Biologically Relevant Thiol Modifications Effects on Protein Function

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Biologically Relevant Thiol ModificationsEffects
on Protein Function

• Christine Winterbourn
• Christchurch School of Medicine, University of
  Otago, New Zealand
• Society for Free Radical Biology and Medicine
• Workshop on Thiol Groups in Oxidative Stress and
  Redox Signaling
• Denver, November 2006

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Thiols are among the most oxidant-sensitive
biological molecules.

• All thiols are not equally reactive.
• All oxidants do not give the same products.
• Identification of biological targets.
• Consequences of thiol protein oxidation.

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Most oxidants react with the anionic form of
thiols.Compounds with low pKa are more reactive.
Hydrogen Peroxide Taurine Chloramine
Hypochlorous Acid
kGSH H2O2 1 M-1s-1 TauCl 115
HOCl gt107
Winterbourn Metodiewa (1999) FRBM
27322 Peskin et al (2001) FRBM 30572
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Relative sensitivities of creatine kinase (pK
5.5) and glyceraldyde-3-phosphate dehydrogenase
(pK 7.5)
SDS-PAGE after labelling reduced thiols with
iodoacetamide-fluorescein
Peskin Winterbourn (2006) FRBM 4045
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Identifying Physiological Targets

• Determined by reaction rates and concentrations.
• Reactions occur in competition.
• Reactions that occur in isolation will not all be
  sufficiently favorable to occur in cells.

For two substrates, the ratio of the amounts of
oxidant reacting with each is given by k1
substrate 1 k2 substrate 2
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A physiological example for H2O2

• kH2O2 M-1s-1 Conc. of H2O2 reacting
  with target
• GSH 1 2 mM 0.04
• Protein tyrosine phosphatases
• PTP1B 20 1 µM 0.0004
• Cdc25 150 1 µM 0.003
• GAPDH 500 100 µM 1
• Peroxiredoxins gt100,000 50 µM 99

Some proteins that are easily oxidized by H2O2 in
isolation are unlikely to be directly oxidized in
a cell -until more favorable targets are oxidized
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Diffusion Distance and Site Localisation
Diffusion distance
For H2O2 D 2x10-5 cm2/s
Diffusion distance of H2O2 in 50 µM
peroxiredoxin 20 µm
Requirements for localized oxidant
action Local area of high substrate
concentration or Association between oxidant
generator and target or Barrier to diffusion eg
membrane
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Some cellular thiol proteins become oxidized even
though they are not particularly
reactive.Could this oxidation be indirect?
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Thiol Oxidation

• 1 electron
• RSH RS?
• thiyl radical
• 2 electron
• RSH RSOH RSO2H RSO3H
• sulfenic sulfinic sulfonic
• RSH
• RSSR
• disulfide

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Products of GSH oxidation LC/MS total Ion
Chromatograms
H2O2
Hypochlorite 11
GSH
GSSG
GSSG
GSSG is the major product but not necessarily the
only one. A Sulfinic and sulfonic acids B
dehydroglutathione C Sulfonamide D Various
C
D
B
Taurine chloramine 0.51
Peroxynitrite 11
D
B
D
A
C
Harwood et al Biochem J 399,161 (2006)
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Products of Protein Thiol Oxidation

• Usual candidates
• Mixed disulfide R-S-S-G
• Sulfenic acid R-S-OH
• Interchain disulfide R-S-S-R
• Internal disulfide (vicinal thiol) R
• S S
• More recently recognized
• Nitrosothiols -S-NO
• Sulfinic (or sulfonic) acid
  eg overoxidized
  peroxiredoxins -SO2H
• Sulfenamide eg PTP1B S-N(amide)
• Sulfinamide eg HOCl modification S-N(amine)

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Distinguishing Different Oxidation Products

• Reversible
• Anti-GSH antibodies
• Use selective reductants
• Arsenite -SOH
• Ascorbate -SNO
• Glutaredoxin -SG
• Irreversible

