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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.

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slide1

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

[email protected]

outline
Outline
  • Introduction: Reactive thiol proteome
  • MS screening for reactive thiols with ICAT
  • S-glutathiolation labeling and detection methods
  • S-glutathiolation in signaling
slide3

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

slide4

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

slide5

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

slide7

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)

slide8

Nature Biotechnology 1999, 17, 994-999

Aebersold, R. and Coworkers

slide9

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

slide10

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.

slide11

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

slide12

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

slide13

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%
slide14

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

slide16

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

slide17

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

slide18

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

slide19

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

slide20

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)

slide21

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
slide22

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

slide23

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

slide24

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

slide25

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

slide26

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

slide27

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

slide28

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

slide29

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

slide30

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

slide31

S-Glutathiolated proteins detected with 35S-GSH

[35S] cysteine

Cycloheximide

IP 2D-Gel

Fratelli, PNAS 2002

slide32

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

slide33

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

slide34

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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