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Reactive Thiols in Redox Signaling SFRBM Worshop November 15, 2006 Denver, CO

This workshop explores the role of reactive thiols in redox signaling and their detection using MS screening and S-glutathiolation labeling methods.

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Reactive Thiols in Redox Signaling SFRBM Worshop November 15, 2006 Denver, CO

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  1. Reactive Thiols in Redox Signaling SFRBM Worshop November 15, 2006 Denver, CO Richard A. Cohen, MD Director, Vascular Biology Unit Boston University School of Medicine BU-NHLBI Cardiovascular Proteomics Center Boston University Medical Center racohen@bu.edu

  2. Outline • Introduction: Reactive thiol proteome • MS screening for reactive thiols with ICAT • S-glutathiolation labeling and detection methods • S-glutathiolation in signaling

  3. Cysteine Thiols Modified by Oxidants Constitute a Thiol Proteome Pathological Physiological Reversible Signaling Irreversible Damage Pr-Cys-SO3H Pr-Cys-SH Pr-Cys-SO2H Pr-Cys-SOH Pr-Cys-S-SG Pr-Cys-SNO ROS

  4. S- NO2 ONOO- H2O2 .OH NO. SNO, SOxH Reactive Cysteine Thiolate Anion SH O O O N H N H O H N H O Cysteine N H

  5. GS. GSOH GSSG GSNO ROS/RNS S- GSH (0.5 - 5 mM) S- SSG Reactive Cysteine Thiolate Anion SH O O O N H N H O H N H S-Glutathiolation: Large - tripeptide Charged - glutamate Reversible GRX,TRX O Cysteine N H

  6. MS screening for reactive thiols with ICAT

  7. Biotin-labeled Iodoacetamide: Thiol-reactive iodoacetamide group Reduced Reactive Thiols Affinity tag (Biotin) Acid-Cleavable ICAT (Applied Biosystems, USA): Thiol-reactive iodoacetamide group Affinity tag (Biotin) Acid-cleavable linker Isotope-coded tag Light (9 12C) Heavy (9 13C)

  8. Nature Biotechnology 1999, 17, 994-999 Aebersold, R. and Coworkers

  9. H2O2 90% H2O2 10% % light heavy light heavy ICAT Approach to Redox Proteomics: Creatine Kinase Non-reactive cysteine Reactive cysteine Peptide with non-reactive cysteine 146 Labeling at pH= 8.5 Normal Light 9 -12C ICAT % m/z Peptide with reactive cysteine 283 LC-MS Oxidant stress Heavy 9-13C ICAT % Ox Mix and trypsin digestion Avidin separation m/z Ox M. Sethuraman et al. Mol Cell Proteomics 3: 273, 2004

  10. ICAT approach to complex protein mixtures Sucrose gradient Isolated rabbit heart SR membrane fraction- reducing agent/protease inhibitor free, 50 mM Tris pH 7.1 200 g protein, 10 mM H2O2 30 mins/RT 200 g protein No H2O2 Light ICAT Heavy ICAT Mixed, Trypsin digestion, Avidin affinity and LC-MS/MS M. Sethuraman et al. J. Proteome Research, 2004, 3(6):1228-33.

  11. Total Ion Chromatogram for LC-ESI-MS/MS of ICAT-labeled Tryptic Peptides from HPLC Cation Exchange Fraction 2 of Membrane Proteins 24.89 100 29.00 31.38 35.70 16.34 17.86 39.18 % 21.60 52.14 13.57 44.87 46.81 54.47 0 10.00 20.00 30.00 40.00 50.00 Time in minutes

  12. MS of ions eluted at 29.002 min Val232-Arg245 of GAPDH 576.319 100 579.325 576.651 579.659 576.984 577.307 % 579.992 580.316 533.318 0 100 m/z 580 577 578 579 515.306 371.717 736.395 % 981.507 561.317 671.357 773.430 863.979 900.471 570.651 999.469 0 m/z 350 450 550 650 750 850 950 1050 1150

