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1 Proteomics analysis of cellular response to oxidative stress: Evidence for in vivo over-oxidation of peroxiredoxins at their active site Thierry Rabilloud 1* , Manfred Heller 2,5 , Françoise Gasnier 3 , Sylvie Luche 1 , Catherine Rey 3 , Ruedi Aebersold 2,6 , Mohamed Benahmed 4 , Pierre Louisot 3 and Joël Lunardi 1 1: CEA- Laboratoire de Bioénergétique Cellulaire et Pathologique, EA 2943, DBMS/BECP CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France 2: Department of Molecular Biotechnology, University of Washington, Seattle, WA 91815, USA 3: Unité INSERM U189 Faculté de Médecine Lyon-sud, BP 12, F-69921 Oullins Cedex, France 4: Unité INSERM U407 Faculté de Médecine Lyon-sud, BP 12, F-69921 Oullins Cedex, France 5: Present address: Geneprot, 2 rue Pré de la fontaine, CH-1217 Meyrin, Switzerland 6: Present address: Institute for Systems Biology, 4225 Roosevelt way, NW, Seattle, WA 98105, USA (Running title): peroxiredoxins and oxidative stress *: to whom correspondence should be addressed Correspondence : Thierry Rabilloud, DBMS/BECP CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-38-78-32-12 Fax (33)-38-78-51-87 e-mail: [email protected] Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on March 19, 2002 as Manuscript M106585200 by guest on March 18, 2018 http://www.jbc.org/ Downloaded from

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Page 1: 1 Proteomics analysis of cellular response to oxidative stress

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Proteomics analysis of cellular response to oxidative stress:

Evidence for in vivo over-oxidation of peroxiredoxins at their active site

Thierry Rabilloud 1*, Manfred Heller 2,5, Françoise Gasnier 3, Sylvie Luche 1, Catherine Rey 3 ,

Ruedi Aebersold 2,6 , Mohamed Benahmed4, Pierre Louisot 3 and Joël Lunardi 1

1: CEA- Laboratoire de Bioénergétique Cellulaire et Pathologique, EA 2943, DBMS/BECP

CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France

2: Department of Molecular Biotechnology, University of Washington, Seattle, WA 91815, USA

3: Unité INSERM U189 Faculté de Médecine Lyon-sud, BP 12, F-69921 Oullins Cedex, France

4: Unité INSERM U407 Faculté de Médecine Lyon-sud, BP 12, F-69921 Oullins Cedex, France

5: Present address: Geneprot, 2 rue Pré de la fontaine, CH-1217 Meyrin, Switzerland

6: Present address: Institute for Systems Biology, 4225 Roosevelt way, NW, Seattle, WA 98105, USA

(Running title): peroxiredoxins and oxidative stress

*: to whom correspondence should be addressed

Correspondence :

Thierry Rabilloud, DBMS/BECP

CEA-Grenoble, 17 rue des martyrs,

F-38054 GRENOBLE CEDEX 9

Tel (33)-38-78-32-12

Fax (33)-38-78-51-87

e-mail: [email protected]

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 19, 2002 as Manuscript M106585200 by guest on M

arch 18, 2018http://w

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Abbreviations:

2D: two-dimensional; amu: absolute mass units; BHP: tert-butyl hydroperoxide; DTT: dithiothreitol; IEF:

isoelectric focusing; IPG: immobilized pH gradient; LC: liquid chromatography; MALDI-TOF: Matrix-

Assisted Laser Desorption and Ionization, Time Of Flight; MS: mass spectrometry; MS/MS: tandem mass

spectrometry; Prx: peroxiredoxin; TFA: trifluoroacetic acid; TNFα: tumor necrosis factor alpha

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Abstract

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the

modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be

systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the

destruction of peroxides. In so doing, a reactive cysteine in the peroxiredoxin active site is weakly

oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are

used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out in

order to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine

present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of

peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being

both important peroxide-destroying enzymes and peroxide targets. Results obtained in primary culture of

Leydig cells challenged with TNFα suggested that this oxidized/native balance of peroxiredoxin 2 may

play an active role in resistance or susceptibility to TNFα -induced apoptosis.

