peroxisomes and mitochondria, respectively, and areinvolved in lipid metabolism and respiration in Caenorhabditis elegans Elise Petit1,2,3, Xavier Michelet4,5, Claudine Rauch1,2,3, Justi
Trang 1peroxisomes and mitochondria, respectively, and are
involved in lipid metabolism and respiration in
Caenorhabditis elegans
Elise Petit1,2,3, Xavier Michelet4,5, Claudine Rauch1,2,3, Justine Bertrand-Michel6, Franc¸ois Terce´6,7,8, Renaud Legouis4,5and Fabrice Morel1,2,3
1 Inserm U620, Universite´ de Rennes 1, France
2 EA-MDC, Universite´ de Rennes 1, France
3 IFR 140, Rennes, France
4 CNRS Centre de Ge´ne´tique Mole´culaire – UPR2167, Gif-sur-Yvette, France
5 Universite´ Paris-Sud Orsay, Universite´ Paris-6, France
6 IFR150, Institut Fe´de´ratif de Recherche Bio-Me´dicale de Toulouse, Plateau technique de Lipidomique, France
7 INSERM, U563, Toulouse, France
8 Universite´ Toulouse III Paul Sabatier, De´partement Lipoprote´ines et Me´diateurs Lipidiques, IFR150, France
Keywords
Caenorhabditis elegans; fatty acids;
glutathione transferase kappa; mitochondria;
peroxisomes
Correspondence
F Morel, INSERM U522 ⁄ EA MDC, Hoˆpital
Pontchaillou, 35033 Rennes, France
Fax: +33 299540137
Tel: +33 299543737
E-mail: fabrice.morel@inserm.fr
R Legouis, Centre de Ge´ne´tique
Mole´culaire – UPR2167 CNRS Baˆtiment 26,
Avenue de la terrasse, 91198 Gif-sur-Yvette
Cedex, France
Tel: +33 169824374
Fax: +33 169824386
E-mail: legouis@cgm.cnrs-gif.fr
(Received 28 May 2009, revised 3 July
2009, accepted 7 July 2009)
doi:10.1111/j.1742-4658.2009.07200.x
To elucidate the function of kappa class glutathione transferases (GSTs) in multicellular organisms, their expression and silencing were investigated in Caenorhabditis elegans In contrast with most vertebrates, which possess only one GST kappa gene, two distinct genes encoding GSTK-1 and GSTK-2 are present in the C elegans genome The amino acid sequences
of GSTK-1 and GSTK-2 share around 30% similarity with the human hGSTK1 sequence and, like the human transferase, GSTK-1 contains a C-terminal peroxisomal targeting sequence gstk-1 and gstk-2 genes show distinct developmental and tissue expression patterns We show that GSTK-2 is localized in the mitochondria and expressed mainly in the phar-ynx, muscles and epidermis, whereas GSTK-1 is restricted to peroxisomes and expressed in the intestine, body wall muscles and epidermis In order
to determine the potential role(s) of GST kappa genes in C elegans, specific silencing of the gstk-1 and gstk-2 genes was performed by an RNA inter-ference approach Knockdown of gstk-1 or gstk-2 had no apparent effect
on C elegans reproduction, development, locomotion or lifespan By con-trast, when biological functions (oxygen consumption and lipid meta-bolism) related to peroxisomes and⁄ or mitochondria were investigated, we observed a significant decrease in respiration rate and a lower concentra-tion of the monounsaturated fatty acid cis-vaccenic acid (18:1x7) when worms were fed on bacteria expressing RNA interference targeting both gstk-1and gstk-2 These results demonstrate that GST kappa, although not essential for the worm’s life, may be involved in energetic and lipid meta-bolism, two functions related to mitochondria and peroxisomes
Abbreviations
Dsba, protein disulfide isomerase A; FAME, fatty acid methyl ester; GFP, green fluorescent protein; GST, glutathione transferase; PTS1, peroxisomal targeting signal 1; RNAi, RNA interference.
