Measuring the poise of thiol/disulfide redox in vivo

Dean P. Jones, Ph.D.Department of Medicine/Division of Pulmonary, Allergy

and Critical Care MedicineEmory University, Atlanta

Emory Clinical Biomarkers Laboratory

The redox state of GSH/GSSG provides a measure of the balance of prooxidants and antioxidants

Jones Meth Enzymol 2002

GSHGSSG

Redox states of different couples can be compared by expression as redox potentials

Eh = Eo + * ln [Ox]_ [Red]

RTnF

Low molecular weight thiols and disulfides are measured by HPLC

Reversible oxidation of thiols alters protein structure and function

Active site

Reduced Trx1 Oxidized Trx1

Watson et al. 2003

Active site

Redox "OFF" switch

Dimerization site

S-Glutathiylation site, S-Alkylation site

S-Nitrosylation site

ASK-1 dockingregulatory site

All cysteines in Trx1 are important in Trx1 function

Protein thiol/disulfide redox states are measured by Redox Western blot analysis

Trx1-Ox2Trx1-Ox1Trx1-Red

Trx1-Ox2Trx1-Ox1Trx1-Red

0 2 10 30 60 120

Time (min) after H2O2

Nuclei

Cytoplasm

µM tBH 0 50 200 300 400

Trx1R

Trx1O

Trx2R

Trx2O

Watson, Jones FEBS Lett 2003Watson et al, JBC 2003

Chen et al FEBS Lett 2006

SDS-PAGE separation by mass following treatment with AMS

Native gel separation by charge following treatment with IAA

Cytoplasm

Mitochondria

Quantification of thiol/disulfide redox in biologic systems has provided 3 general conclusions

1. At the cellular level, GSH redox becomes oxidized as cells progress through the life cycle, and cells regulate extracellular thiol/disulfide redox state

2. At the systemic level, plasma GSH redox becomes oxidized with oxidative stress and is oxidized in association with aging and chronic disease

3. In cells and plasma, GSH redox is NOT equilibrated with thioredoxin or Cys/CySS, providing the basis to consider discrete redox circuits for redox signaling and control

Re

dox

Sta

te (

ER

ed

ox S

tate

(E

h, m

V)

Proliferation

Apoptosis

Differentiation

Redox of GSH/GSSG becomes progressively oxidized in the life cycle of cells

-250

-200

-150

-250

-200

-150

Kirlin et al, FRBM 1999; Nkabyo et al, Am J Physiol 2002

1:1

1:10

10:1

100:1

-(SH)2:-SS-

Extracellular Cys/CySS pool in culture is regulated to a value very similar to that in human plasma

Ext

rac

ellu

lar

Eh (

Cy

s/C

ySS

) (m

V)

0 4 8 12 16 20 24

Time (h)

-120

-100

-80

-60

-40

-20

+200 M Cysteine

+100 M Cystine

HT29 cells

0-50-100-150-200

120

80

40

0

Eh (mV)

Eh, Cys/CySS

Mean=-72.4StDev=12.8

Eh, GSH/GSSG

Mean=-130.9StDev=22.9

Fre

que

ncy

Plasma, 740 subjects

Go and Jones, Circulation 2005Jonas et al, FRBM 2002

Interorgan GSH/Cysteine balance

TissuesPlasma

GSH/GSSGGSH/GSSG

Cys/CySSCys/CySS

Major poolMost reduced

Major poolMost oxidized

-138 mV-220 mV

-150mV

-80 mV

Quantification of thiol/disulfide redox in biologic systems has provided 3 conclusions

1. At the cellular level: Cells regulate extracellular thiol/disulfide redox state. Cellular GSH redox becomes oxidized as cells progress through the life cycle

2. At the systemic level: Plasma GSH redox becomes oxidized with oxidative stress. Plasma redox is oxidized with aging, nutritional deficiency, toxicity and chronic disease

3. Relationship of redox couples: GSH redox is NOT equilibrated with thioredoxin or Cys/CySS. This provides the basis to consider discrete redox circuits for redox signaling and control

