Báo cáo khoa học: Seleno-independent glutathione peroxidases More than simple antioxidant scavengers pptx

REVIEW ARTICLE
Seleno-independent glutathione peroxidases
More than simple antioxidant scavengers
Ste
´
phane Herbette
1
, Patricia Roeckel-Drevet
1
and Joe
¨
l R. Drevet
2
1 UMR 547-PIAF, INRA ⁄ Universite
´
Blaise Pascal, Aubie
`
re Cedex, France
2 UMR 6547-GEEM, CNRS ⁄ Universite
´
Blaise Pascal, Aubie
`
re Cedex, France
Introduction
Glutathione peroxidase (GPX; EC 1.11.1.9) catalyses
the reduction of H
2
O
2
or organic hydroperoxides to
water or the corresponding alcohols using reduced

glutathione. GPX was discovered in 1957 [1] as an
enzyme that protects erythrocytes against oxidative
damage. Later, several additional types of mammalian
GPX were isolated, and those enzymes were shown to
be expressed in a wide range of organisms. In mam-
mals, together with superoxide dismutases (EC
1.15.1.1) and catalases (EC 1.11.1.6), GPX constitutes
the enzymatic antioxidant system which recycles active
oxygen species (AOS) and limits their toxicity. The
mammalian GPX family is divided into six clades
according to their amino-acid sequence, substrate
specificity and subcellular localization (Table 1): classi-
cal or cytosolic (GPX1), the first mammalian GPX to
be identified [1–3]; gastrointestinal (GPX2); plasma
(GPX3); phospholipid hydroperoxide (PHGPX or
GPX4); epididymal (GPX5); olfactory epithelium
(GPX6).
Except for GPX5 and GPX6, all mammalian GPX
proteins contain a selenocysteine (SeCys) residue
instead of a Cys residue (Table 1). SeCys is considered
to be the 21st amino acid. Its cotranslational incorpor-
ation into protein is mediated by a SeCys tRNA, the
presence of a stem loop structure located downstream
from its UGA codon and designated as the SECIS ele-
ment (SeCys insertion sequence), and by the recruit-
ment of a specific elongation factor. This SeCys
insertion system recognizes the opal codon UGA as a
Keywords
free-radical scavenger; glutathione
peroxidase; oxidative stress; selenocysteine;

thioredoxin
Correspondence
J. Drevet, Universite
´
Blaise Pascal, CNRS
UMR 6547 GEEM, 24 avenue des Landais,
63177 Aubiere cedex, France
Fax: +33 4 73 40 52 45
Tel: +33 4 73 40 74 13
E-mail:
(Received 16 January 2007, revised 2 March
2007, accepted 7 March 2007)
doi:10.1111/j.1742-4658.2007.05774.x
Glutathione peroxidases (GPXs, EC 1.11.1.9) were first discovered in mam-
mals as key enzymes involved in scavenging of activated oxygen species
(AOS). Their efficient antioxidant activity depends on the presence of the
rare amino-acid residue selenocysteine (SeCys) at the catalytic site. Nonse-
lenium GPX-like proteins (NS-GPXs) with a Cys residue instead of SeCys
have also been found in most organisms. As SeCys is important for GPX
activity, the function of the NS-GPX can be questioned. Here, we highlight
the evolutionary link between NS-GPX and seleno-GPX, particularly
the evolution of the SeCys incorporation system. We then discuss what
is known about the enzymatic activity and physiological functions of
NS-GPX. Biochemical studies have shown that NS-GPXs are not true
GPXs; notably they reduce AOS using reducing substrates other than
glutathione, such as thioredoxin. We provide evidence that, in addition to
their inefficient scavenging action, NS-GPXs act as AOS sensors in various
signal-transduction pathways.
Abbreviations
AOS, activated oxygen species; GPX, glutathione peroxidase; NS-GPX, nonselenium GPX; PHGPX, phospholipid hydroperoxide GPX;

SeCys, selenocysteine; TPx, thioredoxin peroxidase.
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2163
signal to insert a SeCys residue. Selenoproteins have
been found in archaebacteria, eubacteria and eukaryo-
tes, but not in all organisms. Although SeCys is rarely
used in protein synthesis, it appears to be essential for
selenoprotein function, and is found at the catalytic
sites of many selenoenzymes [4]. This has been demon-
strated, for instance, by point mutations of SeCys to
Cys in GPX1 as well as in GPX4, which both led to a
dramatic fall in enzymatic activity [5,6]. SeCys and Cys
differ in a single chalcogen atom (Se versus S). The
selenol group is entirely ionized at physiological pH
(pK
a
¼ 5.2), whereas the thiol group of Cys is only
partially ionized under similar conditions (pK
a
¼ 8).
Once ionized, the thiol or selenol group is able to react
with H
2
O
2
or hydroperoxides. The catalytic triad of
amino-acid residues, i.e. Trp, Glu, Cys or SeCys, is
common to all GPXs (Fig. 1, alignment).
Although GPX-like proteins have been found in
most organisms studied, expression of SeCys-contain-
ing GPX is restricted to only a few of them. Besides

mammals, SeCys-containing GPXs have been found
in other vertebrates such as Gallus gallus [7] and
Danio rerio [8]. Outside the vertebrate clade, SeCys-
containing GPXs have been reported in the parasitic
helminth Schistosoma mansoni [9], the nematode
Setaria cervi [10], the arthropod Boophilus microplus
[11], the alga Chlamydomonas reinhardtii [12], and
also in the DNA sequence of the virus HIV-1 [13]. In
addition to the seleno-dependent GPXs, nonselenium
GPXs (NS-GPXs) are found in these organisms and
are widely represented in mammals [14,15]. NS-GPXs
have been found in higher plants [16–19], yeast [20],
the protozoan Trypanosoma cruzi [21], the nematode
Brugia pahangi [22] and the cyanobacterium Synecho-
cystis [23]. To our knowledge, neither selenoproteins
nor SeCys insertion systems have been found in these
organisms. In the prokaryotes Escherichia coli and
Neisseiria meningitidis and in the eukaryote Plasmo-
dium falciparum, only NS-GPXs have been found,
although these organisms can express selenoproteins
[24,25].
Although many physiological functions of the thor-
oughly investigated seleno-dependent GPXs have been
elucidated, especially in mammals [26,27], NS-GPXs
have, so far, been the subject of few studies. This is
essentially because they have been found to be a lot
less efficient at detoxifying AOS and peroxides than
seleno-dependent GPXs. In this study, we conducted
an analysis of the evolutionary relationships between
GPXs, particularly the evolution of the SeCys incor-

poration system. We then focused on the enzymatic
activity and proposed physiological functions of the
NS-GPXs.
Table 1. Characteristics of GPX proteins from mammals. The tissue expression given for GPX1, GPX3 and GPX4 are the most representative, as the protein or the mRNA has been found
in all tissues investigated. For GPX6, the presence of SeCys was confirmed in man and pig [50], and invalidated in rat and mouse [15].
Numeric nomenclature GPX1 GPX2 GPX3 GPX4 GPX5 GPX6
Literature
denomination
cGPX (cytosolic or
classical)
GI-GPX
(gastro-intestinal)
pGPX (plasmatic) PHGPX (phospholipid
hydroperoxide)
eGPX (epididymal) OMP (olfactory-metabolizing
protein)
Tissue distribution Red cells, liver,
lung, kidney, …
Stomach, intestine,
liver
Kidney, lung, epididymis,
vas deferens, placenta,
seminal vesicle, heart,
muscle, …
Testis, spermatozoa,
liver, kidney, heart,
brain, …
Epididymis, spermatozoa Olfactory epithelium
Cellular localization Cytosol, nucleus,
mitochondria