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1 Introduction

Organisms living under aerobic conditions need to protect themselves against the damage caused by

reactive oxygen species (O2• -, H2O2, OH• ), arising from either the incomplete reduction of oxygen

during cellular respiration or exposure to external agents such as light, ionizing radiations or some redox

drugs [1, 2]. These reactive oxygen species can damage various components of living cells such as

unsaturated lipids (giving rise to deleterious organic peroxides), proteins or nucleic acids. To counter

these deleterious processes, cells use several protective systems, either repairing the various types of

damages (e.g.DNA repair enzymes) or destroying the reactive oxygen species. One of the latter systems

depends upon superoxide dismutases, which destroy O2• -, but in turn produce hydrogen peroxide.

Hydrogen peroxide can be destroyed by catalase, but this requires that the hydrogen peroxide reaches the

peroxisomes, where catalase is present. For destruction of hydrogen peroxide and organic peroxides

without transport to the peroxisomes, there exists various peroxidases, which are present in many

cellular compartments. While catalase destroys H2O2 to produce water and molecular oxygen,

peroxidases destroy peroxides by their reduction to the corresponding alcohol (or water) with the

simultaneous oxidation of a specific cosubstrate. A typical example is glutathione peroxidase, which in

destroying peroxides oxidizes glutathione from its thiol form to its disulfide form. The oxidized

glutathione is then reduced back to its thiol form by glutathione reductase, using NADPH as the reducing

agent. The final overall reaction is thus the destruction of peroxides to the corresponding alcohols (and/or

water) and consumption of NADPH.

Among the cellular enzymes using a peroxidase-like mechanism, peroxiredoxins represent a special case.

These proteins constitute both the peroxidase and the cosubstrate, as the enzyme itself is oxidized upon

reaction with the peroxide. While many peroxidases use either heme or selenocysteine in their active site,

peroxiredoxins have a cysteine at their active site. The presence of additional conserved cysteines in the

sequence is variable, and provides the basis for the classification of the peroxiredoxins into two

peroxiredoxin subfamilies that are differentiated by the presence of one or two conserved cysteine

residues in their sequence (1-Cys and 2-Cys forms) [3]. The active site cysteine can be oxidized by the

peroxide to either one of two forms: cysteine sulfenic acid in 1-Cys peroxiredoxins [4] or disulfide in the

2-Cys peroxiredoxins [5]. In order to complete the enzymatic catalytic cycle, the peroxiredoxins are then

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reduced back to their active thiol form, for example by the thioredoxin-thioredoxin reductase system for

2-Cys peroxiredoxins [5, 6].

Although they were described rather recently, the list of identified peroxiredoxins is rapidly growing and

their ubiquitous nature is apparent. In addition to the classical cytosolic 2-Cys peroxiredoxins, named

Prx1 and Prx2 (but a variety of other names are also encountered), a third isoform (Prx3, also named

AOP or SP22) is present in mitochondria [5]. Other peroxiredoxins have been described more recently,

and microsomal-secreted [7] , peroxisomal [8] and chloroplastic isoforms [9] are now known, in

addition to 1-Cys peroxiredoxin [10].

This ubiquitous distribution suggests that these enzymes play an important role in the anti-oxidant

defense of the cell. This hypothesis has received support by inactivation of the PRX1 gene in yeast [11].

Furthermore, a mutation in a murine peroxiredoxin correlates with predisposition to atherosclerosis [12].

However, little is known at the protein level in mammalian cells about the response to challenges by

oxidative stress, as most studies are carried out at the RNA level (e.g. in [13]) . Recently, a proteomic

approach has been quite successfully used in yeast cells [14]. It showed a major reorientation in

metabolism and an increase in the synthesis of antioxidant proteins. We therefore decided to investigate

the response to oxidative stress in mammalian cells using a proteomics approach, which also detects

putative post-translational responses. An example of such a study can be found recently [15]. In this

study, some changes in peroxiredoxins were shown to occur upon oxidative stress, but these changes

were not characterized.

2. Materials and Methods

2.1.Cell culture and oxidative stress

Jurkat T cell lymphoma cells were cultured in suspension in RPMI1640 medium containing 1mM

pyruvate, 10µM mercaptoethanol, 10mM Hepes-NaOH pH 7.5 and 10% fetal calf serum. Cell viability

was assessed by Trypan blue exclusion.

Various oxidative stresses were applied prior to harvesting and cell lysis: i) the cells (either attached or in

suspension) were cultured for 0.5 to 6 hours with 75 to 150 µM tert-butyl hydroperoxide (BHP). ii) The

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cells were treated with 14 U/L of glucose oxidase for 18 hours in fresh DMEM with the supplements

described above [16]. Genotoxic stress was carried out by culture of the cells in the presence of 1µM

daunomycin for 18 hours.

