Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Article abstract of DOI:10.1074/jbc.M112.374520

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis ofMetOreduction in proteins, the physiological substrates ofMSRAand MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeastMSRAandMSRBusing three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K_m lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereasMSRBwas specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantificationandidentificationofMetOreducedinthesubstrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.

Full text of DOI:10.1074/jbc.M112.374520

Protein Structure and Folding:
Methionine Sulfoxide Reductases
Preferentially Reduce Unfolded Oxidized
Proteins and Protect Cells from Oxidative
Protein Unfolding
Lionel Tarrago, Alaattin Kaya, Eranthie
Weerapana, Stefano M. Marino and Vadim N.
Gladyshev
J. Biol. Chem. 2012, 287:24448-24459.
doi: 10.1074/jbc.M112.374520 originally published online May 24, 2012
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 29, pp. 24448–24459, July 13, 2012
2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Methionine Sulfoxide Reductases Preferentially Reduce
Unfolded Oxidized Proteins and Protect Cells from Oxidative
□
S
*
Protein Unfolding
Received for publication, April 23, 2012, and in revised form, May 23, 2012 Published, JBC Papers in Press, May 24, 2012, DOI 10.1074/jbc.M112.374520
‡1
‡
‡
§
‡
Lionel Tarrago , Alaattin Kaya , Eranthie Weerapana , Stefano M. Marino , and Vadim N. Gladyshev
‡
§
From Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the Department of
Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467
Background: Methionine sulfoxide reductases have previously been studied mostly using low molecular weight substrates.
Results: Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins.
Conclusion: These enzymes serve a critical function in protein folding by repairing oxidized nascent polypeptides and unfolded
proteins.
Significance: Understanding precise functions of methionine sulfoxide reductases will help define mechanisms of protein
repair and identify their physiological substrates.
2
Reduction of methionine sulfoxide (MetO) residues in proteins S-diastereoisomers of Met sulfoxide (MetO). This modifica-
is catalyzed by methionine sulfoxide reductases A (MSRA) and B tion is reversible, as MetO can be reduced back to Met by
(
MSRB), which act in a stereospecific manner. Catalytic properties methionine sulfoxide reductases (MSRs) A (MSRA) and B
of these enzymes were previously established mostly using low (MSRB), which are specific for the S- and R-forms of MetO,
molecular weight MetO-containing compounds, whereas little is respectively. Theses enzymes are present in almost all living
known about the catalysis of MetO reduction in proteins, the phys- organisms and catalyze the reduction of their substrates at the
iological substrates of MSRA and MSRB. In this work we exploited expense of NADPH using thioredoxin (Trx) or glutaredoxin
an NADPH-dependent thioredoxin system and determined the systems (1, 2).
kinetic parameters of yeast MSRA and MSRB using three different
Whereas the catalytic mechanisms of MSRs are well charac-
MetO-containing proteins. Both enzymes showed Michaelis-Men- terized (2, 3), their physiological functions remain elusive,
ten kinetics with the K lower for protein than for small MetO- mainly due to insufficient information on the identity of their
m
containing substrates. MSRA reduced both oxidized proteins and cellular targets. The absence of clearly established substrates
low molecular weight MetO-containing compounds with similar also limits studies on specificity of Met oxidation and MetO
catalytic efficiencies, whereas MSRB was specialized for the reduc- reduction in proteins. Met oxidation has been reported for pro-
tion of MetO in proteins. Using oxidized glutathione S-transferase teins that could be classified into three groups (4): (i) enzymes
as a model substrate, we showed that both MSR types were more activated by Met oxidation, such as the calcium/calmodulin-
efficient in reducing MetO in unfolded than in folded proteins and dependent protein kinase II (5), (ii) proteins not impaired by
that their activities increased with the unfolding state. Biochemical Met oxidation, which could fulfill together with MSRs a protec-
quantificationandidentificationofMetOreducedinthesubstrates tive (antioxidant) function though cyclic oxidation and reduc-
by mass spectrometry revealed that the increased activity was due tion of Met (6), and (iii) proteins damaged by Met oxidation,
to better access to oxidized MetO in unfolded proteins; it also such as those involved in neurodegenerative diseases (7, 8). The
showed that MSRA was intrinsically more active with unfolded consequence of Met oxidation is determined by the effects of
proteins regardless of MetO availability. Moreover, MSRs most this modification on structure and function of these proteins;
efficiently protected cells from oxidative stress that was accompa- however, these effects have been characterized only for a hand-
nied by protein unfolding. Overall, this study indicates that MSRs ful of proteins. For example, in human prion, oxidation of two
serve a critical function in the folding process by repairing oxida- Met residues converts a cellular ␣-helix-rich form to the infec-
tively damaged nascent polypeptides and unfolded proteins.
tious ␤-sheet-rich form by perturbing the network of stabilizing
interactions (9). In addition, oxidation of two solvent-accessible
Met residues in a human growth hormone increases its suscep-
Proteins may undergo various post-translational modifica- tibility to thermal denaturation (10), and oxidation of two Met
tions altering their structure and function. Their sulfur-con- residues in calmodulin prevents protein-protein interaction
taining residue, methionine (Met), can be oxidized to R- and due to incompatibility of MetO for stable ␣-helixes (11).
MSRs, which repair oxidative modifications, play important
roles in the protection of proteins from oxidative stress in var-
*
Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant AG021518.
S
This article contains supplemental Tables S1–S3 and Figs. S1–S4.
To whom correspondence should be addressed: Division of Genetics, Dept
of Medicine, Brigham and Women’s Hospital and Harvard Medical School,
ious eukaryotes (12–14). Similar protective effects were also
□
1
2
The abbreviations used are: MetO, methionine sulfoxide; MSR, MetO reduc-
7
7 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5122; E-mail:
tase; ANS, 8-anilinonaphthalene-1-sulfonate; MRP, methionine-rich pro-
tein; Trx, thioredoxin.
vgladyshev@rics.bwh.harvard.edu.
2
4448 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 29•JULY 13, 2012

MSRs Preferentially Reduce Unfolded Oxidized Proteins
observed in prokaryotes, particularly in the case of the stress
Expression and Purification of Recombinant Proteins—
TM
induced by hypochlorite, a strong antimicrobial agent found in SoluBL21 E. coli (Gelantis, San Diego, CA) cells were trans-
household bleach that is also produced by mammalian neutro- formed with the expression vector and grown in Luria-Bertani
Ϫ1
phils to kill invading microorganisms (15). For instance, a Heli- containing ampicillin or kanamycin (50 ␮gꢁml ) at 37 °C.
cobacter pylori strain deficient in the expression of MSRs could When the A reached ϳ0.6, production of the recombinant
6
00
not survive in a neutrophil cell culture (16). Hypochlorite is protein was induced by the addition of 100 ␮M isopropyl ␤-D-
known to have a dual effect in provoking oxidation and unfold- 1-thiogalactopyranoside. After overnight incubation at 25 °C,
ing of proteins and trigger chaperone activation (17). These cells were harvested by centrifugation. For thioredoxin reduc-
studies imply that MSR might directly participate in the protec- tase 1, Trx1, MSRA, MSRB, and MRP4 containing His tag,
6
tion from oxidative and unfolding stress through the reduction pellets were resuspended in PBS containing 25 mM imidazole in
of MetO in proteins.