Cytosol, nucleus Secreted, cytosol Nucleus, cytosol,
mitochondria,
membrane-bound
Secreted
Molecular mass (kDa) 21 22 22.5 19 24 22.5
Multimerization 4 4 4 1 2 4
SeCys Yes Yes Yes Yes No Yes ⁄ No
First reference Mills, 1957 [1] Chu et al. 1993 [144] Takahashi et al. 1987 [145] Ursini et al. 1985 [35] Ghyselink et al. 1989 [146] Dear et al. 1991 [15]
Selenium-independent glutathione peroxidases S. Herbette et al.
2164 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
Phylogenetic evaluation of NS-GPXs
Most NS-GPXs belong to the PHGPX group
We compared GPX amino-acid sequences from var-
ious organisms (Fig. 2). This analysis was carried out
independently of the presence ⁄ absence of SeCys in the
proteins. The tree shows that the GPX family can be
subdivided into three broad groups. One group
includes the mammalian GPX1 and GPX2 and iso-
forms from other vertebrates such as the zebra fish
D. rerio [8]. Another group comprises the mammalian
GPX3, GPX5 and GPX6 proteins. A third group, the
PHGPX group, includes the mammalian GPX4 and
most of the GPXs from various organisms. This analy-
sis is in agreement with previous phylogenetic evalua-
tions based on gene structures of mouse GPX1, GPX3,
GPX4 and GPX5 [28] or based on amino-acid
sequences from various mammalian GPXs [28,29].
Except for some higher vertebrate GPXs, most of
the GPX proteins investigated belong to the PHGPX
group. An exception to this situation is, for example, a

GPX protein from the parasitic nematode B. pahangi
(persists in the human lymphatic system and is respon-
sible for lymphatic filariasis), which shows the highest
similarity to GPX3 from Homo sapiens [22]. Proteins
similar to this B. pahangi GPXs have also been found
in other parasitic nematodes [30,31]. One possible
explanation is that the secreted GPX3 has been trans-
ferred from vertebrates to nematodes by horizontal
gene transfer. Genetic analyses have revealed the
importance of horizontal gene transfer between organ-
isms, especially between a host and its parasitic or
symbiotic organism [32]. Another observation in
favour of the hypothesis of horizontal gene transfer is
the close similarity of GPX from HIV-1 to human
GPX3 [13].
In mammals, it has been postulated that the GPX
gene family has evolved from a common gene ancestor
by duplication events followed by random integration
in the genome [28,29]. The ancestor was proposed to
be represented by the GPX4 ⁄ PHGPX sequence. After
a first duplication event, the ancestral PHGPX would
have diverged into two groups, one group represented
by genes encoding the intracellular GPX1 and GPX2
proteins, respectively, and the second represented by
genes encoding the secreted GPX3, GPX5 and GPX6
proteins. On the basis of the low similarities found
Fig. 1. Alignment of GPX amino-acid
sequences from various organisms. The
sequences were compared and aligned
using

CLUSTALW software [143]. Nucleotide ⁄
protein sequence accession numbers
(GenBank ⁄ SWISS-PROT): C. reinhardtii
(AB009083), C. sinensis (Q06652), E. coli
(P06610), L. esculentum (Y14729), Mus
musculus GPX1 (P11352), GPX2
(BC054848), GPX3 (U13705), GPX4
(O70325), GPX5 (P21765) and GPX6
(NP_663426), P. falciparum (Z68200),
S. cerevisiae-1 (P30614), Sch. mansoni
(Q00277), Synechocystis PCC 6803
(NP401201). Amino-acid residues of the
catalytic triad are marked with an asterisk.
Residues common to all proteins are indica-
ted by white letters on a black background,
whereas those shared by more than seven
proteins are shadowed.
S. Herbette et al. Selenium-independent glutathione peroxidases
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2165
between PHGPXs and other GPX proteins, the phylo-
genetic divergence of the PHGPX gene and the genes
encoding other GPX types has been estimated to have
happened approximately one billion years ago [33]. In
fact, mammalian PHGPX appears to be more closely
related to GPXs from various organisms than to all
mammalian GPX types. Hence, the PHGPX group
must be rated as a phylogenetically old achievement of
the GPX family. This suggests that PHGPX proteins,
including most NS-GPXs, would fulfil an important
function conserved in various organisms and distinct

from other mammalian GPX functions. PHGPX can
also be distinguished from other GPX proteins, as they
are the only ones that are monomeric (Table 1).
PHGPX proteins do not possess the subunit interac-
tions sites identified by X-ray crystallography, for
example, in the bovine GPX1 protein [34]. Large gaps
in the PHGPX sequences are observed when aligned
with sequences of other GPX types (Fig. 1). Accord-
ingly, PHGPX proteins have been shown to be
expressed as monomers in several species including
mammals, plants and nematodes [9,35,36]. However, a
PHGPX from Populus trichocarpa was recently shown
to be expressed as a homodimer [37], despite the pres-
ence of gaps in its sequence. Determination of the
structural components responsible for the dimerization
of this plant PHGPX would help to clarify the extent
of oligomerization in the PHGPX group. Taken
together, these data support the fact that most
NS-GPXs form a separate clade from the mammalian
PHGPX.
Although the GPX4 ⁄ PHGPX gene is present as a
single copy in mammals and organisms such as E. coli,
several PHGPX genes have been found per haploid
genome in many species. For instance, seven and six
PHGPX genes were isolated from Arabidopsis thaliana
and P. trichocarpa [37], respectively, and were the only
GPX type found. For Arabidopsis, comparison of their
structures showed that the number of exons is similar
(five to seven), the exon–intron structure is well con-
served between some gene pairs, and neighbouring