For recovery studies, the cells were stressed for 0.5 hours with 75 µM tert-butyl hydroperoxide. The

cells were then washed twice in complete medium without BHP, and re-cultured for the desired period of

time in BHP-free medium.

Leydig cells were prepared from immature porcine testes ( from 2-3 weeks-old animals) by collagenase

treatment as described in [17]. The recovered cells were cultured in 10 cm Petri dishes (20 x 106

cells/dish) at 32°C in a humidified atmosphere of 5% CO2, 95% air in DME/ Ham’s F-12 medium (1:1)

containing sodium bicarbonate (1.2 mg/ml), 15 mM Hepes and gentamycin (20µg/ml). This medium was

supplemented with insulin (2 µg/ml), transferrin (5 µg/ml) and α-tocopherol (10 µg/ml). Cells were

treated with 20 ng/ml TNFα (Preprotech, Canton, MA) for 65 h.

2.2. Sample preparation

Cells were harvested by centrifugation, rinsed in PBS and resuspended in homogenization buffer (0.25

M sucrose, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA). A buffer volume approximately equal to the packed

cell volume was used. The suspension was transferred to a polyallomer ultracentrifuge tube and the cells

were lysed by the addition of 4 volumes (relative to the suspension volume) of 8.75 M urea, 2.5 M

thiourea, 25 mM spermine base and 50 mM DTT. After 1 hour at room temperature, the nucleic acids

were removed by ultracentrifugation (30 minutes at 200,000xg). The protein concentration in the

supernatant fraction was determined by a Bradford assay, using bovine serum albumin as a standard.

Carrier ampholytes (0.4 % final concentration) were added and the protein extracts were stored at -20°C.

2.3. Gel electrophoresis and analysis

Proteins were separated by 2D electrophoresis, using home-made immobilized pH 4-8 gradients with pH

plateaus at both ends [18]. The sample was loaded on the IPG strip by in-gel sample rehydration [18],

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using a urea-thiourea mixture as solubilizing agent [19]. 120 µg of total extract were loaded on analytical

gels (4mm-wide IPG strips) and up to 2mg were loaded on 6mm-wide strips for micropreparative

purposes. The IEF was performed over 24 hours for a total of 55,000 Vhrs. After equilibration of the

IEF strips [20], SDS electrophoresis was performed on 10% gels. After 2D gel electrophoresis, proteins

were stained with silver [21], or with zinc imidazole for subsequent mass spectrometry [22]. Silver-

stained gels were analyzed with the MELANIE software (Genebio). The analysis consisted of spot

detection and quantification after noise and background removal. For each gel, the spots abundances

were expressed in ppm of the sum of the volumes of all the spots detected on the gel. This compensated

for the variations in protein loading from one gel to another.

Statistical analysis was carried out using a heteroscledastic T-test.

2.4. Mass spectrometry analysis

Excised, chopped and dehydrated protein spots from gels were rehydrated on ice for 45 min in 50 mM

ammonium bicarbonate containing 12.5 µg/µl sequencing grade pig trypsin (Promega, Madison, WI).

Digestion was carried out at 37°C for 15 hours. Resulting peptides were extracted by sequential

extraction for 20 min each, in 50 µl aliquots of 20 mM ammonium bicarbonate, followed by 1% TFA,

0.1% TFA in 50% acetonitrile, and finally 5% acetic acid in 50% acetonitrile. Combined extracts were

concentrated in a speed vac to approximately 5 µl, redissolved in 45 µl 0.1 M acetic acid and centrifuged

for 2 min at 14,000xg. The supernatant fractions were carefully transferred into a fresh tube and

concentrated to approximately 5 µl. Samples were loaded from a stainless steel chamber pressurized to

1000 psi onto fused silica capillaries (75 µm i.d.) slurry packed to 8 cm with Magic C18 beads

(Michrom, Auburn, CA). The column was developed with a linear gradient of 5-50% acetonitrile in

0.4% acetic acid, 0.005% hepta-fluorobutyric acid over 25 minutes at 300-400 nl/min. Electrospray

ionization was conducted by applying 1.0 kV to a Valco stainless steel union. Capillary columns

terminated inside the Valco union and a fused silica capillary (5cm x 75µm x 360µm) tapered to

approximately 5 µm i.d. served as an emitter.

