FORUM REVIEW ARTICLE
Protein Glutathionylation in the Regulation of Peroxiredoxins:
A Family of Thiol-Specific Peroxidases That Function As
Antioxidants, Molecular Chaperones, and Signal Modulators
Ho Zoon Chae,1,*Hammou Oubrahim,2,*Ji Won Park,3Sue Goo Rhee,4and P. Boon Chock2
Significance: Reversible protein glutathionylation plays an important role in cellular regulation, signaling
transduction, and antioxidant defense. This redox-sensitive mechanism is involved in regulating the functions of
peroxiredoxins (Prxs), a family of ubiquitously expressed thiol-specific peroxidase enzymes. Glutathionylation of
certain Prxs at their active-site cysteines not only provides reducing equivalents to support their peroxidase
activity but also protects Prxs from irreversible hyperoxidation. Typical 2-Cys Prx also functions as a molecular
chaperone when it exists as a decamer and/or higher molecular weight complexes. The hyperoxidized sulfinic
derivative of 2-Cys Prx is reactivated by sulfiredoxin (Srx). In this review, the roles of glutathionylation in the
regulation of Prxs are discussed with respect to their molecular structure and functions as antioxidants, mo-
lecular chaperones, and signal modulators. Recent Advances: Recent findings reveal that glutathionylation
regulates the quaternary structure of Prx. Glutathionylation of Prx I at Cys83converts the decameric Prx to its
dimers with the loss of chaperone activity. The findings that dimer/oligomer structure specific Prx I binding
proteins, e.g., phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), regulate cell
cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling path-
ways. Critical Issues: Knowing how glutathionylation affects the interaction between Prx I and its nearly 20
known interacting proteins, e.g., PTEN and MST1 kinase, would reveal new insights on the physiological
functions of Prx. Future Directions: In vitro studies reveal that Prx oligomerization is linked to its functional
changes. However, in vivo dynamics, including the effect by glutathionylation, and its physiological significance
remain to be investigated. Antioxid. Redox Signal. 16, 506–523.
tabolism are regulated by reversible covalent modifications.
These post-translational modification reactions are catalyzed
by converter enzymes; for example, protein kinase and
phosphatase catalyze phosphorylation and dephosphoryla-
tion reactions, respectively, and thioltransferase/glutaredox-
in (Grx) catalyzes glutathionylation/deglutathionylation
reactions (20, 102, 119). These signaling pathways involve the
action of one enzyme upon another to constitute a cyclic
cascade system, which has been shown to possess enormous
capacity for integrating biological information and for signal
he activities of many key enzymes and regulatory
proteins in cell signaling pathways and in cellular me-
and rate amplification (19, 51, 124). Cyclic cascades involving
reversible protein glutathionylation are mediated by redox
signals that are produced, at least in part, in response to ag-
onist binding to various cell surface receptors (4, 5, 102, 125).
The physiological importance of this regulatory mechanism is
substantiated by glutathione (GSH), the donor molecule, be-
ing a major redox buffer maintaining a reduced state of pro-
tein thiols in cells and being present in the millimolar
concentration range in most cell types (39, 52). Several in vitro
investigations revealed that protein glutathionylation leads to
functional changes in nearly 100 proteins (27, 71, 79, 102).
However, only a fewstudies haverevealed glutathionylation-
dependent changes in enzyme-specific activity or protein
cytoskeletal function in a physiological context (79). They
1School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea.
2Laboratory of Biochemistry, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health,
3Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
4Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.
*These authors contributed equally to this article.
ANTIOXIDANTS & REDOX SIGNALING
Volume 16, Number 6, 2012
ª Mary Ann Liebert, Inc.
include studies on the protein tyrosine phosphatase 1B (5, 60),
ras (1), mitochondria complex II (17), glyceraldehyde-3-
phosphate dehydrogenase (107), peroxiredoxin (Prx) I (89),
and actin (129, 130). These findings suggest protein glu-
tathionylation is involved in regulating a wide range of
physiological processes. Furthermore, protein glutathionyla-
tion can also protect the protein thiol moiety from being hy-
peroxidized to its irreversible sulfinic acid derivative. The
regeneration of the deglutathionylated protein is catalyzed by
Grx. Thus, reversible protein glutathionylation has emerged
as a major cellular regulatory mechanism in cell signaling and
metabolism, as well as a major antioxidant defense to counter
Free radicals and reactive oxygen species (ROS) play a
major role in normal biological functions. They include the
bactericidal action of neutrophils and macrophages, cellular
signal transduction, and activation of transcriptional fac-
tors, and they are formed as intermediates in many enzyme-
biomacromolecules such as nucleic acids, lipids, and proteins
and lead to the loss/change of their biological functions.