genes are also conserved between these pairs [38].
These observations support the idea of duplication
events leading to the A. thaliana PHGPX gene family.
The seven Arabidopsis PHGPX genes have been shown
to be differently regulated at the transcriptional level,
and, on the basis of sequence criteria, the proteins
have been proposed to display different cellular locali-
zations in A. thaliana [38]. Functionality of the differ-
ent PHGPX isoforms has been investigated in some
species. In Saccharomyces cerevisiae, three PHGPX-
encoding sequences have also been found to be differ-
entially regulated and to encode functional enzymes
[20]. The existence of two PHGPX genes and the
Fig. 2. Phylogenetic tree of GPX proteins
from mammals and various organisms. The
amino-acid sequences were compared using
CLUSTALW software [143], and the PHYLIP
program was used to construct the tree.
Nucleotide ⁄ protein sequence accession
numbers (GenBank ⁄ SWISS-PROT):
B. pahangi (X69128) C. reinhardtii
(AB009083), C. sinensis (Q06652), D. rerio
GPXa (AY215589) and PHGPXa (AY216590),
D. melanogaster (AAO41409), E. coli
(P06610), Homo sapiens GPX1 (P07203),
GPX2 (P18283), GPX3 (P22352), GPX4
(P36969), GPX5 (NM_001509) and GPX6
(NM_182701), L. esculentum (Y14729),
Mus musculus GPX1 (P11352), GPX2
(BC054848), GPX3 (U13705), GPX4

(O70325), GPX5 (P21765) and GPX6
(NP_663426), P. falciparum (Z68200),
S. cerevisiae-1 (P30614), Sch. mansoni
(Q00277), Synechocystis PCC 6803
(NP401201). A bar (0.1) indicates branch
lengths. Proteins containing a SeCys are
shown by a star.
Selenium-independent glutathione peroxidases S. Herbette et al.
2166 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
biochemical properties of the corresponding proteins
have also been reported in Synechocystis [23]. In Try-
panosoma cruzi, the two existing PHGPX genes have
been shown to encode proteins with different cell local-
ization [39]. Together these data suggest that each indi-
vidual PHGPX gene has evolved with specific
functions via tissue-specific and cell-specific expression.
In support of this hypothesis, a PHGPX isoform often
displays more similarities to GPX isoforms from other
species than to another isoform from the same species,
provided that the two species are closely related. This
has recently been illustrated in plants by a phylo-
genetic analysis of the amino-acid sequences of the
PHGPX gene family from several species [40]. For
example, a PHGPX from Oryza sativa shares more
homology (up to 98%) with PHGPX proteins from
several monocotyledons and eudicotyledons than with
any other rice PHGPX (65%).
It should be mentioned that, although most
NS-GPXs belong to the PHGPX family, a few, such
as GPX5 in mammals and a GPX in nematodes, are

not included in this gene family but are in fact closer
in sequence to the GPX3 subgroup. Conversely, some
seleno-dependent GPXs belong to the PHGPX family
(Fig. 2). This raises the question of the evolution of
seleno-dependence or seleno-independence in the GPX
family. In other words, have GPXs evolved by the
acquisition of SeCys or by loss of SeCys?
No SeCys in GPX – remnant or innovation?
Sequences that encode putative GPX-like proteins can
be found in eukaryotes and eubacteria, suggesting that
GPX evolved before the separation of eukaryotes and
eubacteria. Although most NS-GPXs belong to the
PHGPX gene family, some, such as GPX5, GPX6 in
rodents and a GPX in nematodes, are not included in
this gene family, and conversely some seleno-depend-
ent GPXs belong to it (Fig. 2). Hence, the evolution of
GPX seleno-dependence is unclear. NS-GPXs are pre-
valent in living organisms, although SeCys is import-
ant for the activity of SeCys-containing GPXs. This
paradox led to the question of whether GPXs have lost
SeCys or whether others have gained it.
Selenoproteins have been found in archaea, eubacte-
ria and eukaryotes [4] with common SeCys incorpor-
ation features, such as the use of the same UGA
codon and the use of a tRNA for SeCys that is ini-
tially aminoacetylated with a serine [4]. In addition,
SeCys tRNA and selenophosphate synthase, which
provides the selenium donor, are conserved in all
selenoprotein-encoding genomes. These observations
suggest that the insertion of SeCys in the genetic code

occurred before the separation of archaea, eubacteria
and eukaryotes. As several organisms appear to lack
selenoproteins, it has been proposed that SeCys may
be a relic of the primordial genetic code [41,42].
According to these authors, UGA was initially a sense
codon for SeCys, which was used in many enzymes in
the primordial world. Later, when oxygen concentra-
tion increased in the atmosphere, evolutionary proces-
ses selected against the use of SeCys because of the
sensitivity of this amino acid to oxidation. The use of
SeCys progressively decreased and UGA became a
nonsense codon. In contrast, other authors argue that
SeCys was added to the genetic code and that its use
increased during evolution culminating in vertebrates
[43]. Selenoproteins, such as GPX, formate dehydroge-
nase and iodothyronine deiodinase, would take
advantage of the redox properties of SeCys, superior
to those of Cys, for their specific functions [44,45].
Finally, an independent origin of the prokaryotic and
eukaryotic selenoproteomes has been proposed as there
is no direct relation between the two selenoproteomes
[46]. With the exception of selenophosphate synthetase,
no homology can be found between prokaryote and
eukaryote selenoproteins. Eubacterial and archaeal
selenoproteins are primarily involved in catabolic pro-
cesses, whereas eukaryote selenoproteins participate in
antioxidant and anabolic processes. A study based on
a comparative genomic approach has revealed a scat-
tered phylogenetic distribution of selenoproteins in
eukaryotes [47], suggesting a dynamic SeCys ⁄ Cys evo-

lutionary exchange instead of the contradictory images
of the SeCys evolution described above. That some
organisms prefer selenoproteins whereas others prefer
Cys-containing homolog proteins suggests a different
history for each protein and for each species, in which
evolutionary events and functional constraints play a
key role.
Several selenoproteins including two PHGPX pro-
teins have been identified in the plant C. reinhardtii
[12,48]. These selenoproteins, as well as the SeCys
insertion system, were found to be similar to those of
mammals, indicating a common origin for plant and
animal selenoproteins ⁄ SeCys insertion systems. Cys-
containing homologues of these selenoproteins have
also been found in higher plants and other animals.
Phylogenetic analyses led to exclusion of horizontal
gene transfer between C. reinhardtii and mammals,
suggesting that the selenoproteins evolved early and
were independently lost from higher plants and some
animals [48]. Recently, an analysis of sequences
derived from several marine organisms supports the
hypothesis that SeCys utilization has been lost by
many groups of organisms during evolution [49].
S. Herbette et al. Selenium-independent glutathione peroxidases
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2167
In mammals, NS-GPXs and SeCys-containing GPXs,
both originating from a unique gene ancestor as dis-
cussed above, are expressed. The GPX5 protein, a
NS-GPX, shows the closest similarities to GPX3, a
SeCys-containing protein derived from the GPX1 gene,