The peptides eluting from the column were directly analyzed on a Finnigan tsq7000 mass spectrometer

equipped with an in-house built microspray device. Data dependent MS/MS spectra were acquired

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automatically by an Instrument Control Language procedure. Acquired MS/MS spectra were searched

with SEQUEST [23] against the OWL protein database.

For simple MALDI-TOF analysis, standard procedures were followed [24].

2.5. Identification of the porcine peroxiredoxin 2

Porcine peroxiredoxin 2 was identified in porcine Leydig cell maps by image matching with a porcine

erythrocyte 2D map. This assignment was confirmed by comigration of 100 µg of total Leydig cell

extract with 1 µg of porcine peroxiredoxin purified from pig erythrocytes by standard methods [25].

Further confirmation was obtained by MALDI-TOF mass fingerprinting [24] on the putative porcine Prx2

spots obtained from gels loaded with Leydig cell extracts.

3. Results

3.1. Acidic peroxiredoxin spots appear upon oxidative stress

In a search for protein modification occurring after oxidative stress, we used a proteomic approach on

Jurkat cells, stressed either mildly with glucose oxidase, or strongly with butyl hydroperoxide. Typical

results are shown in figure 1. In the complete 2D gel shown in figure 1, one of the most striking

phenomena was the marked induction of an acidic satellite spot to peroxiredoxins. This phenomenon was

most prominent for peroxiredoxin 2, which is the major enzyme of this family in Jurkat cells, and

occurred for various types of oxidative stress. This spot is called acidic because its pI (5.45) is

significantly more acidic than the theoretical pI of peroxiredoxin 2 (5.7), while the pI of the major spot

present in normal lymphocytes and in Jurkat cells under normal culture conditions (5.8) fits the

theoretical pI within less than 0.1 pH unit. The identity of the proteins in the acidic and basic spots was

ascertained by MS/MS, and in both cases proved to be peroxiredoxin 2. As also shown in figure 1,

appearance of an acidic spot also occurred for the mitochondrial peroxiredoxin, peroxiredoxin 3. These

acidic spots appeared upon BHP treatment, but cell viability was severely hampered under these

conditions, as cells died within 6 hours of treatment. We therefore investigated whether the acidic spots

were associated with oxidative stress or just with cell death. These acidic satellite spots did not appear

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when other, non-oxidative stresses were used, e.g. treatment with genotoxic agents (1 µM Daunomycin,

that induced complete cell death in 48 hours), as shown in figure 2. In addition, when a strong oxidative

stress was applied (BHP), the normal spots disappeared at the profit of the acidic ones within 30

minutes, while cell viability was still above 90%. Conversely, under moderate stress conditions (glucose

oxidase), the normal spots were still present after 24 hours of treatment (as shown in figure 2C), and cell

viability was still around 85%.

3.2. The acidic peroxiredoxin 2 spot is oxidized at the active cysteine site.

In order to characterize the modifications taking place in the acidic spots, both the normal and acidic spots

from peroxiredoxin 2 were analyzed by LC/MS/MS. A modified peptide was found at the LC/MS stage

as a peak occurring only in the acidic spot and not in the basic one (figure 3). The mass of this peak

could correspond to the 30-61 peptide (i.e. with two missed trypsin cleavage sites at lysines 34 and 36)

plus three oxygen atoms. In order to confirm this hypothesis, this peak was analyzed by collision-

induced dissociation (figure 4). The y ion series identified the peptide as the active site region. A mass

difference of 151 amu was detected between y11 and y10 indicative of the presence of a cysteinyl residue

modified by three oxygen atoms. This allowed unequivocal assignment of the oxidation of Cys-51 to

cysteic acid. The precision in the mass determination allowed us to exclude intermediate oxidation states

of cysteine (namely cysteine sulfenic and sulfinic acids) and any other modification on this peptide.

Thus, the acidic spot corresponded to the in vivo oxidation of peroxiredoxin 2 at Cys-51, which is the

active site of the enzyme. This oxidation brought an extra negative charge to the protein, resulting in the

lower pI observed on the gels. It must be noted that this extra negative charge made the analysis of the

peptide by mass spectrometry much more difficult, as shown by the 50 picomoles of modified protein

required for this determination. This elevated levels prevented us from carrying out the same experiments

on peroxiredoxin 3, which gave significantly lower yields than did peroxiredoxin 2 in the required

micropreparative 2D gels (2mg of total extract loaded on the strip, data not shown).

However, due to the high sequence conservation between peroxiredoxin 2 and 3, we speculate that the

acidic peroxiredoxin 3 spot also corresponds to an oxidized form at the active site. This has also been

suggested in previous work [26].