Many people have redox states more oxidized than young healthy individuals

Ext

rac

ellu

lar

Eh (

Cy

s/C

ySS

) (m

V)

0 4 8 12 16 20 24

Time (h)

-120

-100

-80

-60

-40

-20

+200 M Cysteine

+100 M Cystine

HT29 cells

0-50-100-150-200

120

80

40

0

Eh (mV)

Eh, Cys/CySS

Mean=-72.4StDev=12.8

Eh, GSH/GSSG

Mean=-130.9StDev=22.9

Fre

que

ncy

Plasma, 740 subjectsYoung healthy in RED

Go and Jones, Circulation 2005Jonas et al, FRBM 2002

Reduced

OxidizedOxidizedReduced

Plasma redox provides a useful measure of oxidative stress in humans

25 45 65 85

Age (y)

Eh

(mV

)

-150

-100

-50

GSH/GSSG

Cysteine/Cystine

25 45 65 85

Age (y)

Eh

(mV

)

-150

-100

-50

-150

-100

-50

-150

-100

-50

GSH/GSSG

Cysteine/Cystine

GSH & Cys redox oxidized with age

GSH redox is oxidized with chemotherapy Antioxidants decrease Cys oxidation with age

Cys redox oxidized with smoking

Jones, FRBM 2002

Jonas, Am J Clin Nutr 2000

Moriarty, FRBM 2004

Moriarty-Craige, Am J Ophthalmol 2005

536466

Smoking StatusCurrentPriorNever

Eh C

ys (

mV

) - 90

- 80

- 70

- 60

*

536466N =CurrentPriorNever

- 90

- 80

- 70

- 60

*

-100

-110

-120

-130

Eh

(mV

)

p = 0.001, effect of time

Pre

-ch

em

o

Po

st-

ch

em

o

Da

y 3

Da

y 7

Da

y 1

0

Da

y 1

4

-

-110

-120

-130

Eh

(mV

)

p = 0.001, effect of time

Pre

-ch

em

o

Po

st-

ch

em

o

Da

y 3

Da

y 7

Da

y 1

0

Da

y 1

4

-100

-120

-140

70 72 74 76 78

Age (y)

EhG

SH

(m

V)

P = 0.002 for effect of time

Mean age = 71.7

Mean age = 76.3-100

-120

-140

70 72 74 76 78

P = 0.002 for effect of time

Mean age = 71.7

Mean age = 76.3

+Vit C, E, -car

Control

Plasma redox is oxidized in association with disease and disease risk

-110

<60Controls

>60Controls

Type 2Diabetes

**

-135

GS

H/G

SS

G E

h (

mV

)

* *

GSH/GSSG is oxidized in T2 Diabetes

Samiec et al, FRBM 1998

Eh GSH/GSSG predicts IMT

0.590.61

0.67

0.54

0.58

0.62

0.66

Eh GSH/GSSG C

aro

tid

IM

T (

mm

)

< -130 mV

p value 0.009

> -120 mV-120 to -130 mV

Ashfaq et al, Am Coll Cardiol 2006

Increased Carotid Intima Media Thickness

Chemotherapy/BMT

Cigarette Smoking

Type 2 Diabetes

Reversible myocardial perfusion defects

Pathophysiologic correlationLow antioxidants, low dietary cysteine

Health-80 mV

-20 mV(-80 mV)

-50 mV(-110 mV)

Cys/CySS Redox(GSH/GSSG Redox)

(-140 mV)

Jones, Antiox Redox Signal, 2006

Lung transplantation

Alcohol abuse

Aging-62 mV(-122 mV)

Quantification of thiol/disulfide redox in biologic systems has provided 3 general conclusions

1. At the cellular level, GSH redox becomes oxidized as cells progress through the life cycle, and cells regulate extracellular thiol/disulfide redox state

2. At the systemic level, plasma GSH redox becomes oxidized with oxidative stress and is oxidized in association with aging and chronic disease

3. In cells and plasma, GSH redox is NOT equilibrated with thioredoxin or Cys/CySS, providing the basis to consider discrete redox circuits for redox signaling and control