the presence of Complete, EDTA-free, protease inhibitor mix-
The MSR activity was quantified for several protein sub- ture (Roche Applied Science), and for GST, the pellet was resus-
strates, such as bacterial Ffh and several targets of plant MSRBs pended in PBS. Cells were disrupted by sonication, His -tagged
6
(
13, 18, 19). However, little is known about enzyme kinetics of proteins were purified on nickel-containing HisꢁBindꢀ Resin
MSRs with oxidized protein substrates, their physiological tar- (Novagen, Billerica, MA), and GST was purified on glutathione-
gets. The yeast Saccharomyces cerevisiae possesses single Sepharose 4 Fast Flow (GE Healthcare). Protein solutions were
MSRA and MSRB genes as well as a free Met-R-O reductase concentrated using 15-ml Amiconꢀ Ultra concentrators with
(
fRMSR) that is specific for the reduction of the R-diastereoiso- 30- or 10-kDa cutoffs (Millipore, Billerica, MA) and desalted in
TM
mer of free MetO (20, 21). In this work we took advantage of the 30 mM Tris-HCl, pH 8, using 5-ml HiTrap
Desalting col-
NADPH-coupled Trx system and used different forms of umns (GE Healthcare). Protein concentrations were deter-
MetO-containing proteins as substrates to examine functions mined spectrophotometrically using the Pierceꢀ BCA Protein
of yeast MSRA and MSRB.
assay kit (Thermo Scientific) and specific molar extinction
coefficients at 280 nm. Protein purity was verified using SDS-
EXPERIMENTAL PROCEDURES
TM
PAGE gels stained with Imperial
Protein Stain (Thermo
Cloning and Site-directed Mutagenesis—Sequences coding Scientific).
for NADPH-dependent thioredoxin reductase 1, Trx1, and
Protein and N-Acetyl-MetO Preparation—MRP4, GST,
MSRB (from codon 30 to the stop codon), were amplified by bovine ␤-casein (Sigma), and Bacillus sp. ␣-amylase (Sigma) (all
Ϫ1
PCR from the S. cerevisiae genomic DNA using Platinumꢀ Pfx 1 mgꢁml ) were oxidized by incubation with 100 mM H O in
2
2
DNA Polymerase (Invitrogen) and specific pairs of primers PBS overnight at room temperature, concentrated, and then
TM
TM
shown in supplemental Table S1. Similarly, sequences coding desalted using HiTrap
Desalting or Illustra NAP5
col-
for Met-rich protein 4 (MRP4) (from codon 23 to stop codon) umns (GE Healthcare) in 30 mM Tris-HCl, pH 8. For unfolding
and Met-rich protein 5 (MRP5) (from codon 21 to stop codon) assays, oxidized proteins were incubated with urea at a final
were amplified from genomic DNA of Idiomarina loihiensis concentration ranging from 0.25 to 7 M for 1 h at room temper-
L2TR and Pseudomonas putida W619, respectively, using spe- ature before the assays. N-Acetyl-MetO was prepared as
3
cific primers. Amplicons were purified and digested with NdeI described (23).
and BamHI for cloning of thioredoxin reductase 1 and Trx1 in
MSR Absolute Stoichiometry and Activity with Dabsyl-MetO—
pET15b or with BamHI and XhoI or with NheI and NotI for Activities of recombinant MSRs (1 ␮M) were determined by
cloning MSRB and MRP4 in pET21b (EMD Biosciences, Bil- monitoring the reduction of 0.5 mM dabsyl-MetO in the pres-
lerica, MA), respectively. Met-rich protein 1 (MRP1) (22) and ence of 20 mM DTT. The absolute stoichiometry was deter-
MRP5 were cloned similarly in p425 yeast expression vector mined similarly; 100 ␮M reduced and desalted MSRs was
under glycerol-3-phosphate dehydrogenase (GPD) promoter incubated with 1 mM dabsyl-MetO for 1 h at room temperature
3
using BamHI and SalI restriction sites. Site-directed mutagen- without the reducing agent. Dabsyl-Met and dabsyl-MetO were
esis of MSRA and MSRB was made by whole plasmid amplifi- separated by HPLC using a C reverse phase column, Sun-
1
8
TM
cation with Phusionꢀ High-Fidelity DNA Polymerase (Thermo Fire
3.5 ␮m, 3.0 ϫ 50 mm (Waters, Milford, MA) as
Scientific, Billerica, MA) using primers containing mutated described (2).
bases (supplemental Table S1). After amplification, the methy-
MSR Activity Assays—MSR activity was measured after
lated template vector was digested by incubation with DpnI for NADPH oxidation at 340 nm in the presence of the Trx system
1
h at 37 °C. 5 ␮l of the digested PCR product was used to (200–400 ␮M NADPH, 2 ␮M thioredoxin reductase 1, 25 ␮M
transform NEB 5-␣ competent Escherichia coli (High Effi- Trx1) using 1–10 ␮M MSRA or MSRB in the presence of free
ciency) cells (New England Biolabs, Ipswich, MA), and clones MetO (0.25–20 mM), N-acetyl-MetO (62.5 ␮M to 5 mM), oxi-
were selected on Luria-Bertani plates containing ampicillin (50 dized MRP4 (0.8–200 ␮M), oxidized ␤-casein (6.25–200 ␮M),
Ϫ1
␮
gꢁml ). All constructs were validated by DNA sequencing. oxidized GST (9.4–150 ␮M) or urea-treated oxidized GST
The expression vectors pET28a-MSRA (20) and pGEX4T1 (GE (4.8–150 ␮M) as substrates. Reactions were carried out at 25 °C
Healthcare) were used to produce yeast MSRA and glutathione in a 500-␮l reaction volume. MSR activities were calculated
S-transferase (GST) from Schistosoma japonicum, respectively. from the slope after subtracting the background (absence of the
enzyme) considering that 1 mol of oxidized NADPH corre-
3
sponds to 1 mol of MetO reduced. The apparent stoichiometry
was determined similarly using subsaturating concentrations of
X. Liang, Y. Zhang, D. T. Le, D. Hua, and V. N. Gladyshev, submitted for
publication.
JULY 13, 2012•VOLUME 287•NUMBER 29
JOURNAL OF BIOLOGICAL CHEMISTRY 24449

MSRs Preferentially Reduce Unfolded Oxidized Proteins
substrates: 0.8–6.5 ␮M oxidized MRP4, 7–29 ␮M oxidized proteins used in this study. A static modification of ϩ57.02146
␤
-casein, 11–44 ␮M oxidized GST, and 5.6–22 ␮M 4 M urea- on cysteine was specified to account for iodoacetamide alkyla-
treated oxidized GST. The amount of oxidized NADPH was tion, and a differential modification of ϩ16 was specified on
determined after the rate of oxidation reached the basal level. methionine to account for oxidation. SEQUEST output files
To test the activity using the urea-treated oxidized protein, were filtered using DTASelect 2.0 (25). Reported peptides were
1
0–25 ␮M substrate was used, and urea was added in control required to be fully tryptic, and discriminant analyses were per-
assays at the same concentration (less than 50 mM). Kinetic and formed to achieve a peptide false-positive rate below 5%. The
catalytic parameters were calculated from nonlinear regres- percentage of oxidation per Met was calculated using the
sions using GraphPad Prism 4.0 (GraphPad Software, Inc, La redundancy of the peptide containing the specific Met, oxidized
Jolla, CA). In the presence of urea-treated oxidized GST, the or not. The percentage of oxidation values corresponds to the
curves fit sigmoidal regressions described by Equation 1, with number of times a MetO was found divided by the total number
the Hill coefficient (h) Ͼ 1.
of times the peptide was found (coverage), multiplied by 100.