  13. MS/MS Sequenced ICAT-labeled Peptides in Cardiac Membrane Fraction Ladder % Cys Score Oxidation with H2O2 Name Description Start End Sequence KCRS_RABIT Creatine kinase sarcomeric mitochondrial precursor 100 173 182 (R)GLSLPPACSR(A) 9.8 55.294 272 287 (R)LGYILTCPSNLGTGLR(A) 42.3 272 287 52.941 (R)LGYILTCPSNLGTGLR(A) 40.5 72% G3P_RABIT Glyceraldehyde phosphate dehydrogenase 69.863 232 245 (R)VPTPNVSVVDLTCR(L) 26.9 64.835 143 159 (K)IVSNASCTTNCLAPLAK(V) 72.2 721.08 720.74 100 ALFA_RABIT Fructose biphosphate aldolase 49.485 69 86 (R)VNPCIGGVILFHETLYQK(A) 12.3 ICAT ICAT 721.42 a 83.871 201 207 (R)CQYVTEK(V) 24.3 56.364 331 341 (R)ALANSLACQGK(Y) 24.1 IVSNASCTTNCLAPLAK 726.76 727.10 % 721.76 50.633 437 451 (K)VGEATETALTCLVEK(M) 32.5 ATA2_RABIT Sarcoplasmic endoplasmic reticulum Ca2+ ATPase Glyceraldehyde-3-phosphate dehydrogenase 727.43 44.138 372 397 (K)VDGDTCSLNEFTITGSTYAPIGEVHK(D) 15.1 727.94 79.07 468 476 (R)ANACNSVIK(Q) 28.6 0 727 PIGR_RABIT Polymeric immunoglobulin receptor precursor 25.62 353 374 (R)SPPVLKGFPGGSVTIRCPYNPK(R) 24 723 725 m/z 721 16.279 587 595 (K)ARCPVPRRR(Q) 99.6 62% 632.67 633.00 36.082 595 612 (R)RQWYPLSRKLRTSCPEPR(L) 13.7 ICAT 100 KPBB_RABIT Phosphorylase B kinase beta regulatory chain 29.508 276 287 (R)QTLCSLLPRESR(S) <5 633.34 635.68 32.836 832 844 (K)NIIYYKCNTHDER(E) 62.3 NIIYYKCNTHDER b 636.01 K6PF_RABIT Phosphofructose kinase muscle type 59.13 108 128 (R)GITNLCVIGGDGSLTGADTFR(S) <5 % 636.35 633.65 Phosphorylase  kinase 59.223 630 648 (K)CNENYTTDFIFNLYSEEGK(G) 48 636.68 81.967 224 235 (K)YQIDPDACFSAK(V) 45.6 POR1_RABIT Voltage dependent anion selective channel protein 1 66.99 120 138 (R)EHINLGCDVDFDIAGPSIR(G) 30.1 0 m/z 633 635 637 66.019 46 64 (K)SCSGVEFSTSGSSNTDTGK(V) 54.38 POR2_RABIT Voltage-dependent anion-selective channel protein 2 48% 750.91 96.364 64 74 (K)VCNYGLTFTQK(W) 48.6 POR3_RABIT Voltage-dependent anion-selective channel protein 3 751.42 100 28.767 64 77 (K)LDKPSVVNWMCYRK(T) 43.9 LCAT_RABIT Phosphatidylcholine-sterol acyltransferase [Precursor] ICAT RETB_RABIT Plasma retinol binding protein 57.143 172 181 (R)QRQEELCLSR(Q) 28.1 755.42 FA10_RABIT Coagulation factor 47.273 334 344 (R)RNVAPACLPQK(D) 39 VCNYGLTFTQK 755.93 751.92 756.42 752.41 75.676 88 95 (K)LSELHCDK(L) 49.6 HBB_RABIT Hemoglobin beta 1 chain c % 756.92 32.727 442 452 (K)QKKHCLVTVEK(G) 89.6 PRTS_RABIT Vitamin K dependent protein S precurson fragment Voltage-dependent anion channel protein 3 MDHM_RAT Malate dehydrogenase 50.746 92 104 (K)GCDVVVIPAGVPR(K) 26 DHSA_HUMAN Succinate dehydrogenase mitochondrial pre 64.384 233 246 (R)GVIALCIEDGSIHR(I) 26.7 0 m/z 751 753 755 757 56.164 233 246 (R)GVIALCIEDGSIHR(I) 26.7 64.706 7 22 (K)TTGLVGLAVCDTPHER(L) 31.5 NUFM_RAT NADH-ubiquinone oxidoreductase Sethuraman et al. J. Proteome Res, Dec 2004 ODO1_HUMAN 2-oxoglutarate dehydrogenase E1 component, mitochondrial 46.575 278 291 (R)FGLEGCEVLIPALK(T) 8.5 • 60 Cysteines ID’d by LC MS/MS • 9 Oxidized by >50% • 3 Oxidized >70%