-250

-200

-150

Re

dox

Sta

te (

E

-300

-250

-200

-150

Re

dox

Sta

te (

E h, m

V)

-300

Proliferation

Apoptosis

Proliferation DifferentiationTrx

GSH

Differentiation

Apoptosis

Proliferation DifferentiationCys

GSH, Trx and Cys redox systems are not in redox equilibrium in cells

Jones et al FASEB J 2004

H2O2

Trx-300

-200

-150

-250

Cys/CySSGSH/GSSG

(apoptosis)

NADPH

Eh (

mV

)

4

GRTR1

O2

SO

O2

TO

2

5/GPx

GSH/GSSG(differentiation)

6b

1/Prx 6a

GSH/GSSG(proliferation)

Cellular

Extracellular

H2O2

Grx

3

GSH/GSSG, Trx and Cys/CySS provide independent nodes for redox signaling and control

Jones et al, FASEB J 2004

GSH/GSSG

Cys/CySS

GSH/GSSG

Cys/CySS

Trx/TrxSS

EGFR MAPK activation

KEAP-1 Nrf-2 translocation to nucleus

Trx/TrxSS

ASK-1 Apoptosis

Nrf-2 DNA binding

Protein synthesisProtein S-thiylation

Redox-dependent systems are differentially controlled byGSH, Trx1 and Cys redox couples

GSH/GSSG

Trx(-SH)2/SS

Cys/CySS

Trx1(-SH)2/SSCys/CySSGSH/GSSG

Trx2(-SH)2/SS

GSH/GSSG

Plasma/InterstitialCytoplasmic

Nuclear

Mitochondrial

Endoplasmic Reticulum

GSH/GSSGPDI

Hansen et al, Annu Rev Pharm Tox, 2006

GSH/GSSG

Compartmentation of thiol/disulfide redox state

Trx2 is preferentially oxidized by TNF

0 5 10 20 40 1 0 5 10 20 40 1

TNF(ng/ml)

H2O2

(mM)H2O2

(mM)TNF(ng/ml)

0 5 10 20 40 H2O2 0 5 10 20 40 H2O2

Thioredoxin-1 Thioredoxin-2

TNF (ng/ml) TNF (ng/ml)

-300

-280

-260

-240

Red

ox

Po

ten

tial

(E

h)

-380

-360

-340

-320

-300

-280Red

ox

Po

ten

tial

(E

h)

J. Hansen

Mitochondrial redox circuits

NADPH

NADH

Cyt c

O2

GR

GSH

-400

-200

0

+200

+400

+600

Redox Signaling and Control Circuits(low flux)

Metabolic Redox Circuits(high flux)

Eh

PyrMal

Succinate

MPT

TR2

Trx2

O2

NADPH

GPx Prx3

H2O2

PrSSGGrx2

Metabolicsubstrates

ASK1

O2-

RegulatorySignal

MnSOD

O2-

DP Jones, Chem-Biol Interact 2006

CoQ

Summary: Trx2 in Mitochondrial Compartment

1. Mitochondrial Trx2 has a more reduced redox state than cytoplasmic or nuclear Trx1 or cellular GSH

2. Mitochondrial Trx2 is more susceptible to oxidation than the cytoplasmic Trx1

3. Redox western blot analysis of mitochondrial Trx2 provides a useful approach to measure mitochondrial oxidative stress

GSH is difficult to measure in nuclei

Cotgreave, 2003 Bellomo, 1992 Voehringer, 1998

Translocation of Trx from the cytoplasm to the nucleus

Hirota et al, J Biol Chem (1999) 274:27891

-150

-200

-250

-300

-350

0 10 20 30 40 50 60Time (min)

Eh (

mV

)

Trx/TrxSS

GSH/GSSG

Time courses of GSH and Trx1 oxidation are similar

Trx-1 is somewhat more resistantTrx-1 recovers somewhat more rapidly

Trx-Ox2Trx-Ox1Trx-Red

Trx-Ox2Trx-Ox1Trx-Red

0 2 10 30 60 120 Time (min)