This method allows quantifying the oxidation of each Met
found in peptide-containing several Met, which is not possible
using area integration.
h
kcat ϫ [S]
kobs
ϭ
(Eq. 1)
h
h
K
ϩ ͓S͔
m
Yeast Spotting Assays—MSRA expressed under the glycerol-
Fluorescence Analyses—Emission spectra of intrinsic fluores- 3-phosphate dehydrogenase promoter from the high copy
cence were recorded in 200 ␮l of 30 mM Tris-HCl, pH 8.0, number yeast expression vector p425 and MSR null strains was
containing 10 or 25 ␮M non-oxidized, oxidized, or urea-treated described previously (21). WT yeast cells were transformed
oxidized proteins with excitation at 280 nm. As a control, with MRP1, MRP5, and/or yeast MSRA constructs. After col-
amounts of urea used in the urea-treated proteins were added ony formation, single colonies were picked up from the plates
to the oxidized protein samples (less than 50 mM). The same and grown overnight in the media lacking histidine or leucine.
samples were used to determine 8-anilinonaphthalene-1-sul- The following day, these cells were retransformed with MSRA
fonate (ANS) fluorescence. Emission at 466 nm (excitation at and/or empty vectors and incubated on media lacking histidine
3
77 nm) was recorded 15 min after the addition of 50 ␮M ANS. and leucine. This procedure was also applied for empty vector
Fluorescence was recorded in 96-well microplates using a Spec- transformation, which was used as a control. In the spotting
traMax M5 fluorescence microplate reader (Molecular Devices, assay, the indicated strains were grown overnight in appropri-
Sunnyvale, CA).
ate media and diluted to an A of 0.3. Cells were washed with
6
00
Mass Spectrometry Analysis—Oxidized MRP4, oxidized the prewarmed PBS buffer and incubated in 1 ml of PBS and the
GST, and urea-treated oxidized GST (100 ␮M) were incubated indicated concentrations of sodium hypochlorite (NaOCl).
with or without 5 ␮M MSRA or MSRB in the presence of 10 mM Every 5 min, 5 ␮l of each culture was spotted on the selective
DTT in 30 mM Tris-HCl, pH 8, for 1 h at 25 °C. A solution media. Yeast MSR mutant strains lacking MSRA, MSRB, or
containing 50 ␮g of substrate was incubated with 12.5 mM iodo- both genes were also tested as indicated above for NaOCl sen-
acetamide for 30 min at 25 °C in 0.1 M ammonium sulfate then sitivity. All plates were incubated for 3 days at 30 °C and pho-
with 1 ␮g of sequencing grade modified trypsin (Promega, tographed. Halo assays were performed to assess viability of
Madison, WI) and 1 mM CaCl overnight at 37 °C. An addi- MSRA-, MRP1-, and MRP5-overexpressing yeast cells under
2
tional 1 ␮g of trypsin was added, and the solution was incubated conditions of H O stress. Cells were prepared as indicated
2
2
for 2 h at 37 °C. The trypsin digests were frozen and stored at above, and the A was adjusted to 0.5. Cells were washed with
6
00
Ϫ80 °C until mass spectrometry analysis. LC-MS/MS analysis the prewarmed PBS, and 1 ml of each culture was spread onto
was performed on an LTQ-Orbitrap Discovery mass spectrom- agar plates missing the appropriate amino acids for selection.
eter (Thermo Fisher) coupled to an Agilent 1200 series HPLC Plates were dried for 1 h at room temperature, and filter paper
system. Tryptic digest (30 ␮l) was pressure-loaded onto a discs were placed in the middle of each plate. 4 ␮l of 30% hydro-
2
50-␮m fused silica desalting column packed with 4 cm of Aqua gen peroxide were applied onto each paper disc, and the plates
C
reverse phase resin (Phenomenex, Torrance, CA). The pep- were incubated for 3 days at 30 °C and photographed. The
18
tides were then eluted onto a C column (100-␮m fused silica diameter of cleared zones in each plate was measured with a
18
with a 5-␮m tip, packed with 10 cm C ) using a gradient ruler. This experiment was repeated three times.
18
5
–100% buffer B in buffer A (buffer A: 95% water, 5% acetoni-
Modeling, Structural Analysis of Substrate Proteins, and
trile, 0.1% formic acid; buffer B: 20% water, 80% acetonitrile, Determination of MSR Hydrophobicity—The percentage of
0
.1% formic acid) and into the mass spectrometer. The flow rate amino acids included in disordered regions was calculated
Ϫ1
through the column was set to ϳ0.25 ␮lꢁmin , and the spray using SPINE-D (26) and Multilayered Fusion-based Disorder
voltage was set to 2.75 kV. One full MS scan (Fourier Transform predictor (27) web servers. The structural coordinates of MSRA
Mass Spectrometry) (400–1800 M ) was followed by 7 data-de- and GST were obtained from the PDB repository (PDB codes
r
pendent scans (Ion Trap Mobility Spectrometry) of the nth- 3PIL and 1DUG, respectively). To model MSRB, we used a -fold
most intense ions with dynamic exclusion enabled.
recognition algorithm, FAS03, to generate alternative profile-
Peptide Identification—The tandem MS data were searched profile alignments. MSRB were modeled with Modeler (46)
using the SEQUEST algorithm (24) using a concatenated tar- using the alignments generated with FAS03 and 2K8D as tem-
get/decoy variant of the human and mouse International Pro- plate. Detailed atomic exposure calculations were calculated
tein Index databases modified to include the sequences for the with Surface Racer 227 (28). Structural profiles were generated
2
4450 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 29•JULY 13, 2012

MSRs Preferentially Reduce Unfolded Oxidized Proteins
TABLE 1
Kinetic parameters of MSRA and MSRB in the reduction of oxidized proteins
Assays were carried out under steady-state conditions following NADPH oxidation at 340 nm. Apparent stoichiometry (mol of NADPH oxidized per mol of substrate) was
determined after full reduction of the substrate using substoichiometric substrate concentrations as described under “Experimental Procedures.” Data are represented as
the means Ϯ S.D.