  14. Val103 –Arg123 GSSG (0 μM) + 5 mM TCEP b) a) GSSG (0 μM) GSSG (100 μM) + 5 mM TCEP 830.003+ GSSG (100 μM) 830.003+ 833.023+ 100 100 833.023+ % % 0 m/z m/z 0 830 832 834 830 832 834 Reversible ICAT labeling due to S-glutathiolation of recombinant p21ras Cys-118 M. Sethuraman et al., submitted 2006

  15. S-glutathiolation labeling and detection methods • S-glutathiolation in signaling

  16. Reactive thiols bind to GSH in greatest abundance SERCA C674FARVEPSHKSK -GSS- Proteomic Screening for S-Glutathiolated Proteins + Protein-SH +ONOO- -GSH -GSS-Protein +Trypsin -GSS-Peptide +DTT Peptide-SH MALDI or ESI Mass spectrometry Adachi et al. Nature Medicine,10,1200-1207, 2004

  17. Phosphorylation Domain Nucleotide - binding Domain B - Domain Cys-674 P D351 Y294 (1) C674 Hinge Domain Stalk Domain Cytosolic Domain (997) Transmembrane Domain M11 M1 M3 M4 M5 M6 M7 M8 M9 M10 M2 Luminal Domain 674 AQRDACLNARCFARVEP S- SERCA: Sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase Cysteine-674 Olesen C, et al. Science 306:2251-5, 2004

  18. C674S Ctr Wt GSS- SERCA 110kDa- SERCA 110kDa- ONOO- (20 M) GSH Sepharose pull-down of intact SERCA via Cys-674 in transfected HEK cells Reactive thiols bind to GSH in abundance! -SSG- Adachi et al. Nature Medicine,10,1200-1207, 2004

  19. Reactive thiols bind to GSH in greatest abundance SERCA C674FARVEPSHKSK -GSS- Proteomic Screening for S-Glutathiolated Proteins + Protein-SH +ONOO- -GSH -GSS-Protein +Trypsin -GSS-Peptide +DTT Peptide-SH MALDI or ESI Mass spectrometry