which also encodes a selenoprotein [28]. These authors
also showed that GPX1 is probably derived from the
GPX4 gene also encoding a selenoprotein. It seems
unlikely that GPX1, GPX3 and GPX4, but not GPX5,
have independently acquired the SeCys, as incorpor-
ation of this amino acid requires a rather complicated
system. It appears more probable that GPX5 originated
from a gene that encoded a SeCys, which later lost the
SeCys residue. In the fish D. rerio, the GPX family
comprises four selenoproteins: two GPX1 and two
GPX4. These proteins are phylogenetically related to
mammalian GPX1 and GPX4 [8]. It appears most unli-
kely that the GPX1 and GPX4 genes independently
acquired SeCys in fishes and mammals. This observa-
tion supports the idea that the mammalian GPX5 was
first a selenoprotein, which evolved after the separation
of fishes and mammals, by the replacement of SeCys by
Cys. Another example is GPX6, which is a NS-GPX in
rodents but a selenoprotein in other mammals [50].
Like GPX5, GPX6 shows the greatest similarities to
the SeCys-containing GPX3 protein.
Taking into account these observations, we propose
that SeCys has been lost during evolution in some
GPXs in mammals and many eukaryotes. One may
ask why this is so, when this residue greatly increases
potential GPX activity, and what could be the function
of NS-GPXs.
Enzymatic activities of NS-GPXs
Do NS-GPXs display GPX activity?
Global GPX activities have been detected in crude

extracts from several higher plants [51–53], P. falci-
parum [54] and yeast [55]. However, this total GPX
activity also takes into account the activity of conta-
minating glutathione S-transferases or peroxiredoxins
which can metabolize GPX substrates [56]. In addition,
PHGPX activity has to be distinguished from GPX
activity. The latter is more active in reducing H
2
O
2
and various organic hydroperoxides such as t-butyl
hydroperoxide, and the former is more efficient at
reducing phospholipid and lipid hydroperoxides. In
most studies, both activities were measured by enzy-
matic characterization of NS-GPXs, although the
authors related only the PHGPX activity.
A partly purified GPX from Citrus sinensis has been
shown to display PHGPX activity as low as the activ-
ity of a mammalian GPX in which SeCys was replaced
by Cys [35]. We have shown that two recombinant
plant NS-GPXs, expressed in E. coli and purified by
affinity chromatography, also have low PHGPX activ-
ity similar to that of the citrus GPX [57]. These
complementary approaches demonstrate that plant
NS-GPXs have a rather low PHGPX activity. Similar
results have also been found with two recombinant
NS-GPX proteins from T. cruzi produced in E. coli
[21,58]. With the use of the same experimental design,
i.e. tagged protein production in E. coli and affinity
purification, other investigations showed that a recom-

binant NS-GPX from C. reinhardtii express PHGPX
activity that is 36 times higher than those from land
plants [59]. In addition, PHGPX activity of the three
yeast NS-GPXs has been investigated by mutant ana-
lyses and biochemical characterization showing that at
least one of these yeast NS-GPXs is a major PHGPX
enzyme [20,60]. Nevertheless, the specific activities of
yeast, algal and plant NS-GPXs remain remarkably
low compared with the specific activity of mammalian
PHGPX. This observation raised the question of the
physiological importance of their activity in detoxify-
ing hydroperoxides.
The absence of SeCys from the catalytic site is not
the only reason to question the capacity of NS-GPXs
to behave in vivo as expected for a GPX enzyme. Bri-
gelius-Flohe
´
et al. [29] suggested that PHGPX may be
misnamed, as all residues invoked to bind glutathione
are mutated or deleted in PHGPX and PHGPX-like
proteins. Compared with GPX1, PHGPX ⁄ GPX4 has a
lower affinity for glutathione and its activity is also
lower by more than one order of magnitude in most
tissues [61,62]. We have shown that two plant
NS-GPXs exhibit weak affinity for glutathione because
their apparent K
m
values for glutathione correspond to
supra-physiological concentrations of glutathione [57].
P. falciparum NS-GPX also showed weak affinity for

glutathione [63]. Other NS-GPXs distinct from the
PHGPX group, such as mammalian GPX5 and the
NS-GPX from B. Pahangi, also lack four of the five
amino acids involved in the binding of glutathione
[64]. The GPX from B. Pahangi has been shown to
exhibit a low affinity for glutathione [64] together with
low GPX activity [30,64].
Some NS-GPXs can use thioredoxin as reducing
substrate
The question of an alternative reducing substrate to
glutathione was first addressed for mammalian seleno-
GPX. Human GPX3 is secreted into the plasma,
although the plasma glutathione concentration is very
Selenium-independent glutathione peroxidases S. Herbette et al.
2168 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
low (< 0.5 lm [65]), suggesting that the function of
GPX3 might be completely dependent on other elec-
tron donors. Thioredoxin and glutaredoxin have been
shown to be efficient electron donors for human GPX3
[66]. Thioredoxins are small ubiquitous proteins with a
redox-active dithiol ⁄ disulfide in their active site.
Reduced thioredoxin operates together with thioredox-
in reductase and NADPH as a general protein disul-
fide-reducing system [67]. Glutaredoxins have similar
conformation and function to thioredoxins, but they
constitute a distinct protein family as they show no
sequence similarity to thioredoxin and they are
reduced by glutathione [68]. In contrast with GPX1
and GPX2, the mammalian GPX4 can use various
thiol-containing reducing substrates such as cysteine,

dihydrolipoamide and dihydrolipoic acid in addition to
glutathione [6]. Moreover, a thiol oxidase activity of
GPX4 has been demonstrated on different proteins
[69].
The weak affinity of NS-GPX for glutathione has
led many authors to reconsider their reducing sub-
strate. A NS-GPX from P. falciparum was the first
GPX shown to use thioredoxin to reduce H
2
O
2
or
organic hydroperoxides, leading the authors to reclas-
sify it as a thioredoxin peroxidase (TPx) [63]. We, and
others, have demonstrated that NS-GPXs from land
plants, yeast and Drosophila can also exhibit TPx
activity [37,57,70–73]. For these proteins, a strong
affinity for thioredoxin was revealed, compatible with
in vivo concentrations. The Cys residues involved in
enzymatic activity were identified [70–72] and an enzy-
matic mechanism proposed (Fig. 3). The Cys forming
the catalytic triad of GPX is oxidized by H
2
O
2
or by
an organic hydroperoxide, and, when oxidized, it
reacts with another Cys from the well-conserved GPX
PCNQF motif (Figs 1 and 3). The resulting disulfide
bridge is then reduced by thioredoxin. Supporting this