MSRA
MSRB
MetO-containing
substrate
Apparent
Apparent
stoichiometry
k
Km
␮M
kcat/Km
stoichiometry
k
Km
␮M
kcat/Km
cat
sϪ1
cat
sϪ1
Ϫ1
Ϫ1.s (ϫ103) mol NADPH ox. mol sub
Ϫ1
Ϫ1
M
Ϫ1.s (ϫ103)
Ϫ1
mol of NADPH ox. mol sub
M
Free MetO
Measured
7.71 Ϯ 0.03 1,120 Ϯ 16 6.9
0.59 Ϯ 0.02 13,014 Ϯ 752 0.05
6,507 Ϯ 376 0.09
a
Corrected
560 Ϯ 8
13.8
N-Acetyl-MetO
Measured
13.23 Ϯ 1.83 896 Ϯ 282 15.0
448 Ϯ 141 30.0
0.80 Ϯ 0.06 1,348 Ϯ 224 0.6
a
Corrected
674 Ϯ 112
1.2
MRP4
Measured
4.16 Ϯ 0.16
2.49 Ϯ 0.15
1.21 Ϯ 0.02
3.25 Ϯ 0.05
13.01 Ϯ 5.4 33 Ϯ 18
137 Ϯ 76
386.3
95.3
7.18 Ϯ 0.66
3.19 Ϯ 0.34
0.78 Ϯ 0.02
4.66 Ϯ 0.24
1.04 Ϯ 0.16 10 Ϯ 3
106.7
14.9
b
Corrected
70 Ϯ 20
␤
-Casein
Measured
Corrected
1.13 Ϯ 0.11 45 Ϯ 13
113 Ϯ 32
25.1
10.1
0.78 Ϯ 0.05 54 Ϯ 9
172 Ϯ 28
14.4
4.5
b
GST
Measured
Corrected
0.73 Ϯ 0.15 356 Ϯ 105 2.1
428 Ϯ 127 1.7
0.41 Ϯ 0.08 142 Ϯ 48
111 Ϯ 37
2.9
3.7
b
c
Urea-treated GST
Measured
0.97 Ϯ 0.19 78 Ϯ 24
254 Ϯ 78
12.4
3.8
0.52 Ϯ 0.03 55 Ϯ 11
256 Ϯ 51
9.5
2.0
b
Corrected
a
b
c
Considering that only the S- and R-diastereoisomers serve as substrates for MSRA and MSRB, respectively, and assuming that the other diastereoisomer does not act as
inhibitor, the K values were divided by 2.
m
m
For comparison, the K values were multiplied by the apparent stoichiometry, allowing the removal of variation due to the different numbers of MetO reduced in each
substrate.
The regression curves obtained for MSRA and MSRB with the urea-treated oxidized GST fit a sigmoidal described by the equation 1 (“Experimental Procedures”) with the h
values equal to 1.4 Ϯ 0.3 and 1.5 Ϯ 0.1 for MSRA and MSRB, respectively.
Ϫ1
as previously described (29). Briefly, all residues lying with one from ϳ1 to ϳ13 s . The K values were ϳ0.5 mM for free
m
or more of their atoms found within 8 Å from the sulfur atom of MetO, N-acetyl-MetO, and GST and ϳ10-fold lower for MRP4
the catalytic Cys residues of yeast MSRA and MSRB were con- and ␤-casein. The catalytic efficiency (k /K ) was ϳ2.1
cat
m
Ϫ1 Ϫ1
sidered and then separately analyzed for their hydrophobic mM ꢁs for GST, and this value increased ϳ5-, ϳ13-, ϳ15-,
content (implementing the standard Kyte-Doolittle scale,
where each amino acid is described by numeric value ranging
from negative, i.e. hydrophilic, to positive, i.e. hydrophobic
and ϳ200-fold in the case of free MetO, ␤-casein, N-acetyl-
MetO, and MRP4, respectively. For MSRB, the k values were
cat
Ϫ1
ϳ1 s for all tested substrates, but a striking difference was
(
supplemental Fig. S4)) (30) and compositional features (e.g.
observed in K values, which were ϳ6.5 mM for free MetO and
m
content of basic, acidic, or aromatic residues) through in-house
Python (v2.6) scripts.
ϳ10 times lower for N-acetyl-MetO. The differences were
more pronounced for proteins, i.e. the K values were 45–650-
m
fold lower for oxidized proteins than for free MetO (Table 1).
Analysis of these data reinforces the idea that MSRB is far more
efficient in the reduction of MetO in oxidized proteins (e.g. its
catalytic efficiency was ϳ1200-fold higher for MRP4 than for
free MetO), whereas MSRA reduces both proteins and MetO-
containing compounds with similar efficiency.
Quantification of MetO Reduction in Protein Substrates—
The absolute stoichiometry displayed by each MSR was 1 mol of
MetO reduced/mol of enzyme (supplemental Fig. S1A). Two
redox-active Cys were used by each enzyme, and mutation
analyses verified the roles of Cys-25 and Cys-176 in MSRA and
Cys-157 and Cys-97 in MSRB as catalytic and resolving Cys,
respectively (supplemental Fig. S1B). We further estimated the
number of MetO reduced by MSRA and MSRB in oxidized
proteins as the apparent stoichiometry i.e. mol of NADPH oxi-
dized/mol of substrate using subsaturating concentrations of
RESULTS
Yeast MSRA Efficiently Reduces Oxidized Proteins and Free
MetO, Whereas MSRB Is Specialized for the Reduction of Oxi-
dized Proteins—To characterize the kinetics of yeast MSRA and
MSRB, we utilized the NADPH-coupled Trx system, which is
physiologically relevant and allows comparative analyses of
MetO-containing substrates. Catalytic parameters of MSRs are
usually determined using low molecular MetO-containing
compounds, whereas little is known on the catalysis of MetO
reduction in oxidized proteins. We examined MSRA and MSRB
kinetics using three different oxidized protein substrates and
used free MetO and N-acetyl-MetO for comparison. These
proteins were chosen based on their high Met content and
structural features; both Met-rich protein 4 (MRP4) and ␤-ca-
sein are predicted to be completely disordered and possess 31
(
22%) and 7 (3.1%) Met residues, respectively. A third protein,
GST, has Met content similar to that of ␤-casein (9 Met, 3.8%) substrates. Fig. 1 shows the data for MRP4. After the addition of
but is a highly structured protein (supplemental Table S2). For the substrate, NADPH consumption was followed until the rate
all substrates, MSRA and MSRB catalysis followed the Michae- reached the background level, representing the state when all
lis-Menten kinetics (Table 1). MSRA displayed the k values MetO residues reducible by MSR were reduced (Fig. 1A). The
cat
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
FIGURE 1. Apparent stoichiometry of MetO reduction in oxidized MRP4. A, NADPH consumption was followed with 0.8–6.4 ␮M oxidized MRP4 as the
substrate. The initial reaction mixture contained 250 ␮M NADPH, 2 ␮M thioredoxin reductase 1, 25 ␮M Trx1, and 10 ␮M MSRA. After equilibration, the substrate
was added (arrow), and NADPH consumption was followed until it reached the diaphorase level. The final absorbance was subtracted taking into account
the diaphorase activity for each concentration of the substrate (two-sided arrows a, b, c, and d). B, oxidized NADPH was plotted as a function of substrate
concentration for MSRA (F) and MSRB (E).
amount of oxidized NADPH was then plotted as a function of these results indicated that MSRA efficiently reduced both free
substrate concentration (Fig. 1B). The slope of the calculated and protein-bound MetO, whereas MSRB showed a dramatic
linear regression corresponded to the apparent stoichiometry. preference for oxidized proteins compared with low molecular
Using MSRA and increasing concentrations of MRP4, the weight MetO-containing molecules, such as free MetO and
apparent stoichiometry of ϳ4 mol of NADPH oxidized/mol of N-acetyl-MetO.
