/S]DTxVCPxx[Q/S]x[K/R]

  18. Trx = Prx = Gpx Amino acid sequence Prx and Trx have the same fold Trx Prx Choi et al. (1998) Nature Struct. Biol.5: 400 Gpx Prx

  19. Peroxynitrite reductase activity of bacterial Prx Bryk, R., Griffin, P. & Nathan, C. Nature (2000), 407:211-215 Prx OONO - + H+ + Trx NO2 + H2O + Trx 106 M-1 s-1 SH S- S -- S

  20. 1-cys 2-cys típica 2-cys atípica

  21. + DTT (-SS-) 61 125 SH SH 61 125 61 125 61 125 SOH SH SS-DTT-SH SH SSG SH 61 125 S S + DTT ROOH + H2O fast Sulfenic acid Reduced protein disulfide GSSG 61 125 61 125 SO3H SH SO2H SH ROOH GSH GSH ROOH Glutathionylated protein Sulfonic acid (oxidation state = +4) Sulfinic acid (oxidation state = +2)

  22. Versatility of Prx Prx (AhpC) as GSSG reductase Mutation = one amino acid insertion (Phe) Ritz et al (2001) Science294: 158

  23. Crystal structure of oxydized decamer High concentrations Disulfides in yellow Thioredoxin fold: 2x (beta-alfa-beta)

  24. 2-Cys –Prx are also chaperons!! WT Delta prx1/prx2 Delta prx1/prx2 + pRS416/cTPxI Delta prx1/prx2 + pRS416/C47S/C170S Heat shock (43 C por 30’) plating

  25. Proteins analyzed by SEC cTPxI,cTPxII 40-1000 kDa!! MW = 21,5 kDa (2-50 proteínas) Western blot 10% PAGE

  26. Electron Microscopy (EM) FI fraction Particle diameter: 22-28nm

  27. Electron Microscopy (EM) Two views: 409 “End on” (five fold symmetry) 170 “Double dot” FII fraction Particle diameter: 14 nm

  28. R-SH P-S47 - S170-P R-SH P-S47H  R-S47- P-S47OH INATIVA REATIVAINSTÁVEL peróxido P-S47O2H oligomerização

  29. R-SH P-S47 - S170-P R-SH P-S47H  R-S47- P-S47OH INATIVA REATIVAINSTÁVEL srx peróxido P-S47O2H oligomerização

  30. Sulfiredoxin (Srx) – 13kDa Cys 84 – reactive site Homolog in humans Biteau et al. (2003), Nature 425: 980

  31. Number of genes >>>>> Number of folds Trx Grx GSH transferase GSH peroxidase Prx Calsequestrins PDI Methionine sulfoxide reductase Trx fold

  32. Trx X Cytochrome c Maturation Protein • (CMP) • - high amino acid sequence divergence • trx fold • Conserved residues involved in catalysis

  33. Motif analysis Trx, CMPs and Prx common ancestor !!

  34. Ohr = Organic Hydroperoxide Resistance protein • Deletion of Ohr gene: • Bacteria sensitive to organic peroxides (not H2O2!!) • Only organic peroxides induce transcription of Ohr gene New peroxiredoxin from Xylella fastidiosa ?

  35. ROS RNS pathogen PLANT

  36. A. B. TBHP (200 M) H2O2 (200 M) Ohr (2 ng/ul) Ohr (50 ng/ul) Ohr is a thiol — dependent peroxidase Cussiol J.R.R., Alves S.V., Oliveira M.A. e Netto L.E.S. (2003) J. Biol. Chem , 278, 11570—11578

  37. Ohr - Xylella fastidiosa Tratada com t-BOOH (1mM/RT/1h) Tampão – Tris-Cl pH=8,5 0,1MPrecipitante – PEG 4000 25% Complete dataset at 1.9 Å Oliveira et al., (2004) Acta Crystall. D60, 337

  38. Ohr structure No trx fold – alfa-beta fold Ohr  Prx  GSH px. New class of thiol dependent peroxidase

More Related