mechanism, Cys-mediated interactions between endog-
enous plant thioredoxin and NS-GPX were observed
in vivo in two distinct studies [74,75]. Moreover, the
redox state of the yeast NS-GPX in vivo was found to
be linked to that of endogenous thioredoxin [71].
It thus seems that NS-GPXs may act similarly to
some peroxiredoxins, especially peroxiredoxin of the Q
and II types [76]. Conversely, some peroxiredoxins,
known for their TPx activity, can use glutathione to
reduce hydroperoxides [56,77]. This link between GPX
and peroxiredoxin can be explained by common struc-
tural features. Both enzymes belong to the thioredoxin
fold superfamily [78]. This superfamily also comprises
glutathione S-transferase, thioredoxin, glutaredoxin
and DsbA proteins generating disulfide bridges. All
these proteins possess the CxxC motif, in which two
Cys residues are separated by two other amino acids,
or a derivative of this motif: CxxS, SxxC, CxxT or
TxxC [79]. They also possess similar secondary struc-
tures with a-helices and b-sheets. For NS-GPXs, the
Cys residue identified in the CxxT motif is one
involved in both GPX and TPx activity. We have
shown that plant NS-GPXs reduce phospholipid
hydroperoxides using thioredoxin [57], whereas peroxi-
redoxin exhibits weak activity towards these substrates
[80]. The PHGPX structure allows access to these
hydrophobic compounds. Therefore, GPXs and peroxi-
redoxin appear complementary to reduce various
peroxides using thioredoxin as a common electron
donor. In agreement with this statement, the functional

relationship between GPX and peroxiredoxin had
previously been suggested by Rouhier & Jacquot [81].
TPx activity is, however, not a characteristic of all
NS-GPXs, as the NS-GPX from B. pahangi has GPX
activity but not TPx activity [64], in contrast with that
documented for its GPX3 human homologue [66]. In
addition, reducing substrates other than thioredoxin
have been found for some NS-GPXs. Two NS-GPX
proteins from Synechocystis PCC 6803 are NADPH-
dependent peroxidases, but they have no GPX activity
[23]. It has been shown that a NS-GPX from S. cere-
visiae reduces H
2
O
2
using the transcription factor
YAP1 as a reducing substrate [82]. In vivo interac-
tion between the NS-GPX and YAP1 has also been
demonstrated.
These recent results help to clarify the enzymatic
function of NS-GPXs. On the one hand, they demon-
strate that NS-GPXs have antioxidant activities of
Fig. 3. Peroxide-reduction mechanism of NS-GPXs. The mechanism
of H
2
O
2
reduction and GPX regeneration by thioredoxin is proposed
for the NS-GPX from Brassica napus (GenBank accession number
AF411209). The position of the catalytic cysteines is given accord-

ing to the results of Jung et al. [70]. The protein is represented by
pins with the N-terminus as a knob.
S. Herbette et al. Selenium-independent glutathione peroxidases
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2169
physiological relevance. On the other, they suggest that
NS-GPXs may be involved in protein thiol–disulfide
exchanges. The next challenge will probably be identifi-
cation of the reducing substrates of NS-GPXs, i.e. the
proteins targeted in thiol–disulfide exchanges. This will
not be a simple task because the reducing substrate is
expected to be specific to the organism investigated, as
underlined above, and to the NS-GPX isoforms in the
same organism. In addition, the in vivo reducing and
peroxide substrates may depend on cellular localiza-
tion, as it has been shown that the different NS-GPX
isoforms are expressed in different cell compartments
[38,83,84]. Therefore, one cannot rule out the idea that
several physiological reducing substrates are used by
the same NS-GPX. We have recently shown in tomato
stem that a NS-GPX was localized in the cytoplasm or
apoplast depending on the cell type [84]. In the apo-
plast, it is likely that the NS-GPX uses a reducing sub-
strate distinct from thioredoxin or glutathione to
reduce peroxides, as these thiols are either not
expressed or are expressed at very low level. In support
of this, a set of disulfide bond (Dsb) proteins belong-
ing to the thioredoxin fold superfamily has been char-
acterized in the periplasm of prokaryotes [85]. These
proteins could be potential reducing substrates for
NS-GPX, and other extracellular reducing substrates

can be expected in eukaryotes. Alternatively, the
NS-GPX could have different functions in these differ-
ent localizations.
NS-GPXs – more than antioxidant
enzymes
Protection against oxidative damage
Several authors proposed that NS-GPXs could be
involved in protecting the cell from oxidative damage
by scavenging peroxides. This function has been inves-
tigated in several organisms. In C. sinensis cells, salt-
induced gene expression of a NS-GPX has been shown
to depend on AOS accumulation, and oxidative stress
is sufficient to induce gene expression [86]. Various
AOS have been shown to be able to up-regulate gene
expression of several NS-GPXs [87–89]. Overexpres-
sion of GPX5 in mammal cells also rendered them
more tolerant to oxidative stress [90]. In addition, it
has been proposed that an increase in the expression
level of this mammalian NS-GPX would compensate
for a decrease in expression of seleno-GPX isoforms
in mice fed a selenium-deficient diet, in order to
cope with failing seleno-dependent GPX activity [91].
Transgenic tobacco plants overexpressing a NS-GPX
from Chlamydomonas were also found to be more
tolerant to oxidative stress generated by paraquat [92].
Similarly, the expression of a tomato NS-GPX in
S. cerevisiae prevented H
2
O
2

-induced cell death [93].
Conversely, a streptococcus strain mutated for a
NS-GPX was more sensitive to oxidative treatments
than the wild-type strain [94].
Considering their homology with the mammalian
membrane-bound GPX4, most NS-GPXs have been
proposed to be involved in reducing membrane peroxi-
dation. We and others have recently demonstrated that
NS-GPXs from various organisms efficiently reduced
lipid peroxides as well as a broad range of peroxide
in vitro [23,57,59,60,70], but the question of the in vivo
substrate of NS-GPX has rarely been addressed.
Recently, the ability of the two NS-GPXs from
Synechocystis to scavenge lipid hydroperoxides in vivo
has been examined [95]. It has been shown that the
GPX knock-out mutants have a lower fatty acid
hydroperoxidase activity and a higher concentration of
lipid hydroperoxides under normal conditions as well
as after oxidative treatment. In mammals, Utomo
et al. [96] demonstrated that a NS-GPX is essential to
avoid cell death after polyunsaturated fatty acid treat-
ment. These results clearly indicate that plant and ani-
mal NS-GPXs protect cells from oxidative injury at
the membrane level.
Phospholipid hydroperoxidase activity has been
clearly demonstrated in vivo for a yeast NS-GPX [97].
This function appeared to be linked to the PHGPX
structure, supporting the idea that most NS-GPXs
would fulfil the same physiological function, i.e. preser-
ving membrane integrity. In this report, the authors

added GPX1 sequences allowing multimerization into
a functional NS-GPX. They confirmed that the lack of
these sequences in the PHGPX protein is responsible
for the in vitro phospholipid hydroperoxidase activity
as well as for their in vivo role in the protection against
lipid peroxidation.
Signalling function
AOS and peroxides are not only considered to be toxic
molecules but they are also known to be key players in
signalling pathways of several physiological processes.
By regulating their accumulation, NS-GPXs like any
antioxidant enzyme would interfere with these signal-
ling pathways. For example, the mammalian GPX4
has been demonstrated to regulate the production of
leukotrienes [98] and prostaglandins [99], which are
key mediators of inflammation processes, as well as to
reduce the interleukin-1-dependent stimulation of
NF-jB [100]. Another example is the antiapoptotic
function of GPX4 in the mitochondrial death pathway
Selenium-independent glutathione peroxidases S. Herbette et al.
2170 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
[101]. These events depend on lipoxygenase activities,
which are inhibited by GPX4 [102–104]. Analogous
functions can be expected for NS-GPXs of the PHGPX
group.
A signalling function has been clearly demonstrated
for a S. cerevisiae NS-GPX. Delaunay et al. [82] repor-
ted that the NS-GPX called ScGPX3 functions as an
H
2