substrate was found (Table 1; Fig. 1B). With MSRB, this value
was ϳ7 mol of NADPH oxidized/mol of substrate (Table 1; Fig. Quantification of MetO reduced by MSRs in the tested proteins
B). As changes in oxidized NADPH were directly proportional suggested that the various oxidized Met were not equivalent
Differential Reduction of MetO Residues by MSRA and MSRB—
1
to those in MetO (18, 31), the data indicate that MSRA and substrates. We subjected MRP4 and GST to tryptic digestion
MSRB reduced 4 and 7 MetO equivalents, respectively, in the and LC-MS/MS analyses to determine the oxidation state of
oxidized MRP4. Because this protein has 31 Met, 13 and 23% each Met before and after reduction by MSRs (Table 2; supple-
Met equivalents were reduced, respectively. To compare cata- mental Table S3). The coverage of oxidized MRP4 was 64%,
lytic parameters for different substrates, the data were normal- allowing us to determine precisely the oxidation state of 11 Met
ized by multiplying the K values by the apparent stoichiome- among the 31 Met present in the protein (supplemental Table
m
try, yielding values per MetO reduced and thus allowing S3). All detected Met were highly oxidized (more than 90% of
removal of variation due to the different numbers of MetO Met were in the form of MetO), with the exception of Met-66
reduced in each substrate (Table 1). For MSRA, this normaliza- (48% oxidized). After the reduction by MSRA or MSRB, protein
tion gave a k /K ratio similar to that for N-acetyl-MetO and coverage was 89% in both cases, allowing us to estimate the
cat
m
free MetO. For MSRB, the corrected catalytic efficiency was 15- oxidation status of 27 Met, which corresponded to 55 and 64%
and 150-fold higher than that for N-acetyl-MetO and free for the substrate reduced by MSRA and MSRB, respectively.
MetO, respectively (Table 1). Similar analyses with ␤-casein Considering only the 11 Met, for which the oxidation state was
showed that 2.5 and 3.2 MetO equivalents, which correspond to determined in the oxidized MRP4 before MSR reduction,
3
6 and 46% of the 7 Met in the protein, were reduced by MSRA MSRA and MSRB reduced 28 and 21% of MetO, respectively.
and MSRB, respectively. The corrected catalytic efficiency was However, not all MetO were reduced with the same efficiency,
similar to that obtained for MSRA with free MetO and 50-fold and their reduction also depended on the MSR used. For
higher in the case of MSRB (Table 1).
instance, oxidized Met-31 was reduced only by MSRA and oxi-
The analysis of apparent stoichiometries further indicated dized Met-66 only by MSRB. In addition, the last Met, Met-125,
that 1.2 and 0.8 MetO equivalents were reduced per molecule of was completely oxidized but reduced by neither MSR (supple-
oxidized GST, corresponding to 13 and 9% of all Met in the case mental Table S3).
of MSRA and MSRB, respectively (Table 1). In the MSRA-cat-
Met residues were on average 70% oxidized in the oxidized
alyzed reaction, the corrected catalytic efficiency observed with GST, with an oxidation status varying from 16% (Met-154) to
GST as the substrate was 5–50-fold lower than with other 99% (Met-168). Surface accessibility of each Met, calculated
tested substrates. In the MSRB-dependent reduction of oxi- using protein structure, was found to correlate with the oxida-
dized GST, this correction gave a catalytic efficiency 45-fold tion status, with the 4 buried Met residues being less than 80%
higher than that obtained with free MetO but similar to those oxidized (Table 2). The average percentage of reduction by
determined for the two other proteins. It is noteworthy that the MSRA and MSRB was 18 and 24%, respectively, but the data
oxidized GST was a better substrate for MSRB; its catalytic varied significantly, indicating that, as in the case of MRP4, not
efficiency was 2-fold higher than the MSRA value. Altogether, all MetO served as substrates for MSRs. For instance, oxidized
2
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
TABLE 2
Surface accessibility of Met residues in GST and their differential oxidation as revealed by mass spectrometry analyses
2
Surface accessibility of each Met to the solvent in Å was determined using protein structure, and the percentage of oxidation of individual Met in the substrate was
determined by mass spectrometry as described under “Experimental Procedures.” Met position, position of the Met in the primary sequence. Oxidized, the GST was
oxidized with 100 mM H O before tryptic digestion. Oxidized ϩ MSRA, the oxidized GST was reduced by MSRA before tryptic digestion. Oxidized ϩ MSRB, the oxidized
2
2
GST was reduced by MSRB before tryptic digestion.
Surface
accessibility
Oxidized
Oxidized ؉ MSRA
Oxidized ؉ MSRB
Met
Secondary
structure
% Oxidation
% Oxidation
% Oxidation
Å2
(coverage )
a
(coverage )
a
% Reductionb
(coverage )
a
% Reductionb
position
Oxidized GST
d
1
coil
ND
93 (25)
57 (65)
39
14
79 (29)
15
2
6
8
9
1
1
1
1
1
9
␣-helix
coil
8.025
2.071
0
67 (47)
57 (110)
100 (22)
52 (383)
0 (11)
66 (58)
31 (29)
50 (326)
50 (4)
c
1
97 (12)
0
68
5
4
␣-helix
␣-helix
␣-helix
coil
53 (348)
80 (4)
1
29
32
54
65
68
0
100
38
d
d
d
d
9.977
0
100 (1)
ND (0)
ND
ND (0)
ND
c
16 (50)
19 (79)
0
6 (52)
64
coil
0
26 (65)
23 (96)
10
26 (70)
0
coil
37.720
7 Ϯ 12
99 (67)
99 (100)
0
99 (70)
1
Average
70 Ϯ 33 (69 Ϯ 108)
51 Ϯ 36 (96 Ϯ 115)
18 Ϯ 37
51 Ϯ 30 (71 Ϯ 99)
24 Ϯ 29
4
M urea-treated oxidized GST
d
1
6
8
9
1
1
1
1
1
coil
ND
100 (2)
53 (15)
47
43 (7)
59 (37)
0 (6)
57
1
c
9
␣-helix
coil
8.025
2.071
0
60 (30)
68 (31)
0
1
100 (5)
50 (4)
50
100
7
4
␣-helix
␣-helix
␣-helix
coil
57 (367)
100 (3)
54 (393)
67 (3)
5
53 (374)
50 (6)
29
32
54
65
68
0
33
50
d
d
9.977
0
100 (1)
100 (1)
0
ND (0)
ND
38 (24)
8 (64)
79
9 (22)
76
coil
0
63 (27)
21 (87)
67
15 (26)
76
coil
37.720
100 (28)
99 (94)
1
88 (26)
12
Average
7 Ϯ 12
80 Ϯ 25 (54 Ϯ 118)
58 Ϯ 31 (77 Ϯ 124)
30 Ϯ 33
40 Ϯ 30 (56 Ϯ 120)
47 Ϯ 37
a
b
c
Coverage represents the number of times the peptide in which Met, oxidized or not, was found.
The percentage of reduction was calculated using the formula described in supplemental Table S3.
Due to experimental approximation, calculation gave a slightly negative percentage of reduction when no reduction activity was observed.