  20. p21ras is a Redox-Sensitive Signaling Molecule TKR Shc Grb Sos Ras Raf Rac PI3K MEKK NADPH oxidase Cys-118 MEK1 MEK4 MEK6 ERK1/2 JNK P38 Akt (Mallis RJ, Thomas JA, Biochem J 2001) Proliferation Hypertrophy ONOO- -induced Glutathiolation of Proteins in Rabbit Aortic Homogenate Peptide masses peptides Protein MW Measured Mass Computed Mass Error Start End Sequence Da Da ppm 861.0504 1318.672 titin 110.05 1317.664 1317.707 -33 552 563 ATKTPVSDLRCK Na-K-ATPase Alpha subunit 114.3 1317.664 1317.732 -52 525 535 ILAAQGCK IAA 1359.659 PP2A subunitA, PR65 regulatory beta 60.97 1358.651 1358.64 9 334 345 CVSSPHFQVAER L-Ca-channel 149.73 1358.651 1358.724 -53 1186 1196 RASFHLECLKR 1638.793 MLCK 125.7 1637.785 1637.771 8 1049 1063 DLEVVEGSAARFDCK Na+ Channel alpha 225.73 1637.785 1637.801 -10 1159 1173 RFPCCQVSIESGKGK 1766.626 ROCK-1 158.33 1765.618 1765.833 -122 740 754 QCSMLDVDLKQSQQK 1791.699 RYR3 550.32 1790.691 1790.926 -131 1956 1970 CPTTLKELISQTMIR 2343.926 PP2A subunitA, PR65 regulatory beta 60.97 2342.918 2343.222 -130 494 514 RKSELPQDPHTKKLEAHCR 2383.89 IgG 11.71 2382.882 2383.137 -107 37 59 GLEWIACIRGGGSGSTNYASWAK Ras 20.91 2382.882 2383.166 -119 168 188 QHKLRKLNPPDESGRGCMSCK nNOS 160.85 2382.882 2383.104 -93 920 939 MREGDELCGQEEAFRTWAKK ROCK-1 158.33 2382.882 2383.154 -114 447 465 DEMEQKCRTSNIKLDKIMK IAA, MO 2441.977 IgG 11.71 2440.969 2441.142 -71 37 59 GLEWIACIRGGGSGSTNYASWAK IAA Ras 20.91 2440.969 2441.171 -83 168 188 QHKLRKLNPPDESGRGCMSCK IAA nNOS 160.85 2440.969 2441.109 -57 920 939 MREGDELCGQEEAFRTWAKK IAA 2490.252 Myosin 2489.244 2489.179 26 109 129 YAAWMIYTYSGLFCVTVNPYK Na-K-ATPase Alpha subunit 114.3 2489.244 2489.195 20 673 694 ACVINGMQLKDMDPSELVEALR IAA 2705.113 Ras 20.91 2704.105 2704.297 -71 102 126 VKDSDDVPMLNKCDVGNKCDLAAR L-Ca-channel 149.73 2704.105 2704.352 -92 18 39 NCELDKNQRQCVEYALKARPLR IAA nNOS 160.85 2704.105 2704.335 -85 829 850 CALMEMRHPNSLQEERKSYKVR RYR3 550.32 2704.105 2704.358 -94 3376 3399 KMLPIGLNMCTPGDQELISLKSR IAA, MO RYR3 550.32 2704.105 2704.16 -21 2086 2106 SQIAFPKMVASCCRFLCYFCR IAA(4)

  21. Techniques available for S-glutathiolation detection in cells • Biotin labeled glutathione ester • Radiolabeled glutathione • Anti-GSH antibody • Mass Spectrometry • In situ visualization of glutathione adducts

  22. Biotinylated GSH Ester Labeling of S-Glutathiolated Proteins in Intact Cells GSH-ester b membrane GSH b ROS SA beads Thiol reduction Western blot Proteomic studies Pr-S- GS-SPr b

  23. GSH ethyl ester GSH ethyl ester GSH ethyl ester 50 mM NaHCO3 pH 8.5 50 mM NH4HCO3 pH 8.5 --NH2 + --N-CO- Biotin --N-CO- Biotin Biotin Sulfo-NHS Biotin Sulfo-NHS 2h, RT 30 min, RT + + Biotin -CO-HNH Biotinylated GSH Ester Synthesis

  24. Protocol for Detection of Cellular S-glutathiolated Proteins with Biotinylated GSH Ester 250 µM BGSH ester 1h incubation Treatment X X min incubation Cell Lysis and elimination of low MW components on PD-10 size exclusion columns Affinity Purification: Streptavidin… Washing step Protein Recovery SDS-PAGE 1h to overnight, 4°C 5 times: 0.5 M NaCl 0.1% SDS 10 mM DTT