O
2
receptor and as a redox transducer for the tran-
scriptional activator YAP1. ScGPX3 interacts in vivo
with YAP1 and oxidizes two Cys residues using H
2
O
2
.
Oxidation of these residues leads to the nuclear accu-
mulation of YAP1 [105], which can activate transcrip-
tion of defence genes such as antioxidants [106]. In
contrast, reduction of the Cys residues of YAP1 by
thioredoxin leads to its inactivation by cytoplasmic
sequestration. This regulatory function of ScGPX3 has
been demonstrated to depend on its PHGPX structure,
especially the ‘gap sequences’ distinguishing mono-
meric PHGPX proteins from the multimeric GPXs
[97]. The phospholipid hydroperoxidase and the
YAP1-mediated signalling activities have been shown
to be independent. ScGPX3 was also recently shown
to interact in vivo through the formation of an inter-
molecular disulfide bond with a methionine sulfoxide
reductase [107]. This interaction, inhibiting the activity
of methionine sulfoxide reductase, was compromised
by treatment with H
2
O
2
, leading the authors to suggest

that ScGPX3 functions as a redox-dependent regulator
of enzyme activity.
Hence, ScGPX3 has at least two independent func-
tional roles: protection from membrane peroxidation
and signalling of oxidative stress.
A NS-GPX from A. thaliana has also been shown to
function as a redox transducer in response to drought
stress and abscisic acid [108]. The NS-GPX was shown
to interact physically with a 2C-type protein phospha-
tase from the abscisic acid signalling pathways and to
regulate its phosphatase activity. For this, NS-GPX
modulated the redox state of the protein phosphatase
using H
2
O
2
.
Regarding these recent data, we speculate that most
NS-GPXs could fulfil such signalling activities based
on thiol redox exchange with protein partners. As dis-
cussed above, such interactions between NS-GPXs
from various organisms and thioredoxins have been
shown in vivo and in vitro. In addition to their partici-
pation as electron donors in the fight against oxidative
stress, thioredoxins are involved in redox regulation of
several physiological processes. Therefore, it is plaus-
ible that NS-GPXs participate in the regulation of
these physiological processes by acting on thioredoxin-
mediated signalling pathways. In plants, several types
of thioredoxin exist and participate in seed germina-

tion, cell division, reproduction, cell communication
and photosynthesis [109,110]. In animals, thioredoxins
regulate the activity of very basic stress-response tran-
scription factors such as NF-jB and AP1 [111]. They
also fulfil a specific function in the inhibition of apop-
tosis, immunomodulation, and pregnancy [112]. In
prokaryotes, thioredoxins are necessary for DNA syn-
thesis or sulfate reduction and are required for the
assembly and export of invasive phages such as T7, f1
or M13 [112].
A PHGPX-like protein from the hymenopteran
endoparasitoid Venturia canescens has been shown to
lack the conserved Cys or SeCys catalytic residue
found in GPX [113]. Except for this residue, this pro-
tein displays all the conserved regions characteristic of
GPX proteins and shows high homology to the
NS-GPX from Drosophila melanogaster. This extracel-
lular NS-GPX is not an active enzyme but may retain
the capacity to interact with membrane lipids. Given
its high expression levels in the calyx lumen, the
authors proposed that this NS-GPX binds to oxidized
phospholipids on the membrane, thereby masking or
otherwise removing their potential immune-eliciting
properties. This study indicates the capacity of PHGPX
protein, i.e. most NS-GPXs, to function in a way other
than as a simple antioxidant.
We undertook a proteomic analysis of transgenic
tomato plants overexpressing a NS-GPX to determine
whether this overexpression would interfere with gene
expression [114]. The accumulation of two proteins

involved in the Calvin cycle and the signalling protein,
RanBP1, was found to be affected in NS-GPX-over-
expressing plants, suggesting that the NS-GPX interferes
with the photosynthetic process and the GTPase-medi-
ated signalling pathways. In addition, in the same
study, we showed that NS-GPX-overexpressing plants
exposed to chilling conditions had greater photosyn-
thetic activity because of greater activity of the
enzymes involved in this process. Similarly, Yoshimura
et al. [92] reported that transgenic tobacco plants
overexpressing a NS-GPX from Chlamydomonas had
higher photosynthetic activity in chilling conditions
than control plants.
Function in structural organization
A structural function has clearly been demonstrated
for the seleno-dependent GPX4 in mammals. GPX4
has been found to be expressed as an enzymatically
inactive, oxidatively cross-linked, insoluble protein in
mature spermatozoa [115]. It has been found to be
responsible for the polymerization of a sperm mito-
chondrion-associated cysteine-rich protein (SMPC), a
S. Herbette et al. Selenium-independent glutathione peroxidases
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2171
major component of the sperm mitochondrial capsule
[116,117]. During this polymerization process, GPX4
catalyzed the formation of cystine from adjacent
SMPC cysteine residues, followed by a reshuffling
[117]. Sperm cell GPX4 was also found to be associ-
ated with sperm nuclei where it promotes disulfide
bridging on thiol-containing protamines, allowing

increased compaction of the sperm nuclei [118].
Although, this was first demonstrated for a seleno-
GPX, a function in structural organization through
disulfide bridging can also be expected for some
NS-GPXs. As discussed above, several NS-GPXs were
able to oxidize thioredoxin through the formation of a
disulfide bridge from two adjacent cysteine residues
(Fig. 3). All these results argue for a function of
NS-GPXs in disulfide bridging. Protein targets of
NS-GPXs remain to be discovered to understand
clearly the role of NS-GPX in disulfide bridge-mediated
structural remodelling of cell structures.
Physiological processes for which NS-GPXs
have been proposed
Role in defence ⁄ response to adverse conditions
In many organisms, especially plants, NS-GPXs have
been shown to be involved in the response to environ-
mental stress. Numerous studies have reported that
various stress conditions alter the steady-state level of
mRNA encoding NS-GPXs in plant species, including
tobacco [16,119], Arabidopsis [120], tomato [18], sun-
flower [19], pea [83], citrus [86] and barley [121]. Tested
stress conditions included osmotic pressure, gentle
mechanical stimulation, wounding, salt and herbicide
treatments, and exposure to ozone, UV, sulfur dioxide,
heat and strong light. In some cases, the increase in
mRNA accumulation was confirmed by an increase in
accumulation of NS-GPX protein [17,84].
In a few reports, transgenic approaches have been
used to investigate the role of NS-GPXs in plant