Trang 2Glutathione transferase (GST) kappa is a 26.5 kDa
protein that was initially isolated from the rat liver
mitochondrial matrix and classified as a class theta
GST [1] The determination of the three-dimensional
structure of the class kappa enzyme from rat
(rGSTK1-1), complexed with glutathione (GSH),
showed a different folding topology from that of the
other GST classes, and revealed that the enzyme shows
similarity with the protein disulfide bond isomerase,
DsbA, from Escherichia coli and a bacterial
2-hydroxy-chromene-2-carboxylate isomerase, an enzyme involved
in the naphthalene degradation pathway [2,3]
Although class kappa GST showed an activity towards
aryl halides, such as 1-chloro-2,4-dinitrobenzene, and
can reduce cumene hydroperoxide and
(S)-15-hydro-peroxy-5,8,11,13-eicosatetraenoic acid [4], this activity
remained quite low when compared with that of other
soluble GSTs Interestingly, a recent study has shown
that GST kappa might also possess a function
inde-pendent of its glutathione conjugation activity in
adi-pose tissue [5] Indeed, Liu et al [5] have identified
GSTK1 as a key regulator for the multimerization of
adiponectin, which is an adipocyte-derived hormone,
in both human and rodent
Tissue distribution, analysed by RT-qPCR, showed
that the hGSTK1 gene is expressed in the 24 different
human tissues examined [4] In the mouse, the
mGSTK1 protein is present in large amounts in the
liver, kidney, stomach and heart, and its association
with liver and kidney mitochondria has been
demon-strated by electron microscopy [6] GSTK1 transcript
tissue expression is similar in the rat and in the mouse
[7] With regard to subcellular localization, in contrast
with soluble GSTs, which are mainly present in the
cytosol, GST kappa is localized in peroxisomes and
mitochondria [4] Although the process of GST kappa
targeting to mitochondria is unclear, it has been
reported to associate with the Hsp60 chaperone [3],
and a possible cleavage site for a mitochondrial
pre-sequence exists at the N-terminus A peroxisomal
tar-geting sequence (tripeptide ARL) has been identified in
the C-terminus of the hGSTK1 subunit [4]
The recent demonstration of GST kappa as a
regu-lator of protein multimerization and its particular
sub-cellular location have led to questions about its further
role(s) and substrate(s) [5] Common peroxisomal and
mitochondrial functions are related to lipid
metabo-lism, including a- and b-oxidation of fatty acids that
generate acetyl-CoA and different acyl-CoA
intermedi-ates [8,9] Thus, the presence of GSTK1 in both
organ-elles suggests that it may be specifically involved in the
b-oxidation of fatty acids, either through its catalytic activity, a certain transport function or interaction with other proteins Interestingly, its role in adipo-nectin regulation is also related to lipid and glucose metabolism
The nematode Caenorhabditis elegans is a genetically well-characterized model organism [10] which presents several advantages: (a) small size; (b) rapid reproduc-tion as a self-fertile hermaphrodite; (c) large number of offspring (250–300 progeny); (d) growth on a solid sur-face medium; and (e) transparent body allowing the observation of cells in mature and developing animals Furthermore, as about 60% of C elegans genes show similarity to human genes, and transient RNA interfer-ence (RNAi) allows specific gene silencing, this model organism represents a powerful tool for gene function analysis
The aim of our study was to characterize GST kappa gene(s) and proteins in C elegans and, by means of RNAi, to investigate the effects of gene silencing on the nematode phenotype Our results showed that the C elegans genome contains two GST kappa genes encoding GSTK-1 and GSTK-2, which localize in peroxisomes and mitochondria, respectively Double inactivation by RNAi affects the worm’s metabolism through a reduction in its rate of respiration and modification of its lipid content
Results
The C elegans genome contains two GST kappa genes
We have previously described a C elegans protein showing 33% homology with the human GST kappa, hGSTK1, amino acid sequence [4] Database analyses revealed the presence of two genes previously named ZK1320.1 and D2024.7 in the C elegans genome These two genes are located on chromosomes II (ZK1320.1) and IV (D2024.7) and have probably arisen by gene duplication Both genes are composed
of three exons and two introns (Fig 1A), the nucleo-tide sequence at the splice junctions is consistent with the canonical GT–AG rule and the corresponding encoded amino acid sequences comprise 226 and 225 residues, respectively, and share 32% identity Ortho-logues of these genes are observed in other nematode species, including C briggsae, C remanei, C japonica, Ancylostoma ceylanicum, Heterorhabditis bacteriophora and Meloidogyne Three arguments strongly suggest
Trang 3that C elegans ZK1320.1 and D2024.7 genes belong to
the kappa class of GSTs and share a common
ances-tral gene with rat GSTK1 Firstly, there are conserved
amino acids in the protein sequences of rat GSTK1
and C elegans ZK1320.1 and D2024.7 Secondly, we
showed common exon–exon junctions in the translated
amino acid sequences of the two C elegans genes
and the rat GST kappa gene (Fig 1B) Finally, using
the psortii program (http://psort.ims.u-tokyo.ac.jp),
the presence of a C-terminal peroxisomal targeting
signal 1 (PTS1), composed of the three amino acids
serine-lysine-leucine (SKL), was demonstrated in some,
but not all, nematode species and rat GST kappa
(tripeptide ARL) amino acid sequences (Fig 1C)
Furthermore, psortii also predicted a mitochondrial
presequence with a putative cleavage site after residue
5 (MPNRK⁄ VV) at the N-terminus of the GSTK-2
sequence For these reasons, ZK1320.1 and D2024.7
genes were renamed gstk-1 and gstk-2
GSTK-1 and GSTK-2 are localized in peroxisomes and mitochondria, respectively
The analysis of GST kappa transcript levels has shown
a ubiquitous expression in human [4] and mouse [6] tissues In order to determine the spatial and temporal expression patterns of gstk-1 and gstk-2 genes in
C elegans, we constructed gfp::gstk-1 and gstk-2::gfp fusions under the control of approximately 1 kb of the 5¢ regulatory gstk-1 and gstk-2 sequences, respectively
In these reporter fusion proteins, green fluorescent protein (GFP) was inserted in frame immediately upstream or downstream of gstk-1 and gstk-2 sequences, respectively Transgenic strains were obtained by microinjection, and the localization of fusion proteins in animals was revealed by the exami-nation of GFP fluorescence (Fig 2)
The expression of the gfp::gstk-1 transgene was first detected in 100 cell embryos, as shown in Fig 2A
gstk-1 (zk1320.1) 1 2 3
gstk-2 (d2024.7)
rGSTK1
GSTK-1
GSTK-2
A
B
C
Fig 1 Genomic structure and intron posi-tions in C elegans gst kappa genes (A) Genomic structure of hGSTK1 Exons are represented as black boxes and introns are represented by lines; the numbers indicate the size in nucleotides The gene structure is drawn to scale (B) Intron positions in rat and C elegans amino acid sequences Filled and open triangles mark common and unique intron positions, respectively (C) Amino acid sequence alignment of rat GSTK1 with GST kappa of nematodes The aligned sequences are listed below, followed by the species’ names and accession numbers in parenthe-ses rGSTK1 (Rattus norvegicus, UniProt: P24473), CelGSTK-1 (Caenorhabditis ele-gans, UniProt: Q09652), CbrGSTK-1 (Caenor-habditis briggsae, UniProt: A8XB52), CelGSTK-2 (Caenorhabditis elegans, UniProt: Q18973), CbrGSTK-2 (Caenorhabditis brigg-sae, UniProt: A8X1K2), CreGSTK-2 (Caenor-habditis remanei, WormBase: RP16274), CjaGSTK-1 (Caenorhabditis japonica, WormBase: JA07681), AcGSTK (Ancylos-toma ceylanicum, GenBank: CB175111.1), MhGSTK (Meloidogyne hapla, GenBank: EX007447.1), HbGSTK (Heterorhabditis bacteriophora, GenBank: BM883827.1).