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3.3 Recovery after transient oxidative stress

Chemically speaking, oxidation of cysteine to cysteic acid is likely to be irreversible under biological

conditions. In order to investigate cell recovery after oxidative stress, we first stressed the cells with

BHP for 30 minutes, and then let the cells recover in a BHP-free medium for various periods of time.

The cellular extracts were then analyzed by 2D gel electrophoresis.

The results are shown on figure 5 and in Table 1. It must be noted that the amount of the modified spots

remained unchanged during at least 3 hours, while that of the normal spots increased back to the original

levels. After 24 hours of recovery, the cells showed the normal levels of normal peroxiredoxin 2, but still

showed elevated levels of the oxidized form. Although we cannot formally exclude that the retro

reduction of the oxidized form of Prx 2 may play a role during recovery of the normal levels of the

normal form, the fact that there is a significant increase in the total Prx 2 (i.e. normal + oxidized), at least

in the early phases of the recovery process (p< 0.01 at 1 and 6 hours, p< 0.001 at 3 hours) strongly

suggested that the recovery of the normal spots occurred mainly through de novo synthesis. This is

further evidenced by the persistence of high levels of oxidized Prx2 during the early phases of the

recovery process (the variation in oxidized Prx2 is not statistically significant during the first 3 hours of

recovery). However, when we tried to block de novo synthesis with cycloheximide or emetine during the

recovery period to confirm this hypothesis, massive cell death occurred and precluded any analysis by 2-

D gel electrophoresis. This was not the case when these protein synthesis inhibitors were used on

unstressed cells for the same period of time.

Interestingly enough, the recovery kinetics were quite different for peroxiredoxin 2 (cytosolic) and

peroxiredoxin 3 (mitochondrial). While peroxiredoxin 2 recovery was >60% complete in 3 hours and

>80% complete in 6 hours (see table 1), peroxiredoxin 3 recovery was barely visible after 6 hours, and

was not complete even after 24 hours. In addition, the level of oxidized Prx3 steadily decreased during

the recovery process. This decrease is significant as early as 1 hour (p< 0.03 at 1 hour, p< 0.01 at 3

hours and p < 0.001 at 6 hours), while the recovery of the normal form is significant only at 24 hours.

This means that the level of the oxidized form decreased while that of the normal form did not increase in

parallel. These data strongly argue against regeneration of Prx3 by a retro reduction of the oxidized form.

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They are also in agreement with the previous observation that Prx3, probably in its oxidized form, is a

substrate for the mitochondrial ATP-dependent protease [26].

3.4 Peroxiredoxin 2 in normal cells

As the results described above were obtained in transformed cells undergoing an experimental oxidative

stress in vitro, we decided to investigate if the same phenomena could occur under more physiological

conditions, and in a system where cell viability issues would not bias the results. As a model, we chose

the porcine Leydig cells in primary culture, which have been shown to be completely resistant to TNF-

induced cell death [27], and thus remain fully viable under these conditions. This provided us a mean to

eliminate any interference that could result from cell death or mortal wounding, without needing to

strongly overexpress peroxiredoxins to restore viability [28, 29] during the assays with oxidative stress-

related challenges such as TNFα [30, 31]. We used porcine peroxiredoxin 2 extracted from erythrocytes

to carry out the assignment by comigration (figure 6A). This assignment was further confirmed by mass

spectrometry (data not shown). The position of the oxidized form was further confirmed by treatment of

the cells in culture with 0.15 mM BHP for 2 hours (figure 5B). This allowed the identification of the

normal and acidic peroxiredoxin 2 spots in control and TNFα -treated cells (figure 6C and D). Here

again, an increase in the amount of the acidic, oxidized spot could be seen upon TNFα treatment (from

680 to 1710 ppm). This showed that the TNF signal led to an increase of the modified, inactive form of

peroxiredoxin 2. However, the level of the normal spot in TNFα -treated cells remained similar to the

one observed in the control cells, and cell death was not observed, as in the case of glucose oxidase-

treated Jurkat cells. Here again, cell death was observed after BHP treatment, correlating with a massive

decrease of the normal PRX 2 spot ( figure 6B vs. 6C).

