Biochemical studies have shown that NS-GPXs are not true GPXs; notably they reduce AOS using reducing substrates other than glutathione, such as thioredoxin.. A third group, the PHGPX gr
Trang 1Seleno-independent glutathione peroxidases
More than simple antioxidant scavengers
Ste´phane Herbette1, Patricia Roeckel-Drevet1and Joe¨l R Drevet2
Introduction
Glutathione peroxidase (GPX; EC 1.11.1.9) catalyses
the reduction of H2O2 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: joel.drevet@University-bpclermont.fr
(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.
Trang 2signal 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 (pKa¼ 5.2), whereas the thiol group of Cys is only partially ionized under similar conditions (pKa¼ 8) Once ionized, the thiol or selenol group is able to react with H2O2 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
Literature denomination
GI-GPX (gastro-intestinal)
mitochondria, membrane-bound
Trang 3Phylogenetic 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
protein sequence accession numbers
(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.
Trang 4between 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 sequpres-ence 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.
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.
Trang 5biochemical 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, evoluconcentra-tionary 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]
Trang 6In 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 H2O2
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 Kmvalues 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
Trang 7low (< 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 protethioredox-in
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 H2O2 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 H2O2 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 H2O2 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
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.
Trang 8physiological 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 H2O2-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
Trang 9[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
H2O2receptor and as a redox transducer for the
tran-scriptional activator YAP1 ScGPX3 interacts in vivo
with YAP1 and oxidizes two Cys residues using H2O2
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 multimono-meric 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 H2O2, 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 H2O2
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, reproducgermina-tion, 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
Trang 10major 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 O1
produced in photosystem II, suggesting a special func-tion for this GPX in protecfunc-tion against O1 [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