*Residues involved in glutathione binding site.#Residues involved in dimer interface.
Trang 4Expression was increased during morphogenesis of the
embryo Fluorescence was observed as strong punctate
staining in intestinal cells and in the epidermis The
number and intensity of fluorescent structures
increased strongly in the intestine during larval
develop-ment (Fig 2D), whereas the epidermal fluorescent
punctata weakened (Fig 2B) In addition, a diffuse
localization of GFP::GSTK-1 was observed in the
body wall muscles (Fig 2C) and in the rectal gland
cells in larvae and adults (Fig 2D) The presence of a
peroxisomal targeting signal in its C-terminus (Fig 1)
and the punctate localization pattern suggest that
GSTK-1 is a peroxisomal protein To confirm the
per-oxisomal localization, we masked PTS1 by fusing the
GFP at the C-terminus of GSTK-1 Transgenic worms
for GSTK-1::GFP only presented diffuse staining (data
not shown), further supporting a peroxisomal
localiza-tion of GSTK-1
In the GSTK-2::GFP transgenic strain, fluorescence
was first detected during the second half of
embryo-genesis (Fig 2E) A strong signal was detected as
punctate staining in the pharynx (Fig 2G) and the
body wall muscles (Fig 2F), and a weaker signal was
also observed in the intestine In muscle cells, the strong GSTK-2::GFP punctata were part of a tubular network which was weakly fluorescent (Fig 2H) Inter-estingly, similar staining has been observed previously
by the expression of GFP fused to specific subcellular targeting sequences [11], strongly suggesting the presence of GSTK-2 in the mitochondria To confirm this mitochondrial localization, we stained both GFP::GSTK-1- and GSTK-2::GFP-expressing worms with MitoTracker Red (Fig 3) Although GFP:: GSTK-1 did not show any colocalization with Mito-Tracker Red staining (Fig 3A¢¢), GSTK-2::GFP fully colocalized with the mitochondrial dye (Fig 3B¢¢) Together, these data indicate that GSTK-1 and GSTK-2 have a peroxisomal and mitochondrial locali-zation, respectively
Impairment of oxygen consumption and lipid content in gstk-1 and gstk-2 double-knockdown worms
Post-transcriptional gene silencing of specific genes by RNAi is a well-established method in C elegans [12]
A
B
C
G F
Fig 2 Analysis of expression pattern of gstk-1 and gstk-2 in embryo and adult C elegans Projection of confocal images of GFP::GSTK-1 (A–D) and GSTK-2::GFP (E–H) at different developmental stages (A) GFP::GSTK-1 is first detected at mid-embryogenesis in the primordium
of the intestine (white arrow) and in the epidermis (arrowheads) A¢ is the corresponding Nomarski picture (B) During larval development, GFP::GSTK-1 is very strongly expressed with a vesicular localization in the intestine (arrows) (C) A weaker expression of GFP::GSTK-1 is present in the muscles (arrowheads) and the epidermis (arrows) (D) Faint diffuse expression is detected in the rectal gland cells (compare with intestinal signal indicated with an arrow) (E) GSTK-2::GFP is first detected in muscle quadrants (arrowheads) during morphogenesis of the embryo E¢ is the corresponding Nomarski picture (F) In larvae, a strong punctate staining is present in the pharynx (arrows) and the body wall muscles (arrowheads), and a weaker signal is observed in the intestine (G) In the pharynx, a very regular expression of GSTK-2::GFP in myo-epithelial cells (arrow) is characteristic of a mitochondrial localization (H) In body wall muscle cells, GSTK-GSTK-2::GFP is detected
as a tubular network with stronger punctata (arrows) typical of the mitochondrial system Scale bar, 10 lm.