response to environmental stress. Transgenic tobacco
plants overexpressing a Chlamydomonas NS-GPX were
found to be more tolerant to chilling and salt [92]. A
NS-GPX from Lycopersicon esculentum expressed in
S. cerevisiae cells has been shown to function as a
cytoprotector, preventing Bax-induced and heat stress-
induced cell death and delaying yeast senescence [93].
Transient expression of this NS-GPX in Nicotiana
tabaccum also produced tolerance to salt and chilling
and suppressed the apoptotic-like features associated
with these stress conditions. In addition, we have
shown that, after chilling, the photosynthetic activity
of transgenic tomato plants overexpressing a NS-GPX
was not affected, whereas it was decreased in control
plants [114]. A S. cerevisiae strain with mutations in
the three NS-GPXs was found to be more sensitive to
aluminium treatment than the wild-type or any single
NS-GPX mutant, indicating that the NS-GPX genes
may collectively contribute to tolerance to aluminium
[122]. Taken together, these results support the idea
that NS-GPXs are definitively involved in resistance to
various environmental stress conditions.
More precisely, the different NS-GPX isoforms are
likely to have different functions in the stress response,
as suggested by differences in gene regulation. In
L. esculentum, we observed that the highest transcript
level of the GPXle-1 isoform was observed 1–2 h after
rubbing of an internode, whereas a significant accumu-
lation of mRNA of the GPXle-2 isoform was observed
later, 2–6 h after stimulation [18]. In fact, the GPXle-2

transcript started to accumulate when the concentra-
tion of GPXle-1 mRNA was back to normal. More-
over, GPXle-1 mRNA also accumulated in the roots
of rubbed plants, whereas GPXle-2 mRNA did not,
underlying differences in terms of inducibility between
the two isoforms. Furthermore, we have shown that
messengers of two NS-GPXs from Helianthus annuus
accumulated differentially in response to various com-
ponents of stress signalling pathways [89]. In Hordeum
vulgare, the expression of two isoforms was shown to
be induced by salt and osmotic stress and by paraquat
treatment, whereas expression of a third isoform was
repressed in these conditions [121]. According to the
authors, these results could be explained by the differ-
ent subcellular localizations of the NS-GPXs. Simi-
larly, another report indicates that the genes of the
GPX family of A. thaliana were differently regulated
through diverse signalling pathways and that the pro-
teins would be localized in distinct cell compartments
[38]. A specific response was observed for a C. rein-
hardtii NS-GPX gene that was shown to be transcrip-
tionally up-regulated by the oxygen singulet O
1
2
produced in photosystem II, suggesting a special func-
tion for this GPX in protection against O
1
2
[88,123].
Taking into account these observations, we suggest

that the NS-GPX isoforms fulfil different functions,
especially in response to stress, and that this speci-
ficity is closely related to their regulation pathways
and ⁄ or their tissue restriction and⁄ or their subcellular
localization.
From the data collected to date, it seems that
NS-GPXs are involved in stress responses as well as in
specific functions in normal conditions. Although
NS-GPXs are known to be induced and expressed in
stress conditions, their expression has often been shown
to be constitutive, suggesting that they also have basal
functions in nonstress situations. Supporting this idea
Selenium-independent glutathione peroxidases S. Herbette et al.
2172 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
are our observation that a tomato NS-GPX was
expressed in the cytoplasm of collenchyma in rubbed
internode, whereas it was expressed in the cytoplasm
of the same cell type in an unstressed internode [84].
Function in sexual reproduction
In mammals, a NS-GPX (GPX5) was first discovered
in the male genital tract [124]. Recently, its expression
and putative functions in the epididymis and spermato-
zoa have been reviewed [125]. Its expression is tightly
controlled by androgen and is exclusively restricted to
epididymis [126]. The protein was found to be both
free in the epididymal fluid and associated with transi-
ting spermatozoa in the epididymal lumen [127–129].
GPX5 association with spermatozoa is limited to the
acrosome region [128]. This is another example of a
NS-GPX that is part of the antioxidant strategies in

the epididymis, more particularly involved in protec-
tion of spermatozoa from oxidative damage [130].
Spermatozoa are highly sensitive to oxidative attack
because of the high polyunsaturated fatty acid content
of their plasma membrane. Spermatozoa themselves
are known to produce AOS, which are involved in the
regulation of their ultimate maturation (i.e. capacita-
tion and acrosomic reaction). The secreted NS-GPX
(GPX5) plays a role in the fine tuning of AOS in the
sperm environment during epididymal maturation.
Sex-specific expression and specific tissue distri-
bution in sex organs have also been observed for
other NS-GPXs, suggesting a role in reproduction. A
NS-GPX from Sch. mansoni has been shown to be spe-
cifically expressed in vitteline cells of female worm and
absent in male worm tissues [131]. Its expression
requires a mature reproductive system and the protein
has therefore been proposed to be involved in the
formation of eggs. Conversely, the expression of a
NS-GPX from D. melanogaster was found to be
greater in male than in female flies, probably because
of its strong expression in testes [113]. In the male
reproductive tissues, the protein was detected in the
testicular duct whereas expression was low in other
tissues. Although lower in female flies, specific expres-
sion of the NS-GPX was also observed in the follicle
and nurse cells from ovaries [113].
Function in host–pathogen interactions
In plants and animal phagocytic cells, the generation
and release of AOS are thought to be important com-

ponents of the host’s immunity against bacterial infec-
tions. Pathogens have developed effective systems to
counteract the resulting oxidative stress (for review, see
[132]). For example, mutant bacteria defective in resist-
ance to oxidative stress have been shown to be aviru-
lent [133]. Neisseria meningitidis and Stretptococcus
pyogenes mutant strains defective in a NS-GPX were
more sensitive to oxidative stress than the respective
wild-type strains, suggesting that NS-GPXs are import-
ant components of the bacterial antioxidant system
[94]. The contribution of NS-GPXs to bacterial
virulence has been investigated in a St. pyogenes
mutant strain defective in the unique NS-GPX [134].
In this study, it was demonstrated that the NS-GPX
was essential for bacterial pathogenesis in several
murine models of streptococcal diseases. However, the
NS-GPX was not necessary for the St. pyogenes viru-
lence in a zebrafish model of streptococcal disease,
characterized by the absence of inflammatory process.
Taken together, these observations suggest that bacter-
ial pathogenesis requires NS-GPX for defence against
the oxidative stress that accompanies the inflammatory
response of the host. Furthermore, in B. pahangi,a
lymphatic nematode parasite, a NS-GPX was found to
be the major cuticular glycoprotein [22]. This NS-GPX
was expressed at low level in mosquito-derived infec-
tion larvae, whereas after infection of the mammalian
host, its expression was up-regulated and the protein
was secreted in the parasite cuticle. Once again, the
NS-GPX may protect the parasite against the AOS