ND, not determined.
d
Met-69 and Met-81 were efficiently reduced by MSRA and urea-treated oxidized GST, which showed a 2.8- and 3.6-fold
MSRB, respectively, whereas oxidized Met-94, Met-165, and increase in MSRA and MSRB activity, respectively, compared
Met-168 were reduced by neither enzyme. These results indi- with the non-treated protein (Fig. 2A). Although the proteins
cate that MetO residues in the tested proteins were not equiv- unfolded by 4 M urea were rapidly assayed after the transfer to
alent MSR substrates and that sequence and structure proper- the MSR activity assay mixture, whereby diluting urea, we could
ties influenced the capacity for their reduction. The data also not exclude a possibility of spontaneous partial refolding of
suggest that the complete reduction of MetO in the protein proteins during the enzymatic reaction. To monitor the sub-
substrate would require opening of the structure to give access strate folding state during activity measurements, we charac-
to MSRs, as low reduction capacities were recorded for buried terized their intrinsic fluorescence and used a ANS probe,
MetO.
which emits fluorescence upon binding to hydrophobic areas of
MSRs Preferentially Reduce Unfolded Proteins—To examine proteins (Fig. 2B; supplemental Fig. S2). We assayed non-oxi-
the reduction of MetO present in the hydrophobic core of pro- dized, oxidized, and urea-treated oxidized proteins. For all
tein substrates, we assayed MSRA and MSRB activities with the tested proteins, oxidation induced changes in intrinsic and
three oxidized Met-rich proteins described above and Bacillus ANS fluorescence (with the exception of MRP4, for which no
subtilis ␣-amylase that contains 12 Met (2.3%) and is highly intrinsic fluorescence could be detected), indicating modifica-
structured (supplemental Table S2) after treatment with 4 M tion of protein structure. However, when oxidized proteins
urea, a chaotropic agent that leads to protein unfolding (Fig. 2). were compared with the urea-treated oxidized proteins,
These proteins were treated with urea in a small volume fol- changes were observed in intrinsic and ANS fluorescence in the
lowed by dilution of the chaotropic agent when the treated pro- case of ␣-amylase and GST. No changes were observed in the
teins were transferred to the reaction mixture. As a control, the case of MRP4 and ␤-casein, indicating that urea treatment did
MSR activities were determined with untreated oxidized pro- not induce changes in the folding state as these proteins are not
teins added to the reaction mixture containing the same final structured (supplemental Table S2). In contrast, after urea
amount of urea. No significant differences in MSRA or MSRB treatment, ␣-amylase and GST remained significantly changed
activities were observed in the case of urea-treated or untreated under conditions used in the MSR activity assays (Fig. 2B; sup-
oxidized MRP4 and ␤-casein, both of which are unstructured plemental Fig. S2). Altogether, these results indicate that for the
proteins. In contrast, when oxidized ␣-amylase was used as a tested proteins, MSRA and MSRB activities were higher with
substrate, the MSRA and MSRB activities were significantly unfolded oxidized than with folded oxidized proteins.
increased by 1.5- and 2.3-fold, respectively, with the urea-
GST was further used to investigate MSRA and MSRB activ-
treated substrate compared with the non-treated protein. ities with unfolded proteins in detail. The kinetics determined
These activity increases were even more dramatic in the case of using increasing concentrations of urea-treated oxidized GST
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
FIGURE 2. MSR activities using oxidized and urea-treated oxidized proteins as substrates and characterization of their folding state by fluorometry.
A, MSRA and MSRB activities were measured using the NAPDH-coupled Trx system with 25 ␮M oxidized protein (Ox.) and the oxidized protein treated with 4 M
urea (Ox ϩ Urea). As a control, for oxidized proteins, the same amount of urea present in the urea-treated oxidized proteins was added to the reaction mixture
(less than 50 mM final concentration). *, **, and ***, significantly different with p Ͻ 0.05, p Ͻ 0.01, and p Ͻ 0.0001, respectively (t test). B, intrinsic and ANS
fluorescence of 25 ␮M oxidized or urea-treated oxidized protein was determined under conditions similar to those used for MSR activity assays.
FIGURE3.SaturationcurvesofMSRA(A)andMSRB(B)activitiesusingoxidizedandurea-treatedoxidizedGST.MSRAandMSRBactivitiesweremeasured
using the NAPDH-coupled Trx system with 9.4–150 ␮M oxidized GST or 5–150 ␮M oxidized GST treated with 4 M urea. As a control, the amount of urea present
inthesample of the urea-treatedoxidized GSTwasadded inthe reactionmixture(lessthan 50mM finalconcentration). The saturation curves obtained withthe
urea-treated oxidized GST best fit sigmoidal regression as described under “Experimental Procedures” with h ϭ 1.4 Ϯ 0.3 and h ϭ 1.5 Ϯ 0.1 for MSRA and MSRB,
respectively.
could be described by a regression curve similar to an allosteric case of MSRA (Table 1; Fig. 3A), the catalytic efficiency (k
/
cat
sigmoidal (Equation 1), with the h values equal to 1.4 Ϯ 0.3 and K ) was 6-fold higher with the urea-treated oxidized GST than
m
1
.5 Ϯ 0.1 for MSRA and MSRB, respectively (Fig. 3). However, the oxidized GST (Table 1; Fig. 3A). Similarly, for MSRB, the
these values are close to 1, and the known MSR mechanism catalytic efficiency revealed a 3.2-fold increase in activity with
with Trx as a reductant (32) suggest that modification of these the urea-treated oxidized GST compared with the oxidized
kinetics could be due to an experimental factor, especially for protein (Table 1; Fig. 3B). The apparent stoichiometries deter-
the low values, and does not reflect an allosteric behavior. In the mined for MSRA and MSRB revealed that 3.2 and 4.7 MetO
2
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
FIGURE 4. Analysis of the folding state of the urea-treated oxidized GST and MSR activities. 10 ␮M non-oxidized or oxidized GST treated with 0–7 M urea
were incubated in 30 mM Tris-HCl, pH 8.0, in the presence (A) or absence (B) of 50 ␮M ANS. Intrinsic and ANS fluorescence was then recorded. Relative MSRA (C)
and MSRB (D) activities were recorded using 25 ␮M oxidized GST treated with 0–7 M urea. A.U., absorbance units.
equivalents, corresponding to 36 and 52% of the 9 Met, were hydrophobic regions, and then decreased at higher concentra-
reduced, respectively. Normalization of catalytic parameters to tions either due to a decrease in exposed hydrophobic parts or
the stoichiometry revealed that both MSRA and MSRB were the fact that ANS did not bind the protein under conditions of
more efficient in reducing the urea-treated oxidized GST con- high urea (Fig. 4A). The intrinsic Trp fluorescence fitted a sig-
sidering the whole protein as a substrate. Moreover, MSRA was moidal curve with a half-maximum of 2.2 Ϯ 0.1 M of urea (Fig.
more efficient even when the activity values were corrected to 4B). Interestingly, MSRA and MSRB activities using the same
the number of MetO reduced in the protein substrate.
urea-treated GST samples followed similar sigmoidal curves,
The apparent stoichiometries indicated that a higher propor- with the half-maxima of 2.3 Ϯ 0.2 and 2.9 Ϯ 0.3 M urea, respec-
tion of MetO was reduced in the urea-treated oxidized GST tively (Fig. 4, C and D). These results suggest that MSRA and
than in the oxidized protein. To determine the identity of MetO MSRB activities were proportional to the urea concentration
used as substrates after urea treatment, the unfolded protein and increased with the unfolding state of oxidized GST.
was analyzed by mass spectrometry after incubation with
Overall, these results showed that both MSRs were more effi-
MSRA or MSRB (Table 2). In both cases the average percentage cient in the reduction of unfolded proteins due to better access to
of reduction was almost doubled for the urea-treated oxidized MetO in the hydrophobic core of the substrate. Moreover, regard-
GST compared with the native protein. In particular, buried less of MetO exposure, MSRA was intrinsically more efficient in
Met-154 and Met-165 were more efficiently reduced by both the reduction of MetO in the unfolded oxidized GST.