Trang 5In our study, C elegans (strain N2) was fed with
bac-teria producing dsRNA of the gstk-1 and⁄ or gstk-2
coding regions In each experiment, four feeding
condi-tions were defined: one group of worms was fed with
control bacteria containing the empty plasmid pL4440,
one with bacteria expressing gstk-1(RNAi), another
with bacteria expressing gstk-2(RNAi) and one with a
mix (1 : 1) of both RNAi-expressing bacteria To test
the efficiency of RNAi, fluorescence levels in
RNAi-treated GSTK::GFP transgenic worms were compared
with the levels observed in control worms Figure S1
(see Supporting information) shows that RNAi
directed against either gstk-1 (Fig S1A) or gstk-2
(Fig S1E) was efficient, with the exception of the
pharynx in gstk-2::gfp transgenic worms, where
silenc-ing was incomplete (Fig S1E,F) It is noteworthy that
gstk-2(RNAi) had no effect on the transgenic strain
expressing GFP::GSTK-1 (Fig S1B), and
GSTK-2::GFP expression remained unchanged in transgenic
animals fed with bacteria producing gstk-1 dsRNA
(Fig S1D), indicating the specificity of these RNAi
forms and the absence of compensatory adaptation
[i.e upregulation of gstk-1 in gstk-2(RNAi) worms]
RNAi silencing of gstk-1 or gstk-2 had no apparent
effect on C elegans reproduction or development (data
not shown) This absence of obvious phenotype has
been reported previously in wide RNAi screens (http://
www.wormbase.org) We also found that these RNAi forms did not affect the C elegans lifespan (Fig 4) The first animal died after 5 days and all animals were
Fig 3 GSTK-2, but not GSTK-1, localizes in mitochondria Single confocal images of GFP::GSTK-1 (A) and GSTK-2::GFP (B) in adult animals GSTK-2::GFP fully colocalizes with the mitochondrial-specific marker MitoTracker (A¢), whereas GFP::GSTK-1 is not localized in the mitochondrial network (B¢) Scale bar, 10 lm.
60 70 80 90
100 Control
gstk-1 (RNAi) gstk-2 (RNAi) gstk-1/k-2 (RNAi)
10 20 30 40 50
0
Days
Fig 4 gstk-1(RNAi) and gstk-2(RNAi) do not affect the C elegans lifespan Effects of RNAi-mediated knockdown of gstk-1 and ⁄ or gstk-2 on the lifespan in wild-type worms Worms were fed either with control bacteria not expressing any dsRNA or bacteria expressing dsRNA that targets gstk-1, gstk-2 or both gstk-1 and gstk-2 Nematode survival was analysed by the Kaplan–Meier method using Graphpad Prism 5 The same software was used to test the equality of survival with the log-rank (Wilcoxon) test Each experimental condition was tested in triplicate.
Trang 6dead after 32 days The mean lifespan for wild-type,
gstk-1(RNAi), gstk-2(RNAi) and gstk-1+gstk-2
(RNAi) were 14, 14, 13 and 13 days, respectively
These results indicate that gst kappa genes are not
essential for the survival of C elegans
As our expression data for GSTK-1 and GSTK-2
supported peroxisomal and mitochondrial
localiza-tions, we further investigated cellular funclocaliza-tions, such
as lipid metabolism and oxygen consumption, which
are closely related to these two organelles The control
worms showed an oxygen consumption of 12.2 nmolÆ
min)1 per 1000 worms Interestingly, a significant
decrease in oxygen consumption of about 40% (7.5 nmolÆmin)1 per 1000 worms) was observed when worms were fed on bacteria expressing dsRNAs target-ing both gstk-1 and gstk-2 (Fig 5) However, no significant decrease in oxygen consumption was observed in worms fed with 1(RNAi) or gstk-2(RNAi) alone compared with the control condition Thereafter, in order to investigate the effect of gstk-1 and⁄ or gstk-2 silencing on lipid metabolism, we measured the following lipid fractions: phospholipids, diglycerides, triglycerides, free or esterified cholesterol, free fatty acids and total fatty acids Although most lipid concentrations were unchanged (Tables S1–S4, see Supporting information) between wild-type and gstk-1(RNAi), gstk-2(RNAi) and gstk-1⁄ gstk-2(RNAi) worms, a difference was observed for cis-vaccenic acid (18:1x7) for the fatty acid methyl ester (FAME) frac-tion, which was decreased significantly in worms depleted for both gstk-1 and gstk-2 (Fig 6) These data strongly suggest that GSTK-1 and GSTK-2 have overlapping functions, as oxygen consumption and cis-vaccenic acid levels were unchanged in gstk-1(RNAi) and gstk-2(RNAi) worms and decreased only
in double-knockdown animals
Discussion
In this study, we investigated the localization and potential role(s) of GST kappa in C elegans In contrast with most vertebrates, the C elegans genome contains two GST kappa genes, gstk-1 and gstk-2 The two genes are located on different chromosomes and contain three exons Interestingly, orthologues of
C elegans gstk-1 and gstk-2 genes are also found in
0
5
10
15
Control gstk-1
(RNAi)
gstk-2 (RNAi)
gstk-1/k-2 (RNAi)
O 2
RNAi Strain
Fig 5 gstk-1 ⁄ gstk-2(RNAi) animals present an altered respiratory
rate Oxygen consumption was assessed in the fourth larval stage
of animals fed either with control bacteria not expressing any
dsRNA or bacteria expressing dsRNA that targets gstk-1, gstk-2
or both gstk-1 and gstk-2 Results are the mean of six
val-ues ± standard deviation, and are expressed as nmoles of O 2 per
minute per 1000 worms Student’s t-test was applied for statistical
studies between RNAi-fed worms and control worms (*P £ 0.05).