4. Discussion

While examination of the response to oxidative stress in yeast cells showed major changes, including

several affecting core metabolism [14], we detected only very limited changes in the mammalian cell

system, as have other authors [15]. The most prominent change was observed as a post-translational

modification of peroxiredoxins. Although only described rather recently, the importance of

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peroxiredoxins for the control of the oxidative status of cells is rapidly emerging. As an example, the

peroxiredoxin-based system (peroxiredoxin-thioredoxin-thioredoxin reductase) is the major

mitochondrial antioxidant system [32, 33] together with Mn superoxide dismutase. This enzyme has also

been shown to be induced under mild oxidative stress conditions in bovine aortic cells [34]. In addition,

several transfection experiments have shown that the overexpression of various peroxiredoxins is able to

counteract several pro-apoptotic signals [28, 29] thereby also indicating the importance of the oxidative

status of cells in the onset of apoptosis.

However, the precise response of the peroxiredoxin systems in mammalian cells under either oxidative

stress or in response to pro-apoptotic signals was not known. Using a proteomics approach, we detected

an alteration of the peroxiredoxin pattern upon oxidative stress. Two-dimensional electrophoresis

showed an increase in satellite, acidic spots of peroxiredoxins upon oxidative stress. Analysis of tryptic

peptides generated from the basic and acidic peroxiredoxin protein spots by MS and MS/MS showed that

the pI shift was caused by the oxidation of the active site cysteine into cysteic acid, thereby adding a

negative charge to the protein. This charge shift was detected by a mobility shift of the protein to a more

acidic pI in the 2D gel. As the cysteic acid corresponds to a strong overoxidation of the cysteine, this

acidic form must be considered as an inactive form of the peroxiredoxin. Analysis of the recovery phase

showed that the oxidized form persisted for several hours after arrest of oxidative stress, but seemed to

be eventually degraded. This degradation has been previously described for peroxiredoxin 3 [26]. This

cysteic acid form has also been previously described, but following in vitro oxidation of the protein with

massive amounts of hydrogen peroxide [35]. Lower cysteine oxidation states have also been described

for another peroxiredoxin (1-cys peroxiredoxin) but here again only in vitro [36]. From our study, it

appears that this form is encountered in vivo following even a moderate oxidative stress, and is

constitutively present in normal erythrocytes [37]. It must be mentioned, however, that our analysis takes

place under reducing conditions, so that lower oxidation states of peroxiredoxins (e.g. the disulfide

bridge or sulfenic acid states) will not be analyzed by our method.

Another interesting input of the 2D gel analysis lies in its quantitative description of the deconvoluted

normal and inactive peroxiredoxin forms. Following SDS electrophoresis and blotting [34] or protein

quantitation by antibodies, the peroxiredoxin signal represents the sum of the normal and altered spots.

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As such, it gives the impression that the peroxiredoxin amount is increased by a mild oxidative stress, or

that it remains almost constant during a short, intense oxidative stress. However, our data show that the

situation is more complex. In mild oxidative conditions, the amount of inactivation caused by

peroxiredoxin oxidation can be compensated, most likely by de novo synthesis of the native, active

enzyme. Thus, the cell is able to "fill the gap" and to keep its antioxidant defense level constant. In

contrast, under strong oxidative stress, the normal form of peroxiredoxins almost disappears due to rapid

and uncompensated inactivation by oxidation. This effectively annihilates the peroxiredoxin-based

antioxidant defense, and cell death occurs shortly thereafter. In fact, we have observed a very good

correlation between the state of the peroxiredoxins and cell survival. These data, added to the previously-

described transfection data [28, 29] strongly suggest that peroxiredoxins play a key role in the resistance

to pro-oxidant signals. However, the data obtained by transfection actually describe the effect of a

massive overexpression of peroxiredoxins in transformed cells. We therefore chose to investigate the

peroxiredoxin system in normal, non-transformed cells and without forced overexpression of

peroxiredoxins. We chose as an experimental model the TNF resistance of porcine Leydig cells in

primary culture [27]. This model has the important feature of being naturally totally resistant to TNFα ,

with absolutely no loss in cell viability following challenge with TNFα [27]. We obtained the same result

with TNF that we had with mild oxidative stress (e.g. glucose oxidase). An increase in the oxidized

peroxiredoxin spot was observed upon TNF treatment, but the level of the active form remained high,

probably here again by de novo synthesis. Interestingly enough, Leydig cells treated with TNFα and

cycloheximide died within 48 hours, while cells survived when challenged with only one of the two

drugs. Thus, de novo synthesis of Prx 2 may explain, at least in part, the survival of the cells under TNF

challenge.

