Measuring the poise of thiol/disulfide redox in vivo Dean P. Jones, Ph.D. Department of Medicine/Division of Pulmonary, Allergy and Critical Care Medicine Emory 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 GSH GSSG Redox states of different couples can be compared by expression as redox potentials E h = E o + * ln [Ox]_ [Red] RT nF Low molecular weight thiols and disulfides are measured by HPLC

Reversible oxidation of thiols alters protein structure and function Active site Reduced Trx1Oxidized Trx1 Watson et al. 2003

Active site Redox "OFF" switch Dimerization site S-Glutathiylation site, S-Alkylation site S-Nitrosylation site ASK-1 docking regulatory site All cysteines in Trx1 are important in Trx1 function

Protein thiol/disulfide redox states are measured by Redox Western blot analysis Trx1-Ox2 Trx1-Ox1 Trx1-Red Trx1-Ox2 Trx1-Ox1 Trx1-Red Time (min) after H 2 O 2 Nuclei Cytoplasm µM tBH Trx1 R Trx1 O Trx2 R Trx2 O Watson, Jones FEBS Lett 2003 Watson 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

Redox State (E h, mV) Proliferation Apoptosis Differentiation Redox of GSH/GSSG becomes progressively oxidized in the life cycle of cells Kirlin et al, FRBM 1999; Nkabyo et al, Am J Physiol :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 Extracellular E h (Cys/CySS) (mV) Time (h)  M Cysteine +100  M Cystine HT29 cells E h (mV) E h, Cys/CySS Mean=-72.4 StDev=12.8 E h, GSH/GSSG Mean= StDev=22.9 Frequency Plasma, 740 subjects Go and Jones, Circulation 2005 Jonas et al, FRBM 2002

Interorgan GSH/Cysteine balance Tissues Plasma GSH/GSSG Cys/CySS Major pool Most reduced Major pool Most 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 Extracellular E h (Cys/CySS) (mV) Time (h)  M Cysteine +100  M Cystine HT29 cells E h (mV) E h, Cys/CySS Mean=-72.4 StDev=12.8 E h, GSH/GSSG Mean= StDev=22.9 Frequency Plasma, 740 subjects Young healthy in RED Go and Jones, Circulation 2005 Jonas et al, FRBM 2002 Reduced Oxidized Reduced

Plasma redox provides a useful measure of oxidative stress in humans GSH & Cys redox oxidized with age GSH redox is oxidized with chemotherapyAntioxidants 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 Smoking Status CurrentPriorNever E h Cys (mV) * N = CurrentPriorNever * Age (y) E h GSH (mV) P = for effect of time Mean age = 71.7 Mean age = P = for effect of time Mean age = 71.7 Mean age = Vit C, E,  -car Control

Plasma redox is oxidized in association with disease and disease risk -110 <60 Controls >60 Controls Type 2 Diabetes ** -135 GSH/GSSG E h (mV) * * GSH/GSSG is oxidized in T2 Diabetes Samiec et al, FRBM 1998 E h GSH/GSSG predicts IMT E h GSH/GSSG Carotid IMT (mm) < -130 mV p value > -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 correlation Low 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

Redox State (E Redox State (E h, mV) -300 Proliferation Apoptosis Proliferation Differentiation Trx GSH Differentiation Apoptosis Proliferation Differentiation Cys GSH, Trx and Cys redox systems are not in redox equilibrium in cells Jones et al FASEB J 2004

H2O2H2O2 Trx Cys/CySS GSH/GSSG (apoptosis) NADPH E h (mV) 4 GR TR1 O2O2 SO O2O2 TO 2 5/GPx GSH/GSSG (differentiation) 6b 1/Prx 6a GSH/GSSG (proliferation) Cellular Extracellular H2O2H2O2 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 synthesis Protein S-thiylation Redox-dependent systems are differentially controlled by GSH, Trx1 and Cys redox couples

GSH/GSSG Trx(-SH) 2 /SS Cys/CySS Trx1(-SH) 2 /SS Cys/CySS GSH/GSSG Trx2(-SH) 2 /SS GSH/GSSG Plasma/Interstitial Cytoplasmic Nuclear Mitochondrial Endoplasmic Reticulum GSH/GSSG PDI Hansen et al, Annu Rev Pharm Tox, 2006 GSH/GSSG Compartmentation of thiol/disulfide redox state

Trx2 is preferentially oxidized by TNF  TNF  (ng/ml) H 2 O 2 (mM) H 2 O 2 (mM) TNF  (ng/ml) H2O2 Thioredoxin-1 Thioredoxin-2 TNF  (ng/ml) Redox Potential (Eh) Redox Potential (Eh) J. Hansen

Mitochondrial redox circuits NADPH NADH Cyt c O2O2 GR GSH Redox Signaling and Control Circuits (low flux) Metabolic Redox Circuits (high flux) EhEh Pyr Mal Succinate MPT TR2 Trx2 O2O2 NADPH GPxPrx3 H2O2H2O2 PrSSG Grx2 Metabolic substrates ASK1 O2-O2- Regulatory Signal MnSOD O2-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, 2003Bellomo, 1992Voehringer, 1998

Translocation of Trx from the cytoplasm to the nucleus Hirota et al, J Biol Chem (1999) 274:27891

Time courses of GSH and Trx1 oxidation are similar Trx-1 is somewhat more resistant Trx-1 recovers somewhat more rapidly Trx-Ox2 Trx-Ox1 Trx-Red Trx-Ox2 Trx-Ox1 Trx-Red Time (min) Nuclei Cytoplasm High levels of oxidants are not selective between GSH and Trx1 Watson, Jones (2003) FEBS Lett 543: mM H 2 O 2

Physiologic oxidation in response to EGF is specific to cytosolic Trx Time (min) Nuclear Trx1 Eh (mV) Nuclear Trx Time (min) Cytoplasmic Trx1Eh (mV) Cytosolic Trx Time (min) GSH/GSSG Eh (mV) Cellular GSH P. Halvey et al, Biochem J Time (min) Trx2 Eh (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 Maf ARE Nrf-2 Maf ARE Transcription Transcriptional activation by Nrf2

EmptyTRX1 +TBHQ ControlBSONAC Nuclear Nrf-2 (% Control) Keap-1 Nrf-2 Keap-1 Cytoplasm Nucleus Nrf-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

EmptyTRX1 +TBHQ ControlBSONAC Nuclear Nrf-2 (% Control) Keap-1 Nrf-2 Keap-1 Cytoplasm Nucleus Nrf-2 Trx1(SH) 2 Trx1(SS) Nrf-2 Maf ARE Nrf-2 Maf ARE Transcription ↑ GSH ↓ GSH Empty Trx-1 C35S Trx-1 NLS-Trx-1 C35S NLS-Trx-1 % Control (Luc/B-gal) 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 IkB p50p65 p50p65 + IkB PO 4 cytosol nucleus p50p65 NF-kB-dependent gene (e.g. TNF) p50p65 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 = -280 Cys/CySS = -80 GSH/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

Summary 1.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