*
Control gstk-1(RNAi) gstk-2(RNAi) gstk-1/k-2(RNAi)
*
8
10
12
14
16
18
Fatty acid mono esters
0
2
4
6
16:0 18:0 16:1w7 18:1w9 18:1w7 18:2w6 20:5w3 17 Cyclo
Fig 6 gstk-1 ⁄ gstk-2(RNAi) animals display
an abnormal FAME composition Simplified
FAME composition of control and gstk-1,
gstk-2 and double gstk-1(RNAi) and
gstk-2(RNAi)-treated animals (see also
Tables S1–S4) Depletion of both gstk-1 and
gstk-2 leads to a decrease in the 18:1w7
fatty acid, but does not affect other lipids.
The results are the mean of three
experi-ments ± standard deviation Student’s t-test
was applied for statistical studies between
RNAi-fed worms and control worms
(*P £ 0.05).
Trang 7the nematode species C briggsae and C remanei,
indi-cating that a gene duplication took place before the
speciation of these three Caenorhabditis species
Sequence conservation between the GSTs of C elegans
gstk-1⁄ 2 and those of other nematode species
(C briggsae, C remanei, C japonica, Ancylostoma
ceylanicum, Heterorhabditis bacteriophora and
Meloido-gyne hapla), as well as with rat GSTK1, is also
observed, the most highly conserved residues being
those that contribute to the glutathione-binding site
and dimerization of the protein Interestingly, structure
prediction and molecular modelling studies have
shown that, despite the low sequence similarity
(30%), rGSTK1 and hGSTK1 structures are
recog-nized as the closest structural homologues of C
ele-gans GSTK-1 and GSTK-2 (data not shown)
Together, these observations suggest that C elegans
GSTK-1 and GSTK-2 might have similar activities to
their mammalian orthologues [3,4,7], and may
contrib-ute, at least in part, to detoxification processes
Moreover, a tripeptide sequence (SKL), known as
PTS1, is present at the C-terminal end of the C elegans
GSTK-1 protein PTS1 is involved in protein import
into peroxisomes, and the importance of this signal for
peroxisome targeting in C elegans has been shown
pre-viously by Motley et al [13] Interestingly, GFP fused
to the N-terminus of GSTK-1 was found to localize in
punctate bodies of C elegans cells in several tissues,
strongly suggesting a peroxisomal localization By
con-trast, colabelling with MitoTracker Red showed that
GSTK-2::GFP was present mainly in the mitochondria
It is noteworthy that, in human cells, hGSTK1 is both
peroxisomal and mitochondrial and contains a
C-termi-nal PTS1 [4] This preserved intracellular localization
of GST kappa of both nematodes and vertebrates,
together with sequence conservation and intron
posi-tions in amino acid sequences, indicate that kappa class
genes probably originated from a common ancestral
gene which was present before the protostome⁄
deutero-stome split Interestingly, the presence of two
dupli-cated paralogous genes, gstk-1 and gstk-2, in the
C elegans genome allowed specialization of the
sub-cellular localization for each gene
The use of reporter fusion proteins allowed the
study of tissue expression patterns of gst kappa genes
Although both GFP::GSTK-1 and GSTK-2::GFP
fusion proteins are observed in common tissues, such
as the intestine, they also have a specific localization in
other tissues, such as the epidermis or pharynx for
GFP::GSTK-1 and GSTK-2::GFP, respectively In
C elegans, the intestine and epidermis are at the
interface between the organism and its environment
Therefore, these tissues represent defence barriers
against toxic agents, such as gut-derived oxidants or endogenously generated reactive oxygen species The intestine is also a highly metabolically active organ and represents the tissue in which most C elegans peroxisomes are found, as shown by immunostaining for catalase [14] and by electron microscopy [15] Inter-estingly, GSTK2::GFP is predominantly expressed in muscle cells (pharynx and body wall muscle) The expression of GST kappa genes in body wall muscle and pharynx might be related to the large number of mitochondria in these two tissues, which are associated with high energy consumption
In order to gain further insight into the potential function(s) of GSTK-1 and GSTK-2, RNAi was used
to knock down the expression of the two correspond-ing genes, either separately or simultaneously Knock-down of gstk-1 and⁄ or gstk-2 had no effect on worm lifespan, locomotion or development, suggesting that GST kappa genes are not essential for the worm’s life Because, as in mammals, peroxisomes and mitochon-dria in C elegans play a key role in the production of reactive oxygen species and in lipid metabolism, including fatty acid b-oxidation [16], we investigated the potential role of gstk-1 and⁄ or gstk-2 on the lipid composition of worms For this purpose, phospho-lipids, diglycerides, triglycerides, free and esterified cholesterol, and free and total fatty acid levels were measured in worms fed on gstk-1(RNAi) and⁄ or gstk-2(RNAi) With the exception of cis-vaccenic acid (18:1x7) from the FAME fraction, there was no modi-fication of lipid composition between worms fed on the empty vector control RNAi and those fed on gstk-1(RNAi) and⁄ or gstk-2(RNAi) It is noteworthy that the concentration of cis-vaccenic acid methyl ester was decreased only in double-knockdown (gstk-1 and gstk-2) worms Vaccenic acid is the most abundant fatty acid in phospholipids and triglycerides [17], and