produced by the host phagocytes.
Similarly, a plant nematode parasite expressed a
secreted form of NS-GPX in addition to an intracellu-
lar isoform [31]. This secreted isoform showed anti-
oxidant activity restricted to the hypodermis, which
may protect the parasite from host defences.
The involvement of NS-GPX in the mechanism of
host defence has also been investigated, especially in
plants. One of the earliest responses to avirulent
pathogen attack is the generation of an oxidative burst
that can trigger cell death, also called the hypersensi-
tive response. This is thought to deprive the pathogens
of food and confine them to the infection site. We
have demonstrated that two NS-GPXs from H. annuus
were differently regulated in leaves infected with either
a virulent or an avirulent race of the obligate parasite
Plasmopara halstedii, suggesting that NS-GPX gene
expression level is important for establishment of the
hypersensitive response developed to limit spreading of
the avirulent pathogen [89]. Both NS-GPXs were
up-regulated during the compatible interaction with
the virulent race, whereas they were down-regulated
during the incompatible interaction with the avirulent
strain. Similar results have been obtained with a rice
NS-GPX after infection with the blast pathogen [135].
Interestingly, modification of the expression of the
S. Herbette et al. Selenium-independent glutathione peroxidases
FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS 2173
sunflower NS-GPX is temporally correlated with the
Plasmopara-induced symptoms in plants for both com-

patible and incompatible interactions [89,136].
A decrease in gene expression during an incompat-
ible interaction has also been observed for an ascor-
bate peroxidase, another antioxidant enzyme [137].
This down-regulation of antioxidant genes favours
AOS accumulation, which is necessary to promote the
hypersensitive response. More precisely, down-regula-
tion of NS-GPXs favours the lipoxygenase-mediated
accumulation of lipid hydroperoxides, an enzymatic
peroxidative process that is a major feature of the
hypersensitive response [138]. In contrast, up-regula-
tion of NS-GPX expression would help in fighting
against oxidative damage generated during the disease
progression. Our hypothesis about the function of
NS-GPXs in the hypersensitive response could be tes-
ted quite easily in transgenic plants known to overex-
press a NS-GPX. In agreement with such behaviour
are the data of Yoshimura et al. [92], who have
shown that overexpression of a tomato NS-GPX in
tobacco conferred protection against the phytopatho-
gen Botrytis cinerea. Although this result seems to be
contradictory to our hypothesis, it actually completes
it because this fungus is a necrotrophic pathogen
which utilizes dead plant tissue originating from an
induced oxidative burst [139]. For this special host–
pathogen interaction, we propose that overexpression
of NS-GPXs could reduce AOS or lipid peroxides,
limiting host cell death and hence the propagation of
the pathogen.
Thus, in our opinion, the expression level of the

NS-GPX gene of the host is differently adjusted
depending on the pathosystem, to help mount the
appropriate defence response. Further studies of
NS-GPX gene expression (i.e. level and subcellular
localization of proteins) in the host as well as in the
pathogen would help to clarify NS-GPX function in
the interaction.
Conclusions
Although seleno-GPXs appear to be an efficient means
of AOS scavenging, especially because of the presence
of the SeCys residue, NS-GPXs are prevalent in the
living world, and some of them probably evolved from
seleno-GPXs. The weak GPX ⁄ PHGPX activity found
for most of the NS-GPXs and the fact that they
use substrates other than glutathione, especially thio-
redoxin, led us to conclude that NS-GPXs are not true
GPXs and hence are misnamed. Thioredoxin is prob-
ably not the only potential physiological substrate, as
demonstrated for a yeast NS-GPX which oxidizes
in vivo a transcriptional factor. Hence, these proteins
could be included in a thiol peroxidase (TPx) family.
A reclassification of the NS-GPXs away from the
GPX family has also been proposed by other authors
[81,140]. In support of this reclassification, NS-GPXs
with the mammalian PHGPX form a separate clade
from the other seleno-GPXs (Fig. 2).
We can speculate that NS-GPX ⁄ TPxs, using a pro-
tein as reducing substrate, do not compete with effi-
cient AOS-scavenging enzymes such as catalase and
seleno-GPXs in animals and ascorbate peroxidase in

plants. The concentration of the NS-GPX ⁄ TPx-redu-
cing substrate is lower than the concentrations of
ascorbate and glutathione, which accumulate in the
millimolar range, and this is probably not enough
to scavenge AOS during oxidative stress. Instead,
NS-GPX ⁄ TPx would use AOS for protein disulfide
exchange in signal-transduction pathways as proposed
for yeast and Arabidopsis NS-GPX ⁄ TPx [82,108] or in
structural organization processes. This is in line with
the re-evaluation of the concept of oxidative stress as
an ‘oxidative signalling’ proposed by Foyer & Noctor
[141], that is, a process by which cells sense the envi-
ronment and make appropriate adjustments to gene
expression, metabolism and physiology. By such sig-
nalling functions, NS-GPX ⁄ TPx would be involved in
defence ⁄ response to adverse conditions, in protection
against AOS induced by adverse conditions, and in
host–pathogen interactions. In most organisms, several
genes encode NS-GPX ⁄ TPx (there are, for example,
seven NS-GPX-encoding genes in A. thaliana), and
they can be expressed in the same cell. The idea that
all these peroxidases might simply be backup systems
of other and more efficient antioxidant systems can be
questioned. More likely, they have evolved to play
highly specialized parts in cellular processes. This view
is also based on the emerging knowledge on the speci-
fic roles of the mammalian seleno-GPX4 [142]. To
clarify the precise role of individual NS-GPXs, in vivo
analyses will have to be performed, especially with
regard to their tissue and cell localization. Phenotypic

and functional analyses of knockout organisms must
also be conducted to determine the true roles of these
proteins. Further challenges will be to identify the thiol
protein substrates of the NS-GPXs. The identification
of the YAP1 transcription factor and methionine sulf-
oxide reductase as thiol protein partners in yeast
[82,107] suggests that many other protein partners in
addition to thioredoxin can be expected in various
organisms. In plants, several NS-GPX isoforms are
expressed, and their respective protein partners remain
unknown as well as the components of any signal-
transduction pathways based on disulfide exchange.
Selenium-independent glutathione peroxidases S. Herbette et al.
2174 FEBS Journal 274 (2007) 2163–2180 ª 2007 The Authors Journal compilation ª 2007 FEBS
Such investigations will not only help in understanding
NS-GPX function but will also provide new insights
into the role of AOS in cell physiology.
Acknowledgements
We are indebted to Felicity Vear for correction of
English grammar and syntax.
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