Measuring the poise of thioldisulfide redox in vivo

1
Measuring the poise of thiol/disulfide redox in
vivo
Emory Clinical Biomarkers Laboratory
Dean P. Jones, Ph.D. Department of
Medicine/Division of Pulmonary, Allergy and
Critical Care Medicine Emory University, Atlanta
2
The redox state of GSH/GSSG provides a measure of
the balance of prooxidants and antioxidants
Redox states of different couples can be compared
by expression as redox potentials
Low molecular weight thiols and disulfides are
measured by HPLC
Jones Meth Enzymol 2002
3
Reversible oxidation of thiols alters protein
structure and function
Reduced Trx1
Oxidized Trx1
Active site
Watson et al. 2003
4
All cysteines in Trx1 are important in Trx1
function
5
Protein thiol/disulfide redox states are measured
by Redox Western blot analysis
Native gel separation by charge following
treatment with IAA
Trx1-Ox2 Trx1-Ox1 Trx1-Red
Nuclei
Trx1-Ox2 Trx1-Ox1 Trx1-Red
Cytoplasm
0 2 10 30 60 120
Time (min) after H2O2
Watson, Jones FEBS Lett 2003 Watson et al, JBC
2003
SDS-PAGE separation by mass following treatment
with AMS
Cytoplasm
Mitochondria
µM tBH
0 50 200 300 400
Chen et al FEBS Lett 2006
6
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
7
Redox of GSH/GSSG becomes progressively oxidized
in the life cycle of cells
-(SH)2-SS-
Proliferation
1001
-
250
-
250
, mV)
h
101
Differentiation
Redox State (E
Redox State (E
11
-
200
-
200
Apoptosis
110
-
150
-
150
Kirlin et al, FRBM 1999 Nkabyo et al, Am J
Physiol 2002
8
Extracellular Cys/CySS pool in culture is
regulated to a value very similar to that in
human plasma
HT29 cells
Plasma, 740 subjects
Frequency
Extracellular Eh (Cys/CySS) (mV)
Eh (mV)
Jonas et al, FRBM 2002
Go and Jones, Circulation 2005
9
Interorgan GSH/Cysteine balance
Tissues
Plasma
Major pool Most reduced
-220 mV
-138 mV
GSH/GSSG
GSH/GSSG
Cys/CySS
-150mV
Cys/CySS
-80 mV
Major pool Most oxidized
10
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
11
Many people have redox states more oxidized than
young healthy individuals
HT29 cells
Plasma, 740 subjects Young healthy in RED
-120
200 mM Cysteine
Reduced
-100
-80
Frequency
Oxidized
Reduced
Extracellular Eh (Cys/CySS) (mV)
-60
Oxidized
-40
100 mM Cystine
-20
Eh (mV)
Time (h)
Jonas et al, FRBM 2002
Go and Jones, Circulation 2005
12
Plasma redox provides a useful measure of
oxidative stress in humans
GSH Cys redox oxidized with age
Cys redox oxidized with smoking
-
-
90
90
-
-
80
80
Eh Cys (mV)