is elongated from palmitoleic acid (16:1x7)
Another phenotypic feature of double-knockdown (gstk-1 and gstk-2) worms was the impairment of oxy-gen consumption It is also noteworthy that vaccenic acid synthesis and worm respiration are closely linked
to peroxisomal and⁄ or mitochondrial activities Inter-estingly, vaccenic acid is an important component of cardiolipin in different animal species [18], and this phospholipid plays a key role in mitochondrial func-tion, particularly at the respiratory chain level [19] Although the link between decreased vaccenic acid levels and impairment in oxygen consumption merits further investigation, the presence of altered phenotypes only in double-knockdown worms indicates compensa-tory roles for GSTK-1 and GSTK-2 and suggests over-lapping functions As peroxisomes and mitochondria
Trang 8are metabolically linked, cooperate and cross-talk,
especially in the b-oxidation of various fatty acids and
in the maintenance of homeostasis in cellular reactive
oxygen species [20,21], our results further strengthen
the close relationship between these two organelles It
is noteworthy that specific knockdown of either gstk-1
or gstk-2 was not accompanied by an upregulation of
the paralogous gene Such compensatory responses
have been demonstrated in Gst alpha 4 (Gsta4) or Gst
zeta 1 (Gstz1) knockout mice, where the expression of
other Gst classes and antioxidant enzymes was induced
[22,23] By contrast, knockout of Gst pi1⁄ 2 (Gstp1 ⁄ 2)
did not lead to the upregulation of at least class alpha
and mu transferases, as reported by Henderson et al
[24] As the C elegans genome contains more than 50
genes encoding zeta, sigma, pi and omega class GSTs
[25], further studies are needed to determine whether
GSTs belonging to these classes or other genes are
upregulated in gstk-1 and⁄ or gstk-2 knockout worms
With regard to the potential role of GST kappa,
either direct or indirect, in lipid metabolism and
respi-ration, it stills remain unclear One hypothesis might
be that GST kappa plays a role in the folding of
pro-teins involved in lipid synthesis or respiration Indeed,
it has been demonstrated that GST kappa shares
sequence and secondary structure homology with
E coli protein disulfide bond isomerase (DsbA) and
has the same general folding as DsbA The DsbA
fam-ily is a subfamfam-ily of the thioredioxin famfam-ily and
cata-lyses disulfide bond formation during the folding of
secreted proteins in bacterials [26] Recently, Liu et al
[5] have shown that mouse and human GST kappa,
renamed DsbA-L by these authors, are highly
expressed in adipose tissue and interact with
adiponec-tin Adiponectin is an adipokine specifically secreted
from adipose tissue, which plays a key role in glucose
and lipid metabolism in insulin-sensitive tissues [27]
Overexpression of GST kappa promotes adiponectin
multimerization by the formation of disulfide bonds
between trimers [5] Although there is no adiponectin
gene in the C elegans genome, GST kappa might have
conserved such a role in the regulation of protein
multi-merization or interaction Certain proteins involved in
lipid metabolism can exist as both monomers and
dimers, for example the fatty acid synthase complex,
and it has been demonstrated that some desaturases
also form dimers [28,29] Similarly, several
mitochon-drial proteins form disulfide-linked multimeric
com-plexes [30] Thus, a possible role of GST kappa might
be to favour specific protein–protein interactions, and
the absence of such interactions in double-knockdown
(gstk-1 and gstk-2) worms may lead to lipid metabolism
and respiration impairment Further investigation will
be needed to confirm this hypothesis and to determine which proteins might be regulated by GST kappa
In conclusion, this work has allowed the character-ization of two GST kappa genes, gstk-1 and gstk-2, in the C elegans genome The products of these genes are differentially expressed in worm tissues and show dis-tinct subcellular localizations, namely peroxisomal for GSTK-1 and mitochondrial for GSTK-2 Specific repression of each gene has no consequences on the worm phenotype By contrast, double-knockdown (gstk-1 and gstk-2) worms show decreased vaccenic acid levels and lower oxygen consumption when compared with wild-type worms
Materials and methods
Caenorhabditis elegans strains Caenorhabditis elegans cultures were grown and maintained
at 20C using NGM agar plates supplemented with
5 lgÆmL)1of cholesterol [10] The wild-type reference strain Bristol N2 was used All experiments were performed at
20C
Fluorescent-tagged protein constructs and the production of transgenic animals
Reporter gene constructs were obtained by a PCR fusion-based approach [31] Genomic gstk-2 (D2024.7), with 1.8 kb immediately upstream of the start codon, was PCR amplified from wild-type genomic DNA using a Triple-MasterPCR System (Eppendorf, Hamburg, Germany) This product was then coamplified with a 1.8 kb PCR fragment containing the GFP coding sequence and the 3¢ untrans-formed region (UTR) of unc-54 (from plasmid pPD95.75 kindly provided by A Fire) For gstk-1 (ZK1320.1), a 1.1 kb promoter fragment was amplified and fused with the GFP coding sequence and then coamplified with the gstk-1 geno-mic and 3¢ UTR Sequences were checked and the resulting gfp::gstk-1and gstk-2::gfp fragments were microinjected [32]