MSRA and MSRB, indicating that urea treatment allowed bet-
ter access to buried MetO (Table 2).
MSRs Protect against Oxidative Unfolding Stress Induced by
Hypochlorite—To test if the observed preferential reduction of
We further incubated oxidized GST with increasing concen- unfolded proteins by MSRs is physiologically relevant, we uti-
trations of urea to examine MSRA and MSRB activities as a lized sodium hypochlorite (NaOCl), which leads to concomi-
function of the unfolded state of protein substrate. To charac- tant oxidation and unfolding of proteins (17). First, we exam-
terize the folding state, ANS and intrinsic fluorescence were ined if cells deficient in MSRs were more susceptible to NaOCl
measured for each urea concentration (Fig. 4). After oxidation, stress than WT cells. Growth of MSRA-null yeast cells was dra-
ANS fluorescence increased with the increase in urea concen- matically inhibited by treatment with 20 ␮M NaOCl (Fig. 5A).
tration up to 4 M, presumably due to increased exposure of Although MSRB-deficient cells grew similarly to WT cells
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
FIGURE 6. Overview of MSR targets. Based on the effects of MetO oxidation
on the structure and activity of proteins, three categories of MSR targets were
proposed (4): 1, enzymes in which Met oxidation leads to an increased activ-
ity, such as calcium/calmodulin-dependent protein kinase II; 2, proteins that
support cyclic Met oxidation and MetO reduction thereby providing antioxi-
dant defense; 3, native proteins damaged by formation of MetO, such as
those involved in neurodegenerative diseases. Based on our data, we pro-
pose a fourth type of MSR targets: 4, unfolded proteins and nascent polypep-
tides whose protein core Met are susceptible to oxidation thereby affecting
their folding, structure and function.
Finally, we tested the role of MSRA and MRP in the protec-
tion against H O , which induces oxidative stress but does not
2
2
lead to obvious protein unfolding (17). Although the effects
were less pronounced, overexpression of MSRA protected yeast
cells from this oxidant; however, expression of MRPs did not
offer additional protection (supplemental Fig. S3). Thus,
whereas the Met oxidation/reduction cycle protects yeast dur-
ing an oxidative and unfolding stress, the effect is less clear in
the case of oxidative stress alone. Altogether, these results
showed that both MSR types protected yeast against oxidative
and unfolding stress and that the protection offered by MSRA
was the most efficient.
FIGURE 5. Roles of MSRs and MRP in the protection of yeast cells from
hypochlorite. A, viability of the indicated MSR mutants and WT cells under
conditions of hypochlorite stress is shown. Yeast strains (5 ␮l each) were spot-
tedontoagarplatesandincubatedat30 °C. B, viabilityofcellsoverexpressing
MRPs and/or MSRA is shown. Cells overexpressing an indicated MRP and/or
DISCUSSION
This study describes the first comparative kinetic analyses of
MSRA and MSRB using MetO-containing proteins as sub-
MSRA were treated with different concentrations of NaOCl. Cells were plated, strates. MSRA reduced efficiently both free MetO and oxidized
incubated at 30 °C, and photographed. Cells expressing empty vectors were
proteins, whereas MSRB was specialized for the reduction of
used as controls.
oxidized proteins. Further experiments revealed that both MSR
types were more efficient in the reduction of unfolded than
when treated with NaOCl, deletion of this gene in the context of folded oxidized proteins, and this effect was due to a better
MSRA deficiency made yeast cells more susceptible to NaOCl access to MetO present in the hydrophobic cores of substrate
stress compared with the MSRA knock-out cells. Thus, both proteins. However, MSRA was intrinsically more efficient with
MSRA and MSRB contributed to the protection against NaOCl unfolded proteins irrespective of MetO accessibility. The rele-
stress by reducing MetO formed in cellular proteins upon treat- vance of this finding was verified in vivo as yeast cells deficient
ment with NaOCl. However, MSRA was more efficient in pro- in MSRs were highly sensitive to oxidative unfolding stress pro-
tecting yeast cells.
voked by hypochlorite, whereas overexpression of MSRA and
We further took advantage of previously identified Met-rich Met-rich proteins was highly protective. Our study suggests a
3
proteins (22) to determine if these proteins could protect cells new functional category of natural targets for MSRs, the
through cyclic oxidation of Met and reduction of MetO by unfolded proteins such as nascent polypeptides, proteins in
MSRs. We overexpressed a structured MRP1 possessing 38 Met route for subcellular compartments, and proteins unfolded
3
and a completely disordered MRP5, which has 31 Met residues because of oxidative stress (Fig. 6).
(
supplemental Table S2). Overexpression of MSRA, MRP1, and
Characterization of catalytic mechanisms of MSRA and
MRP5 independently protected yeast cells from oxidation. MSRB was previously carried out using free MetO or its vari-
However, the most dramatic effect was observed when MSRA ants, such as N-acetyl-MetO or dabsyl-MetO (23, 32, 33). The
and an MRP were coexpressed (Fig. 5B). We conclude that catalytic parameters with a protein substrate were only known
MRPs alone or in combination with MSRs protect cellular pro- for bovine MSRA acting on oxidized calmodulin (35). We used
teins through cyclic oxidation of their Met residues during the three oxidized proteins as model substrates; an unstructured
oxidative and unfolding stress provoked by hypochlorite.
MRP4 that has an exceptionally high Met content (21%),
2
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MSRs Preferentially Reduce Unfolded Oxidized Proteins
another unstructured protein ␤-casein (3.1% Met) (36), and also recorded with the urea-treated oxidized ␣-amylase than
GST with 3.8% Met and a well defined structure (37) (supple- with the corresponding untreated oxidized protein (Fig. 3). Fur-
mental Table S2). We showed that both MSRs displayed higher ther characterization revealed that protein unfolding increased
affinity for these proteins than for free MetO and N-acetyl- with the increase in urea concentration, following a sigmoidal
MetO (Table 1). Whereas MSRA was characterized by catalytic curve with the half-maximum at ϳ2 M urea (Fig. 4); the maxi-
efficiencies similar for free MetO, N-acetyl-MetO, and oxidized mum was reached when the oxidized protein was fully
proteins, MSRB displayed a striking preference for oxidized unfolded. In addition, a higher proportion of MetO was
proteins (Table 1). The catalytic parameters for the reduction of reduced in the 4 M urea-treated oxidized protein than in the
free MetO and N-acetyl-MetO by MSRA and MSRB were in the untreated oxidized protein (Table 2). Moreover, unfolding
range of those determined for prokaryotic and plant MSRs (23, allowed an almost complete reduction of MetO residues.