  25. GSS-Proteins in Rat SMC Cont Ang II Cont Ang II -DTT +DTT(10 mM) Streptavidin blot Biotinylated GSH Ester Tracer for S-Glutathiolated Proteins GSH-ester b membrane GSH b ROS Pr-S- GS-SPr b SA Pull-down SA Blot Adachi, JBC 2004

  26. 0 1 30 (min ) PE NO 1 30 Tension, 10 g (min) Bio-GSE 1 h NO S-Glutathiolates SERCA and Relaxes the Rabbit Aorta GSH-ester b membrane GSH b ROS -bGSS-SERCA 110 kDa- Pr-S- GS-SPr b NO (1 mM) SA Pull-down Blot SERCA Adachi et al. Nature Medicine,10,1200-1207, 2004

  27. Tension, 10 g PE NO 1 Bio-GSE 1 h Decreased NO-induced S-Glutathiolation of SERCA in Atherosclerotic Rabbits -bGSS-SERCA -SERCA 20 * N.S. 15 (unit) 10 5 0 PE PE+NO PE PE+NO Normal Atherosclerotic Adachi et al. Nature Medicine,10,1200-1207, 2004

  28. Ascorbate - - - + DTT - - + - ONOO- - + + + IB p21ras --21 kDa Biotinylated GSH Ester Tracer for S-Glutathiolated Proteins GSH-ester b membrane GSH b ROS Pr-S- GS-SPr b SA Pull-down Blot p21ras Clavreul et al. FASEB J, 2006

  29. Ras S-Glutathiolation -GSS-Ras -Ras GSS-Ras Control Ang II 10–7 M Ras -7 -8 -9 Control H2O2 (250 mM) AII log [mol/L] (15 min) p21ras S-Glutathiolation caused by Angiotensin II in RASMC GSH-ester b membrane GSH b ROS Pr-S- GS-SPr b SA Pull-down Blot p21ras Adachi, JBC 2004

  30. 4 ** 3 Fold Increase 2 1 0 Control BGSH BGSH SA Increase in intracellular GSH caused by biotinylated-GSH ester in BAEC Clavreul et al. FASEB J, 2006

  31. S-Glutathiolated proteins detected with 35S-GSH [35S] cysteine Cycloheximide IP 2D-Gel Fratelli, PNAS 2002

  32. 4 GSS-p21ras * 3 Fold Increase 2 1 0 LacZ C118S Grx C118S Grx LacZ oxLDL DTT - - + + oxLDL - + - + p21ras-- --21 kDa S-Glutathiolation of p21ras in Endothelial Cells Exposed to Oxidized LDL: Anti-GSH Antibody IP: p21ras IB: GSH Clavreul et al. ATVB 2006

  33. 953.32 118-CDLAAR-123 953.43 953.43 SSG OxLDL OxLDL + DTT + DTT 150-QGVEDAFYTLVR-161 1397.69 1397.69 1397.63 1397.63 S-Glutathiolated p21ras in Endothelial Cells Exposed to Oxidized LDL: MALDI-TOF IP p21ras 1 1 1 2 2 2 3 3 3 100 100 100 75 75 75 50 50 50 37 37 37 25 25 25 20 20 20 15 15 15 - - - Coomassie -GSH Anti IB GSH Clavreul et al. ATVB, 2006

  34. Acknowledgements: Vascular Biology Unit: Nicolas Clavreul Karlene Maitland Ardelle Jia Ying Chaomei Shi Takeshi Adachi Xuiyun Hou Reiko Matsui Maha Sethuraman Cardiovascular Proteomics Center Bingbing Jiang Mark McComb Mengwei Zang Claire Dauly Xiaoyong Tong Peter O’Connor Markus Bachschmid Cheng Zhao Stefan Schildknecht Cathy Costello Tyler Heibeck Hossein Bayat University of Kansas Rebecca Zee Christian Schoeneich Robert Weisbrod Viktor Sharov Shanqin Xu

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