Nuclei

Cytoplasm

High levels of oxidants are not selective between GSH and Trx1

Watson, Jones (2003) FEBS Lett 543:144

+1 mM H2O2

Physiologic oxidation in response to EGF is specific to cytosolic Trx-1

-285

-275

-265

-2550 10 20 30

Time (min)

Nu

cle

ar

Trx

1 E

h (

mV

)

Nuclear Trx1-285

-275

-265

-2550 10 20 30

Time (min) Cy

top

las

mic

Trx

1E

h (

mV

)

Cytosolic Trx1

-285

-275

-265

-2550 10 20 30

Time (min)

GS

H/G

SS

G E

h (

mV

)

Cellular GSH

P. Halvey et al, Biochem J 2005

-365

-355

-345

-3350 10 20 30

Time (min)

Trx

2 E

h (

mV

) Mitochondrial Trx

Trx1 and PrSH/PrSSG are more reduced in nuclei

Nuclei contain less protein-SH per mg protein than cytoplasm

Nuclear Trx1 and PrSH/PrSSG are more resistant to oxidation than cytoplasmic

pools

Keap-1 Nrf-2

Keap-1

Cytoplasm

Nucleus

Nrf-2

Nrf-2

Maf

ARE Nrf-2

Maf

ARE

Transcription

Transcriptional activation by Nrf2

0

40

80

120

160

200

Empty TRX1 TRX1+TBHQ

0

50

100

150

Control BSO NAC Nu

cle

ar

Nrf

-2 (

% C

on

tro

l)

200

250

300

Keap-1 Nrf-2

Keap-1

Cytoplasm

Nucleus

Nrf-2Nrf-2

Maf

ARE Nrf-2

Maf

ARE

Transcription

↑GSH↓GSH

J. Hansen et al, Tox Sci 2004

GSH controls cytoplasmic activation of Nrf2 translocation to nucleus

0

40

80

120

160

200

Empty TRX1 TRX1+TBHQ

0

50

100

150

Control BSO NAC Nu

cle

ar

Nrf

-2 (

% C

on

tro

l)

200

250

300

Keap-1 Nrf-2

Keap-1

Cytoplasm

Nucleus

Nrf-2

Nrf-2

Trx1(SH)2

Trx1(SS)Nrf-2

Maf

ARE Nrf-2

Maf

ARE

Transcription

↑GSH↓GSH

050

100150200250300

Empt

y

Trx-1

C35S T

rx-1

NLS-T

rx-1

C35S N

LS-T

rx-1

% C

on

tro

l (L

uc

/B-g

al)

J. Hansen et al, Tox Sci 2004

GSH and Trx control different steps in transcriptional activation by Nrf2

Cytoplasmic activation of Nrf2 is dependent upon GSH/GSSG

Nuclear activity of Nrf2 is dependent upon Trx1

Distinct roles for Trx in the cytoplasm and the nucleus

IkBp50 p65 p50 p65

+IkB PO4

cytosol

nucleus

p50 p65

NF-kB-dependent gene (e.g. TNF)

p50 p65

Trx-(SH)2

Ref1 <-- Trx-(SH)2

Ubiquitination,Degradation

endotoxin cytokines oxidants, etc.

GSH/GSSG = -220 to -260

Trx1(-SH)2/SS = -300

Cys/CySS = -160

Trx1(-SH)2/SS = -280Cys/CySS = -80GSH/GSSG = -140

Trx2(-SH)2/SS= -360

GSH/GSSG = -300

Plasma/Interstitial

Cytoplasmic

Nuclear

Mitochondrial

Endoplasmic Reticulum

GSH/GSSG = -150

Hansen et al, Annu Rev Pharm Tox, 2006

Summary1. Redox signaling and control involves discrete

redox circuitry

2. The mitochondrial compartment is most reduced and most susceptible to oxidation

3. Nuclei are more reduced than cytoplasm and contain special mechanisms to protect against oxidative stress

4. Analytic methods are available to elucidate the redox circuitry and compartmentation of oxidative stress