3
1, 33, 34), consistent with the idea that the preference for
Correction of the K for the apparent stoichiometry allowed
m
MetO reduction in proteins is a common feature of MSRBs, estimation of the catalytic efficiency per each reduced MetO.
whereas MSRAs act on any MetO-containing substrates. This MSRA was 3-fold more efficient in the reduction of MetO in the
finding also agrees with in vivo analysis showing that, despite unfolded GST than in the folded oxidized protein. Apparently,
the presence of three MSRB isozymes, human cells could not MSRA is intrinsically more efficient in the reduction of MetO in
reduce the R-diastereoisomer of free MetO (38).
unfolded proteins regardless of the accessibility to MetO. In the
The use of NADPH-coupled Trx system allowed us to deter- case of MSRB, the catalytic efficiency was higher with the
mine the fraction of MetO actually used as a substrate in oxi- unfolded proteins than with the folded one, but this could be
dized proteins. As the overall stoichiometry was 1 mol of explained by improved accessibility of MetO. These observa-
NADPH oxidized per mol of substrate reduced, NADPH con- tions are consistent with the finding that MSRA preferentially
sumption directly reflected MetO reduction using low sub- reduced MetO in a disordered region of oxidized calmodulin
strate concentrations (18, 31). This assay is independent of and that tryptic digestion was required to open up the protein
enzyme concentration, and the use of increasing substrate con- structure for access to all MetO (35).
centrations allows carrying out linear regression whose slope
An analysis of yeast MSR structures showed that hydropho-
represents the apparent stoichiometry and, thus, the number of bicity was particularly pronounced in the active sites, which
MetOs reduced in the protein (Fig. 1). MRP4 possesses 31 Met were also enriched in aromatic residues (supplemental Fig. 4).
residues and the apparent stoichiometries were 4 and 7 mol of These properties could make these enzymes better equipped to
NADPH oxidized/mol of substrate, indicating that MSRA and interact with unfolded proteins, which expose hydrophobic
MSRB reduced 13 and 23% of MetO, respectively (Table 1). residues usually confined to the core regions of folded proteins
Further analysis of the Met oxidation state in oxidized MRP4 by (40, 41). Therefore, the high content of aromatic and aliphatic
mass spectrometry showed that MSRA and MSRB reduced on residues in MSRs could be an important factor promoting the
average 33 and 24% of MetO, respectively (supplemental Table ability of these enzymes to preferentially use unfolded protein
S3). In excellent agreement for MSRB, the value found for substrates through hydrophobic or ␲-stacking interactions.
MSRA was 2.5-fold higher than that calculated by apparent This property should allow them to exhibit a higher affinity for
stoichiometry. This could be due to the lower protein coverage hydrophobic regions of target proteins, such as protein cores
observed in the oxidized sample, with 11 Met not covered, and regions involved in protein-protein interactions through
whereas only 4 were not covered in the sample repaired by hydrophobic interactions, where Met is particularly enriched
MSRA or MSRB, indicating that Met oxidation affects effi- (40, 41). Contrary to the oxidation of surface-exposed Met in
ciency of tryptic digestion as suggested previously (39). Simi- folded proteins, which may have little effect on protein function
larly, the mass spectrometry analysis showed that MSRA and (42), Met oxidation in buried regions should dramatically affect
MSRB reduced 18 and 24% of MetO in GST. These values were folding and function of cellular proteins. Likewise, oxidation of
higher than those determined by the calculation of apparent Met in the regions involved in protein interactions is expected
stoichiometry, i.e. 1.2 (13%) and 0.8 (9%) MetO reduced by to affect protein structure and function as shown for calmodu-
MSRA and MSRB, respectively. This difference was likely due lin (35). MSRs may play a major role in the protein folding
to enrichment of Met-containing peptides, as in almost all process by protecting Met from oxidation in nascent peptides.
cases, the mass spectrometry signal for each peptide was higher
for the MSR-reduced samples than for the oxidized proteins oxidation in catalase, concomitant with inactivation and
Table 2). The amino acid immediately upstream of Met may unfolding of this protein. This study showed that the reduction
Recently, the use of hypochlorite was found to induce Met
(
influence both the susceptibility of Met to oxidation and its of MetO within the hydrophobic core of catalase was required
reduction by MSRs, particularly when a Pro flanks the Met (39). for enzyme refolding by the chaperone GroEL (16). Hypochlo-
Our mass spectrometry analysis corroborated this observation rite treatment leads to both oxidative and unfolding stress as
as we found that the Met residues preceded by Pro residues in demonstrated by HSP33 activation (17). Indeed, whereas the
MRP4 and GST were completely oxidized by hydrogen perox- concomitant treatment with H O and thermal denaturation
2
2
ide but were not substrates for MSRs (Table 2; supplemental were required to activate HSP33, sodium hypochlorite alone
Table S3).
led to its full activation. This could be due to these two oxidants
Treatment of the oxidized GST with urea dramatically oxidizing Met residues in different ways. For example, Met oxi-
affected the MSR catalytic parameters, increasing catalytic effi- dation by NaOCl could result in dehydromethionine interme-
ciency (Table 1; Fig. 4). Higher MSRA and MSRB activities were diates that are then converted to MetO (43).
JULY 13, 2012•VOLUME 287•NUMBER 29
JOURNAL OF BIOLOGICAL CHEMISTRY 24457

MSRs Preferentially Reduce Unfolded Oxidized Proteins
An analysis of yeast cells grown in the presence of NaOCl 12. Minniti, A. N., Cataldo, R., Trigo, C., Vasquez, L., Mujica, P., Leighton, F.,
Inestrosa, N. C., and Aldunate, R. (2009) Methionine sulfoxide reductase
showed an involvement of MSRs in protection against oxidative
A expression is regulated by the DAF-16/FOXO pathway in Caenorhab-
unfolding stress (Fig. 5). Indeed, the MSRA-null mutant and
ditis elegans. Aging Cell 8, 690–705
especially MSRA/MSRB-null cells were more sensitive than
WT cells to hypochlorite treatment, and overexpression of
MSRA protected cells from this stressor. Similar effects were
observed in prokaryotes (15, 16). Interestingly, overexpression
of two Met-rich proteins also conferred protection, very likely
due to reversible Met oxidation/reduction. This observation
shows that the higher MSR activity with unfolded proteins was
relevant in vivo during the NaOCl stress that triggered unfold-
ing of proteins. This property may be particularly important
during the oxidative battle between neutrophils and pathogens
wherein both opponents induce protein oxidation and use
MSRs as a sword and shield strategy (44, 45). Overall, our find-
ings suggest that a major protective function of MSRs in the cell
is to rescue and repair oxidized nascent polypeptides and
unfolded proteins.
1
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Rey, P. (2010) Arabidopsis thaliana plastidial methionine sulfoxide reduc-
tases B, MSRBs, account for most leaf peptide MSR activity and are essen-
tial for growth under environmental constraints through a role in the
preservation of photosystem antennae. Plant J. 61, 271–282
1
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1
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Acknowledgment—We thank Dr. Pascal Rey (CEA-Cadarache) for
the kind gift of dabsyl-MetO.
1
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1
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