progression of a number of diseases and with aging (for re-
as unavoidable byproducts of normal electron transport
processes and as products of reactions between metabolites,
such as hydrogen peroxide or alkyl peroxides and trace
amounts of iron or copper. Thus, it is necessary for a func-
tionally healthy cell to maintain an elegant regulatory mech-
anism to prevent the accumulation of harmful concentrations
of ROS yet allow the accumulation of ROS to mediate their
diverse physiological functions. To this end, cells rely on
several enzymes including peroxiredoxins (Prxs), a family of
peroxidases. These enzymes possess multiple functions in
stress protection as antioxidants, molecular chaperones, and
regulators of signal transduction. Prxs appear to play a role in
maintaining a transient and cellular location specific envi-
of cascades, as well as in eliminating ROS from inducing cy-
Prxs, found in all kingdoms with multiple isoforms, are
among the most abundant proteins in most cells. This family
of antioxidants was initially identified, purified, and charac-
terized as ‘‘protector’’ proteins in Saccharomyces cerevisiae (62,
63). Without knowing its function, one member of this family,
‘‘torin,’’ was isolated from red blood cells early on and later
identified as Prx II (46). The correct annotation for the first
time was ‘‘thioredoxin peroxidase’’–reducing peroxides with
the use of thioredoxin (Trx) (9), and later ‘‘peroxiredoxin’’—
reflecting an expansion of family members using other than
Trx as an electron donor such as GSH, tryparedoxin, and the
57-kDa component of alkylhydroperoxide reductase (AhpF)
(12, 53, 85, 103). Prxs are not confined to cytosol. They are also
extracellular regions (28, 40, 41, 69, 103, 105, 121, 126, 136).
This familyofperoxidases exertstheir antioxidantfunctionby
reducing H2O2, organic hydroperoxides and peroxynitrite
using intracellular reducing equivalents containing thiol (48,
104). Based on sequence and structural homology, Prxs have
been proposed to comprise six subfamilies, namely, Prx A, B,
C, D, E, and F (29). However, another classification, based on
the structure, number, and location of conserved Cys residues
and their contribution to peroxidase catalysis, is more com-
monly accepted and is preferentially applied to mammalian
Prxs. In this classification, Prxs are grouped into three sub-
but all isoforms are not necessarily expressed in one cell at the
same time. The typical 2-Cys Prx subfamily consists of Prx I–
IV. These four 2-Cys Prxs share >70% amino acid sequence
identity (103), and they possess both conserved N- and C-
terminal Cys residues. The conserved N-terminal low pKa
Cys is referred to as the peroxidatic Cys (SP) while the con-
served cysteine residue at the C-terminal region is the re-
solvingcysteine (SR).MostPrx proteinswith theSPinreduced
state form a head-to-tail homodimer, which in turn servesas a
building block for a doughnut-shaped decamer, composed of
five homodimer pairs (Fig. 2) (115, 134). During peroxidase
catalysis, the peroxidatic Cys is oxidized by either H2O2, or-
ganic peroxide (ROOH), or peroxinitrite (ONOO2) to form a
sulfenic acid derivative (SP–OH) with a second order rate
constant in the range of 105to 107M-1s-1at pH 7.4 (36, 87).
This unusually rapid oxidation is attributed to the hydrogen-
bonding network created by the four conserved amino acid
residues (Pro, Thr, Arg, and Glu/Gln/His) present in all Prx
active sites, which stabilize the SPthiolate anion and activate
the peroxide substrate (44) (Fig. 3). The sulfenic derivative,
(SP–OH), in turn forms an intermolecular disulfide bond with
dimer (see Fig. 2A). The intermolecular disulfide formation
requires local unfolding because the distance between the SP
and SRis *13A˚, too far apart for disulfide bond formation
(134). The latter effect weakens the dimer–dimer interaction,
resulting inthedissociation ofthedecamer tofivedimers.The
SP-SRdisulfide bond is reduced specifically by a Trx system
(Trx/TrxR/NADPH) composed ofTrx,thioredoxin reductase
(TrxR), and NADPH (9–11, 56). In contrast, atypical 2-Cys Prx
exhibits about 20% sequence identity with the four typical 2-
the conserved peroxidatic Cys (SP) residue at its N-terminal
region. However, its peroxidase activity requires the partici-
pation of one additional, nonconserved Cys residue in the
same polypeptide. During catalysis, the sulfenic acid deriva-
tive formed at the peroxidatic cysteine in the atypical 2-Cys
Prx reacts with a resolving cysteine residue to form an in-
tramolecular disulfide bond (Fig. 4) (31). This intramolecular
disulfide bond is then reduced either by a cytosolic or a mi-
tochondrial Trx system (65, 118). It should be pointed out,
however, that the atypical 2-Cys Prx from poplar accepts both
Trx and Grx (67, 110), while the atypical 2-Cys Prx from yeast
(Ahp1p) is reduced only by Trx (67). In the case of the 1-Cys
Prx (Prx VI), it also contains one conserved Cys residue at the
N-terminal region, required for its peroxidase activity (103–
105, 136). However, Prx VI does not possess a second cysteine
residue for disulfide bond formation; therefore, its resolving
mechanism is more complex. The sulfenic acid intermediate
formed following its reaction with H2O2or ROOH has been
shown to be reduced by nonphysiological thiols such as di-
thiothreitol (DTT) but not by Trx or Grx (61). It has also been
reported that the physiological reductant could be the GSH in
conjunction with the p isoform of glutathione S-transferase in
the case of mammalian 1-Cys Prx (PrxVI) (34, 75, 100) (to be
discussed later). However, TrxR, Grx, and ascorbate have
REGULATION OF PRXS BY GLUTATHIONYLATION 507
been reported to serve as reductants for yeast mitochondrial
1-Cys Prx (42, 81, 94).