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Sulfenic Acids Low MW forms unstable but can be
stabilised within Protein
Albumin treated with H2O2
Detection 1. Spectral change on reaction with
NBD Thiol lmax 420 nm Sulfenate lmax 350 nm
H2O2-treated
native
2. Reaction with dimedone and MS of tryptic digest
7Cl-4NO2benzo-2-oxa-1,3-diazone
Carballal et al (2003) Biochemistry 429906 Ellis
Poole (1997) Biochemistry 3615013
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Consequences of Protein Thiol Oxidation

• Antioxidant protection
• Cell signaling
• Metabolic regulation
• Toxicity

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Mechanisms
Removal of oxidant Enzyme inactivation Mixed
disulfide formation Conformational
change Crosslinking/aggregation
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Protection
Removal of oxidant Enzyme inactivation Mixed
disulfide formation Conformational
change Crosslinking/aggregation
Metabolic regulation Signaling
Toxicity
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GSH and Thioredoxin

• Ultimate sinks for cellular oxidations
• Antioxidant activity
• Disulfide reduction
• Often not direct targets for oxidants
• Usually not in redox equilibrium and reactivity
  dictated by kinetics
• Recycled by NADPH

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Labeled Oxidized Thiol Proteins in Jurkat Cells
control
200 ?M H2O2
GAPDH
Peroxiredoxin 2
Proteins extracted, reduced, labelled with
fluorescein-iodoacetamide and separated by 2D
SDS-PAGE (Baty et al (2005) Biochem J 389785)
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OxyR A redox-activated genetic switch
Prokaryotic transcription factor Thiol oxidation
to disulfide induces conformational change to
activate DNA binding kH2O2 2x105 M-1s-1
Pomposiello Demple (2001) Trends Biotech 19109
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HSP33 A redox-regulated molecular chaperone
Prokaryote chaperone Activated by oxidative stress
Graf Jakob (2002) CMLS 591624
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Peroxiredoxins

• Ubiquitous class of antioxidant or signaling
  proteins
• Present in high copy numbers
• Highly reactive with H2O2 (kgt105 M-1s-1)
• 2-cys and 1-cys forms

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Prx2 A 2-Cys PeroxiredoxinConventional
peroxiredoxin / thioredoxin cycle
H2O2
H2O2
SpH
SrH
SOH
SrH
SO2H
SrH
SrH
SpH
SrH
SOH
SrH
SO2H
Overoxidation
Thioredoxin, Thioredoxin reductase, NADPH
S
S
S
S
Kang et al (2005) Trends Mol Med 11571
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Model for Mammalian 2-Cys Peroxiredoxin Oxidation
Wood, Poole Karplus, Science 2003
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High reactivity of Prx2 with H2O2
6 µM Prx2
H2O2 ?M 0 0.8 1.5 3
Reagents mixed for 20 s and separated by
non-reducing PAGE
dimer
monomer
A. Peskin et al (2006) submitted
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Prx2 and catalase react with H2O2 at similar rates
Prx2 0.15 mg/ml
H2O2 (5 µM) -
Catalase
(mg/ml) 0 2
catalase Prx2 dimer Prx2 monomer
Catalase rate constant 5 x 106 M-1 s-1
A. Peskin et al (2006) submitted
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Prx2 is abundant (250 µM) in the erythrocyteIt
is oxidized by very low H2O2 concentrations
despite active catalase and GPxIt forms
reversible dimers but does not undergo
irreversible oxidation
H2O2 µM 0 1 2 5 10 20
50 100 200
Prx2 dimer Prx2 monomer
F Low et al (2006) Blood in press
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Summary

• Thiol oxidation is important in antioxidant
  defense and redox signaling.
• Thiols differ in reactivity depending on pKa and
  molecular environment.
• Absolute reactivity and selectivity vary for
  different oxidants.
• For H2O2, only a few proteins have shown
  sufficient reactivity to be direct targets.
• Conformation change as well as enzyme
  inactivation are important regulatory mechanisms.