at 50 ngÆlL)1 into the syncytial gonad of young wild-type adult hermaphrodites, together with 200 ngÆlL)1of the plas-mid pRF4 containing the dominant marker rol-6(su1006) [33] For each construct, at least three independent lines were analysed for expression
Immunofluorescence microscopy Routinely, fluorescence expression patterns and phenotypic analyses were carried out on a Zeiss axioskop 2 plus equipped with Nomarski optics (Zeiss, Le Pecq, France) Confocal stacks of images every 0.3–0.5 lm were captured
on an inverted Leica SP2 confocal microscope (Leica, Rueil-Malmaison, France) Z projections were analysed
Trang 9using Image J software and then processed using Adobe
Photoshop To stain mitochondria, animals were
incu-bated for 10 min with 10 lm of MitoTracker Red
(Invitro-gen Molecular Probes, Cergy Pontoise, France), and then
moved to a fresh plate for 2 h Worms were anaesthetized
in either 1 mgÆmL)1levamisole or 10 lm azide
RNAi experiments
RNAi by feeding bacteria was performed using the N2
strain, as described previously [34,35], with the following
modifications The bacterial HT115(DE3) E coli strains
used for feeding experiments were obtained from J
Ahrin-ger (University of Cambridge, UK) (gstk-1, ref:
WBR-NAi00021881) and OpenBiosystems (Fisher Scientific-Open
Biosystems, Illkirch, France) (gstk-2, ref : RCE1182) In
brief, control and RNAi strain cultures were grown for 6 h
in LB medium containing 100 mgÆmL)1 ampicillin, and
then spread onto NGM agar containing isopropyl
thio-b-d-galactoside (1 mm) and carbenicillin (25 lgÆmL)1) For
dou-ble-RNAi treatment, equal concentrations of both strains
were mixed before seeding The next day, animals at the
fourth larval stage were placed onto RNAi plates, grown
for 2 days and then harvested by rinsing with M9 buffer
(0.1 m NaCl, 0.05 m potassium phosphate, pH 6.0) Adults
were allowed to settle, and eggs were recovered from
hermaphrodites by alkaline hypochlorite lysis (5 min at
room temperature in 0.5 m NaOH, 5% hypochlorite) [36]
The eggs were rinsed with M9 buffer and the resulting L1
larvae were transferred the next day to fresh agar plates
containing the different dsRNA conditions
Lifespan assays
First-generation progeny from RNAi and control
condi-tions were picked at the fourth larval stage and transferred
onto fresh RNAi plates The day of the shift was counted
as day 0 in the adult lifespan assay To prevent mixing test
worms with their progeny during the reproduction period,
adult nematodes were transferred daily to fresh plates
Monitoring of lethality was performed every day and
worms were considered to be dead when they failed to
move, either spontaneously or in response to touch, and
showed no pharyngeal pumping Worms that crawled off
the plate were excluded (considered to have escaped) One
hundred worms per condition were used in each lifespan
experiment, conducted in triplicate Nematode survival was
analysed by the Kaplan–Meier method using Graphpad
Prism 5 The same software was used to test the equality of
survival with the log-rank (Wilcoxon) test
Oxygen consumption assays
Oxygen consumption rates were measured using a DW1⁄ AD
Clark-type oxygen electrode (Hansatech, Norfolk, UK)
Young adult worms that were maintained on NGM agar plates covered with the corresponding RNAi bacteria were washed twice and resuspended in 50 lL of M9, and then transferred into the chamber already containing 450 lL of M9 buffer, and respiration was measured at 20C for at least 10 min All washes and measurements were performed
in oxygenated M9 buffer Samples were carefully recovered from the chamber and the number of worms was counted For each condition, the mean rate was calculated from triplicate experiments
Western blotting
To prepare total extracts, worm pellets were resuspended in Laemmli sample buffer, vortexed three times for 15 s after the addition of broken glass beads, and then denatured for
5 min at 100C and separated by 10% SDS–PAGE Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany) and probed with the mouse anti-GFP IgG1k (Roche Diag-nostics, Meylan, France) Immunoreactive proteins were revealed with a chemiluminescent detection system (Super-Signal Pico Chemiluminescent Substrate; Pierce Inc., Rockford, IL, USA)
Lipid analyses Aliquots of C elegans were crushed in 2 mL of methanol–
5 mm EGTA (2 : 1, v⁄ v) with an Ultra Turax; 100 lL of homogenate were evaporated and the pellet was dissolved
in 0.25 mL of NaOH (0.1 m) overnight for protein mea-surement using the Bio-Rad assay For each analysis, lipids from the homogenate were extracted according to Bligh and Dyer [37] in chloroform–methanol–water (2.5 : 2.5 : 2.1, v⁄ v ⁄ v) in the presence of the internal standards For total fatty acid analysis, lipids from a 200 lL homogenate were extracted and transmethylated with 1 mL
BF3⁄ CH3OH (SUPELCO 10% w⁄ w) for 1 h at 150 C FAMEs were extracted with 2 mL of hexane–1 mL of water The organic phase was evaporated to dryness and dissolved in 20 lL of ethyl acetate One microlitre of FAME was analysed by gas–liquid chromatography [38] on