During the reduction of H2O2or ROOH, the oxidation of
the peroxidatic cysteine in Prxs may not stop at its sulfenic
acid state. When the sulfenic intermediate is further oxidized
to form a sulfinic acid (Cys-SO2H) derivative, or oxidized to
an irreversible sulfonic acid (Cys-SO3H) derivative, Prx loses
its peroxidase activity (98, 138). However, in the case of the
typical 2-Cys Prxs, their sulfinic derivatives can be reduced to
sulfenic acid, catalyzed by the sulfiredoxin (Srx), conserved
only among eukaryotes, in the presence of MgATP (7, 131,
132) (Fig. 5). This unique Srx-mediated reactivation of the
inactive sulfinic derivative of Prx in eukaryotes would con-
stitute a mechanism for cells to accumulate sufficient quanti-
ties of H2O2to induce signal transduction at a specific cellular
location in a time-dependent manner. This notion is some-
times referred to as a floodgate hypothesis (102, 135). In ad-
dition, when typical 2-Cys Prxs are hyperoxidized, they form
higher-order oligomeric structures. These high molecular
weight complexes have been shown to gain a molecular
dimers. Typical 2-Cys subfamily members form two intermolecular disulfide bonds by resolving the sulfenic derivative (Cys-
SOH) of peroxidatic Cys. Atypical 2-Cys Prx forms one intramolecular disulfide bond per each monomer, despite the fact that
it exists in an antiparalleled dimer like typical 2-Cys Prx. Whereas, 1-Cys Prx does not form a disulfide due to an unavail-
ability of proximal Cys residue for resolving. Glutathione with p isoform of glutathione S-transferase (p-GST) is required for
the resolution of peroxidatic Cys-OH.
Peroxidase catalytic mechanisms of peroxiredoxin (Prx). All of the subfamily members of Prx are obligatory
ture of Prxs. (A) Dimeric
structure of rat isoform of
bond between SPand SR(ball
and stick with sulfur atom
colored yellow). This figure
is based on a figure from
Hirotsu et al. (47). (B) Deca-
meric structure of human Prx
I adopted from a figure from
Schroder et al. (115). (To see
this illustration in color the
reader is referred to the web
508 CHAE ET AL.
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Address correspondence to:
Dr. P. Boon Chock
Laboratory of Biochemistry
National Heart, Lung, and Blood Institute
National Institutes of Health
Building 50, Room 2134, MSC-8012
Bethesda, MD 20892-8012
Date of first submission to ARS Central, August 30, 2011; date
of final revised submission, November 21, 2011; date of
acceptance, November 23, 2011.
AhpC¼22-kDa catalytic subunit of alkyl
BioGEE¼biotinylated glutathione ethyl ester
Cdk1¼cyclin B-dependent kinase 1
MST1¼mammalian sterile 20 like 1 kinase
NADPH¼nicotinamide adenine dinucleotide
PABA/NO¼a diazeniumdiolate of structure
Me2NN(O)¼ NOAr (Ar is a 5-substituted-2,4-
dinitrophenyl ring, 5-substituent is
pGST¼p isozyme of glutathione S-transferase
PTEN¼phosphatase and tensin homolog
ROS¼reactive oxygen species
S128W/NTD¼(S128W) mutant of N-terminal domain
of AhpF include residues 1–202
Sp¼sulfur atom of peroxidatic cysteine residue or
SR¼sulfur atom of resolving cysteine residue or
REGULATION OF PRXS BY GLUTATHIONYLATION523