In conclusion, detailed examination of peroxiredoxins by a proteomics approach provided

physiologically-relevant information. The normal spot indicates the level of antioxidant defense by

peroxiredoxins, while the oxidized spot level is more an indicator of the oxidative injury to the cells.

This, coupled to the various subcellular localizations of peroxiredoxins, provides a means to investigate

the intensity of oxidative stress in various cell compartments (e.g. glucose oxidase stress vs. BHP

stress). Thus, parallel, quantitative examination of both forms allows detailed study of the phenomena

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occurring during oxidative, or other, stress and subsequent cell recovery.

Acknowledgments

We would like to thank Ruth Griffin-Shea for critical reading of the manuscript. M.H. was supported by

a fellowship from the Swiss National Science Foundation. Work was supported in part by the NSF

Science and Technology Center for Molecular Biotechnology.

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Legends to figures

Figure 1. Peroxiredoxin 2 and 3 spots in Jurkat cells

Whole cell extracts from Jurkat cells were separated by two-dimensional electrophoresis. The

peroxiredoxin spots (indicated by arrows) were identified by mass spectrometry. The cells were either

control cells (panel A), or treated with 75 µM tert-butyl hydroperoxide (BHP) for 1 hour (panel B).

Note the dramatic increase in the acidic peroxiredoxin spots under oxidative stress, and the

corresponding decrease in the basic spot under BHP treatment. The rectangle shows the zone of the gels

shown on figures 2 and 4.

Figure 2. Peroxiredoxin spots in various cell injury conditions

Whole cell extracts from Jurkat cells were separated by two-dimensional electrophoresis. Only the 20-30

kDa region of the gels is shown. The peroxiredoxin spots (indicated by arrows) were identified by mass

spectrometry. The cells were cultured under normal conditions (panel A) or submitted to oxidative stress

with 75µM tert-butyl hydroperoxide (BHP) for 30 minutes (panel B), or 14mU/ml glucose oxidase for

18 hours (panel C), or treated with 1µM daunomycin for 18 hours (panel D). The increase in the acidic

spots is correlated with oxidative stress.

Figure 3. Mass spectrometry analysis of normal and modified Prx2

The spots corresponding to normal and modified Prx 2 were digested with trypsin, and the digest was

analyzed by LC/MS/MS. The LC/MS trace of the normal and modified spots is shown in A. All the

peaks were analyzed by Collision Induced Degradation and a second MS stage. Despite alterations in the

position in the LC chromatogram, all peaks were similar in sequence in both spots, except for the m/z

1281 peak (arrow), which was present only in the modified form, as shown in B. This m/z 1281 peak

was a triply-charged peak. This led to the tentative identification of this peak as the 30-61 peptide (and

not the theoretical 37-61 peptide), modified by 3 oxygen atoms. The sequence of the 30-61 peptide is

LSDYKGKYVVLFFYPLDFTFVCPTEIIAFSNR

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Figure 4. CID dissociation spectrum of the modified peptide.

The CID spectrum of the m/z 1281 peak provided enough sequence data for unequivocal assignment to

peptide 30-61 (i.e., with two missed trypsin cleavage sites), in a triply-charged state and with a

modification. A partial MS/MS spectrum of this peak is shown, with assignment of some ions, leading to

sequence information. The number in parentheses below or above each amino acid is its position in the

sequence of the protein. Some numbers have been omitted in order to limit the crowding of the figure.

The regular-print series (b and y) corresponds to single-charged fragment ions, while the italics series

corresponds to doubly-charged fragment ions. These assignments, together with the mass of the peptide,

allowed unequivocal assignment of the modification as the oxidation of Cys 51 to cysteic acid.

Figure 5. Recovery after oxidative stress

Whole cell extracts from Jurkat cells were separated by two-dimensional electrophoresis. Only the 20-30

kDa region of the gels is shown. The cells were control cells (panel A) or submitted to oxidative stress

with 75µM tert-butyl hydroperoxide (BHP) for 30 minutes, and analyzed immediately after stress (panel

B) or rinsed and left to recover for 3 hours (panel C), 6 hours (panel D) or 24 hours (panel E).

Arrows indicate the two forms of peroxiredoxin 2 and peroxiredoxin 3 (normal and oxidized).