a 5890 Hewlett Packard system using a Famewax RESTEK fused silica capillary column (30 m· 0.32 mm inside dia-meter, 0.25 mm film thickness) The oven temperature was programmed from 110 to 220C at a rate of 2 CÆmin)1 and the carrier gas was hydrogen (0.5 bar) The injector and detector were maintained at 225 and 245C, respec-tively Finally, 2 lg of glyceryl triheptadecanoate were used
as internal standard
For free fatty acid analysis, 400 lL of homogenate were extracted and dissolved in 1 mL of hexane Free fatty acids were transmethylated in 1 mL of BF3⁄ CH3OH (10% w⁄ w) for 5 min at room temperature and free FAMEs were extracted with 2 mL of hexane–1 mL of water The organic
Trang 10phase was evaporated to dryness and dissolved in 10 lL of
ethyl acetate Analysis was performed as above with 1 lg
of nonadecanoic acid as internal standard All chemicals
were obtained from Sigma-Aldrich, Lyon, France
Acknowledgements
This work was supported in part by the Institut
National de la Sante´ et de la Recherche Me´dicale,
Centre National de la Recherche Scientifique and
Association pour la Recherche sur le Cancer Elise
Petit was founded by the Ligue National Contre le
Cancer and Xavier Michelet by the Association pour
la Recherche contre le Cancer We are grateful to
Pro-fessor A Guillouzo and Drs E Culetto and B
From-enty for critical reading of the manuscript The
Imaging and Cell Biology Facility of the IFR87
(FR-W2251) ‘La plante et son environnement’ is
sup-ported by the Action de Soutien a` la Technologie et la
Recherche en Essonne, Conseil de l’Essonne
References
1 Harris JM, Meyer DJ, Coles B & Ketterer B (1991)
A novel glutathione transferase (13-13) isolated from
the matrix of rat liver mitochondria having structural
similarity to class theta enzymes Biochem J 278,
137–141
2 Ladner JE, Parsons JF, Rife CL, Gilliland GL &
Armstrong RN (2004) Parallel evolutionary pathways
for glutathione transferases: structure and mechanism
of the mitochondrial class kappa enzyme rGSTK1
Biochemistry 43, 352–361
3 Robinson A, Huttley GA, Booth HS & Board PG
(2004) Modelling and bioinformatics studies of the
human Kappa-class glutathione transferase predict a
novel third glutathione transferase family with similarity
to prokaryotic 2-hydroxychromene-2-carboxylate
isomerases Biochem J 379, 541–552
4 Morel F, Rauch C, Petit E, Piton A, Theret N, Coles B
& Guillouzo A (2004) Gene and protein
characteriza-tion of the human glutathione S-transferase kappa and
evidence for a peroxisomal localization J Biol Chem
279, 16246–16253
5 Liu M, Zhou L, Xu A, Lam KS, Wetzel MD, Xiang R,
Zhang J, Xin X, Dong LQ & Liu F (2008) A
disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates
adiponectin multimerization Proc Natl Acad Sci USA
105, 18302–18307
6 Thomson RE, Bigley AL, Foster JR, Jowsey IR,
Elcombe CR, Orton TC & Hayes JD (2004)
Tissue-specific expression and subcellular distribution of
murine glutathione S-transferase class kappa
J Histochem Cytochem 52, 653–662
7 Jowsey IR, Thomson RE, Orton TC, Elcombe CR & Hayes JD (2003) Biochemical and genetic characteriza-tion of a murine class Kappa glutathione S-transferase Biochem J 373, 559–569
8 Bartlett K & Eaton S (2004) Mitochondrial beta-oxidation Eur J Biochem 271, 462–469
9 Poirier Y, Antonenkov VD, Glumoff T & Hiltunen JK (2006) Peroxisomal beta-oxidation – a metabolic path-way with multiple functions Biochim Biophys Acta
1763, 1413–1426
10 Brenner S (1974) The genetics of Caenorhabditis elegans Genetics 77, 71–94
11 Labrousse AM, Zappaterra MD, Rube DA & van der Bliek AM (1999) C elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane Mol Cell 4, 815–826
12 Timmons L & Fire A (1998) Specific interference by ingested dsRNA Nature 395, 854
13 Motley AM, Hettema EH, Ketting R, Plasterk R & Tabak HF (2000) Caenorhabditis elegans has a single pathway to target matrix proteins to peroxisomes EMBO Rep 1, 40–46
14 Togo SH, Maebuchi M, Yokota S, Bun-Ya M, Kawahara A & Kamiryo T (2000) Immunological detection of alkaline-diaminobenzidine-negative peroxisomes of the nematode Caenorhabditis elegans: purification and unique pH optima of peroxisomal catalase Eur J Biochem 267, 1307–1312
15 Yokota S, Togo SH, Maebuchi M, Bun-Ya M, Haraguchi CM & Kamiryo T (2002) Peroxisomes of the nematode Caenorhabditis elegans: distribution and morphological characteristics Histochem Cell Biol 118, 329–336
16 Gurvitz A, Langer S, Piskacek M, Hamilton B, Ruis H
& Hartig A (2000) Predicting the function and subcellu-lar location of Caenorhabditis elegans proteins simisubcellu-lar to Saccharomyces cerevisiaebeta-oxidation enzymes Yeast
17, 188–200
17 Tanaka T, Ikita K, Ashida T, Motoyama Y, Yamaguchi
Y & Satouchi K (1996) Effects of growth temperature on the fatty acid composition of the free-living nematode Caenorhabditis elegans Lipids 31, 1173–1178
18 Wolff RL, Combe NA & Entressangles B (1985) Positional distribution of fatty acids in cardiolipin of mitochondria from 21-day-old rats Lipids 20, 908–914
19 Mileykovskaya E, Zhang M & Dowhan W (2005) Cardiolipin in energy transducing membranes
Biochemistry (Mosc) 70, 154–158
20 Camoes F, Bonekamp NA, Delille HK & Schrader M (2009) Organelle dynamics and dysfunction: a closer link between peroxisomes and mitochondria J Inherit Metab Dis 32, 163–180
21 Schrader M & Yoon Y (2007) Mitochondria and peroxisomes: are the ‘big brother’ and the ‘little sister’ closer than assumed? BioEssays 29, 1105–1114