-
-
70
70
-
-
60
60
53
64
66
53
64
66
N
Current
Prior
Never
Current
Prior
Never
Smoking Status
Jones, FRBM 2002
Moriarty, FRBM 2004
GSH redox is oxidized with chemotherapy
Antioxidants decrease Cys oxidation with age
-
140
-
140
Mean age 71.7
Mean age 71.7
-
120
-
120
Vit C, E, b-car
GSH (mV)
Control
P 0.002 for effect of time
P 0.002 for effect of time
Mean age 76.3
Mean age 76.3
-
100
-
100
h
E
70
72
74
76
78
70
72
74
76
78
Age (y)
Jonas, Am J Clin Nutr 2000
Moriarty-Craige, Am J Ophthalmol 2005
13
Plasma redox is oxidized in association with
disease and disease risk
Eh GSH/GSSG predicts IMT
0.67
p value 0.009
0.66
0.61
0.62
Carotid IMT (mm)
0.59
0.58
0.54
lt -130 mV
gt -120 mV
-120 to -130 mV
Eh GSH/GSSG
Ashfaq et al, Am Coll Cardiol 2006
14
Pathophysiologic correlation
Health
-80 mV
Low antioxidants, low dietary cysteine
(-140 mV)
-62 mV (-122 mV)
Aging
Alcohol abuse
-50 mV (-110 mV)
Type 2 Diabetes
Cigarette Smoking
Increased Carotid Intima Media Thickness
Reversible myocardial perfusion defects
Chemotherapy/BMT
-20 mV (-80 mV)
Lung transplantation
Cys/CySS Redox (GSH/GSSG Redox)
Jones, Antiox Redox Signal, 2006
15
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
16
GSH, Trx and Cys redox systems are not in redox
equilibrium in cells
Jones et al FASEB J 2004
17
GSH/GSSG, Trx and Cys/CySS provide independent
nodes for redox signaling and control
NADPH
TR1
GR
Trx
3
GSH/GSSG (proliferation)
6a
1/Prx
Eh (mV)
GSH/GSSG (differentiation)
2
6b
GSH/GSSG (apoptosis)
Grx
Cys/CySS
4
5/GPx
SO
H2O2
TO
H2O2
O2
O2
Cellular
Extracellular
Jones et al, FASEB J 2004
18
Redox-dependent systems are differentially
controlled by GSH, Trx1 and Cys redox couples
Trx/TrxSS
ASK-1 ? Apoptosis
KEAP-1 ? Nrf-2 translocation
to nucleus
GSH/GSSG
GSH/GSSG
Nrf-2 ? DNA binding
EGFR ? MAPK activation
Cys/CySS
Trx/TrxSS
Cys/CySS
Protein synthesis Protein S-thiylation
19
Compartmentation of thiol/disulfide redox state
Plasma/Interstitial
Cytoplasmic
GSH/GSSG
Trx1(-SH)2/SS
Cys/CySS
GSH/GSSG
Cys/CySS
Mitochondrial
Trx2(-SH)2/SS
Nuclear
GSH/GSSG
Trx(-SH)2/SS
GSH/GSSG
Endoplasmic Reticulum
GSH/GSSG PDI
Hansen et al, Annu Rev Pharm Tox, 2006
20
Trx2 is preferentially oxidized by TNF?
Thioredoxin-1
Thioredoxin-2
TNF? (ng/ml)
H2O2 (mM)
H2O2 (mM)
TNF? (ng/ml)
0 5 10 20 40 1
0 5 10 20 40 1
-300
-380
-360
-280
-340
Redox Potential (Eh)
Redox Potential (Eh)
-320
-260
-300
-280
-240
0 5 10 20 40
H2O2
0 5 10 20 40
H2O2
TNF? (ng/ml)
TNF? (ng/ml)
J. Hansen
21
Mitochondrial redox circuits
Redox Signaling and Control Circuits (low flux)
Metabolic Redox Circuits (high flux)
Metabolic substrates
NADPH
Eh
NADPH
GR
TR2
-400
ASK1
Pyr
NADH
Grx2
Mal
PrSSG
GSH
Trx2
-200
MPT
Succinate
0
GPx
Prx3
CoQ
O2-
200
O2-
MnSOD
Cyt c
H2O2
400
O2
O2
600
Regulatory Signal
DP Jones, Chem-Biol Interact 2006
22
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
23
GSH is difficult to measure in nuclei
Cotgreave, 2003
Bellomo, 1992
Voehringer, 1998
24
Translocation of Trx from the cytoplasm to the
nucleus
Hirota et al, J Biol Chem (1999) 27427891
25
Time courses of GSH and Trx1 oxidation are
similarTrx-1 is somewhat more resistantTrx-1
recovers somewhat more rapidly
High levels of oxidants are not selective between
GSH and Trx1
1 mM H2O2
Trx-Ox2 Trx-Ox1 Trx-Red
Nuclei
Trx-Ox2 Trx-Ox1 Trx-Red
Cytoplasm
0 2 10 30 60 120
Time (min)
Watson, Jones (2003) FEBS Lett 543144
26
Physiologic oxidation in response to EGF is
specific to cytosolic Trx-1
P. Halvey et al, Biochem J 2005
27
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
28
Transcriptional activation by Nrf2
Cytoplasm
Nrf-2
Keap-1
Nucleus
Nrf-2
Nrf-2
Maf
Keap-1
Nrf-2
Transcription
ARE
Maf
ARE
29
GSH controls cytoplasmic activation of Nrf2
translocation to nucleus
Cytoplasm
Nrf-2
Keap-1
Nucleus
?GSH
?GSH
Nrf-2
Nrf-2
Maf
Keap-1
Nrf-2
Transcription
ARE
Maf
ARE
300
250
200
Nuclear Nrf-2 ( Control)
150
100
50
0
Control
BSO
NAC
J. Hansen et al, Tox Sci 2004
30
GSH and Trx control different steps in
transcriptional activation by Nrf2
Cytoplasm
Nrf-2
Keap-1
Nucleus
Nrf-2
?GSH
?GSH
Trx1(SH)2
Nrf-2
Trx1(SS)
Maf
Nrf-2
Keap-1
Nrf-2
Transcription
ARE
Maf
ARE
300
250
200
Nuclear Nrf-2 ( Control)
150
100
50
0
Control
BSO
NAC
J. Hansen et al, Tox Sci 2004
31
Cytoplasmic activation of Nrf2 is dependent upon
GSH/GSSG
Nuclear activity of Nrf2 is dependent upon Trx1
32
Distinct roles for Trx in the cytoplasm and the
nucleus
endotoxin cytokines oxidants, etc.
Ubiquitination, Degradation
PO4

Trx-(SH)2
cytosol
nucleus
Ref1 lt-- Trx-(SH)2
NF-kB-dependent gene (e.g. TNF)
33
Plasma/Interstitial
GSH/GSSG -140
Cytoplasmic
Cys/CySS -80
Trx1(-SH)2/SS -280
GSH/GSSG -220 to -260
Cys/CySS -160
Mitochondrial
Trx2(-SH)2/SS -360
Nuclear
Trx1(-SH)2/SS -300
GSH/GSSG -300
Endoplasmic Reticulum
GSH/GSSG -150
Hansen et al, Annu Rev Pharm Tox, 2006
34
Summary

• Redox signaling and control involves discrete
  redox circuitry

• The mitochondrial compartment is most reduced and
  most susceptible to oxidation

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

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