Figure 6. Peroxiredoxin 2 spots in porcine Leydig cells

Whole cell extracts from Leydig cells were separated by two-dimensional electrophoresis. Only the 20-30

kDa region of the gels is shown. The peroxiredoxin spots (indicated by arrows) were identified by

comigration with purified porcine peroxiredoxin from erythrocytes (panel A), and by mass spectrometry

(data not shown). The cells were treated with 0.15 mM tert-butyl hydroperoxide (BHP) for 2 hours

(panel B), cultured in standard conditions (panel C) or treated with TNF-α for 48 hours (panel D). The

dotted lines show the pI of the acidic and basic peroxiredoxin 1 spots. Note the increase in the acidic

form upon TNF-α treatment.

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Page 20: 1 Proteomics analysis of cellular response to oxidative stress

Table 1: Quantitative variation of the normal and oxidized forms of peroxiredoxins.

spot value prx2 normal prx2 oxidized total prx2† prx3 normal prx3 oxidized total prx 3†(in ppm*)

control 1436±101 53±5.4 1490±104 904±47 330±122 1230±147glucose oxidase‡ 757±135 780±113 1537±168 780±143 330±64 1110±150daunomycin** 1367±109 71±21 1437±88 891±36 219±69 1110±96BHP†† 20±14 1470±102 1490±89 17.5±29 942±34 960±57recovery 1 hr‡‡ 460±59 1354±74 1814±122 8.5±3 852±46 861±48recovery 3 hrs 951±48 1386±56 2337±92 13.5±2.4 835±43 849±43recovery 6hrs 1272±64 597±76 1870±20 32±22 582±22 615±34recovery 24 hrs 1410±80 89±25 1499±87 329±89 469±34 797±78

*: The values in the table are in ppm of the sum of the spots visualized on the entire gels, entered as mean±S.D, rounded at the closest integernumber except when mean<20ppm.†: total prx: sum of normal and oxidized prx spots‡: cells are treated with 14 units of glucose oxidase/liter for 18 hours**: cells are treated with 1µM daunomycin for 18 hours††: cells are treated with 75µM BHP for 0.5 hours‡‡: recovery experiments are expressed by the time of recovery in normal medium after an initial oxidative stress in a medium containing 75µMBHp for 0.5 hours Note the wide S.D in the measurement of weak spots (below 50 ppm). Number of experimental repeats: 4 (except BHP: 5)

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Page 21: 1 Proteomics analysis of cellular response to oxidative stress

A B

Prx2Prx2 Prx2Prx2

Prx3 Prx3

pH4 8Mw

200kDa

20kDa

Figure 1

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Page 22: 1 Proteomics analysis of cellular response to oxidative stress

A

B

C

D

prx2prx3

prx2prx3

prx2prx3

prx2prx3

acidic basic

Figure 2

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100

Retention time (min)

native PrxII

modified PrxII

A

m/z 1281, native Prx II

m/z 1281, modified Prx II

B

Figure 3

Relativeabundance

604020

80

0100

604020

80

0

100

604020

80

0100

604020

80

0

24 28 32 36 40 44

20 24 28 32 36 40 44

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Page 24: 1 Proteomics analysis of cellular response to oxidative stress

0

1020304050

90100

900

y8(949.5)

y9(1050.5)

y10(1146.6)

y11(1297.6)

y12(1397.8)

y13(1545.0)

FVC-SO3HPT

800 120011001000 1300 1400 1500 1600700400 500 600300200100

y7(820.0)

y6(706.5)

y5(593.8)

y4(522.5)

A I I E

y3(376.4)

F80

7060

Relativeabundance

m/z

634.3 863.0

V L F F Y

b9b10b11

b12

b13b14

664.8607.2

477.8b8

V

478.8316.0Y K G

b4 b5 b6b3

(50)(49)

(51)(52)(53)(54)(55)(56)(57)(58)

(33) (34)

(43)(38) (41)

Figure 4

707.5

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A

B

C

D

E

prx2

prx2

prx2

prx2prx3

prx2prx3

prx3

prx3

prx3

Figure 5

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B

C

prx2

D

A

prx2

Figure 6

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Page 27: 1 Proteomics analysis of cellular response to oxidative stress

Ruedi Aebersold, Mohamed Benahmed, Pierre Louisot and Joel LunardiThierry Rabilloud, Manfred Heller, Françoise Gasnier, Sylvie Luche, Catherine Rey,

over-oxidation of peroxiredoxins at their active siteProteomics analysis of cellular response to oxidative stress: Evidence for in vivo

published online March 19, 2002J. Biol. Chem. 

  10.1074/jbc.M106585200Access the most updated version of this article at doi:

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