From the Departments of Forensic Medicine and
¶ Biology, Fukui Medical School, Fukui 910-1193, Japan and the
Department of Legal Medicine, Gunma University School of
Medicine, Gunma 371-8511, Japan
Received for publication, October 24, 2002
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ABSTRACT |
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M-LP (Mpv17-like protein) has been identified as
a new protein that has high sequence homology with Mpv17 protein, a
peroxisomal membrane protein involved in the development of early onset
glomerulosclerosis. In this study, we verified the peroxisomal
localization of M-LP by performing dual-color confocal analysis of
COS-7 cells cotransfected with green fluorescent protein-tagged
M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To
characterize the peroxisomal membrane targeting signal, we examined the
intracellular localizations of several green fluorescent protein-tagged
deletion mutants and demonstrated that, of the three transmembrane
segments predicted, the first near the NH2 terminus
and NH2-terminal half of the following loop region, which
is abundant in positively charged amino acids, were necessary and
sufficient for peroxisomal targeting. To elucidate the function of
M-LP, we examined the activities of several enzymes involved in
reactive oxygen species metabolism in COS-7 cells and found that
transfection with M-LP increased the superoxide dismutase activity
significantly. Quantitative real-time PCR analysis revealed that the
manganese SOD (SOD2) mRNA level of COS-7 cells transfected with
M-LP was elevated. These results indicate that M-LP participates in
reactive oxygen species metabolism.
The Mpv17-like protein
(M-LP)1 gene was identified
on the basis of an expressed sequence tag obtained by differential
display screening of age dependently expressed genes in mouse kidneys (1, 2). The M-LP gene is expressed mainly in the kidney and spleen and
the amount expressed increases steadily during development, reaches its
highest level in adulthood, and decreases gradually with aging. It
encodes 194 amino acids of a polypeptide with a sequence and membrane
topology markedly similar to those of two peroxisomal membrane
proteins, Mpv17 protein (3) (30.4% identity, 66.7% similarity) and
PMP22 (4) (25.0% identity, 72.1% similarity). These results suggest
that M-LP might be embedded in the peroxisomal membrane in a similar
manner to these two proteins.
Peroxisomes are ubiquitous eukaryotic subcellular organelles that play
essential roles in a variety of metabolic pathways, including
H2O2 metabolism and the oxidative degradation
of fatty acids (5). Recently, a number of studies have been carried out
to investigate the mechanisms of peroxisomal biogenesis and protein
import (6-8). Targeting of peroxisomal matrix proteins is mediated by
cytosolic receptors Pex5p and Pex7p that recognize the peroxisomal
targeting signals PTS1, which consists of the sequence SKL (and
conservative variants) at the carboxyl terminus (9, 10), and PTS2,
which consists of the consensus sequence (R/K)(L/V/I)X5(H/Q)(L/A) near the amino terminus
(11, 12), respectively. Whereas the targeting of peroxisomal matrix
protein import has been well characterized, neither conclusive
consensus sequence nor the receptor(s) involved in peroxisomal membrane protein targeting have been determined. Thus, it is important to
characterize the membrane peroxisome targeting signals (mPTSs) in as
many peroxisomal membrane proteins as possible to understand the
biogenesis of peroxisomal membranes.
The Mpv17-negative mouse strain was generated by inserting a defective
retrovirus into the germ line of mice and is characterized by
progressive glomerulosclerosis and neurosensory deafness at a young age
(3, 13). The phenotype results from loss of function of the Mpv17 gene
encoding a 20-kDa peroxisomal membrane protein. The molecular function
of the Mpv17 protein has yet to be elucidated, but it was recently
hypothesized that it plays an important role in peroxisomal reactive
oxygen species (ROS) metabolism and that glomerular damage is because
of overproduction of ROS. In Mpv17 gene-inactivated mice, a significant
increase in ROS and lipid peroxidation adduct production was observed
and oxygen radical scavengers prevented glomerular damage (14).
Moreover, a recent study showed that the Expression Vectors for the Peroxisomal Forms of Fluorescent
Proteins--
All the primers used for expression vector construction
in this study are listed in Table I.
Green fluorescent protein (GFP)-PTS1 was amplified by the PCR using the
primer set exGFPS and GFPPTS1A and pEGFP (Clontech,
Palo Alto, CA) as the template. DsRED2-PTS1 was amplified using the
primer set DsREDS and DsREDPTS1A and pDsRED2 (Clontech) as the template. The PCR products were
cloned into the EcoRI/BamHI sites of a mammalian
expression vector, pcDNA3.1 (Invitrogen, Carlsbad, CA).
Expression Vectors for M-LP--
A DNA fragment encoding
full-length M-LP was created by PCR amplification using the primer set
exMLPS2 and exMLPA2 and first-strand cDNA synthesized using the
total RNAs from the kidneys of 9-month-old mice as the template. The
PCR product was cloned into the EcoRI/BamHI sites
of pcDNA3.1 (the resulting vector was designated pcDNA3.1/MLP). GFP tagging of the COOH or NH2 terminus of M-LP was done
using a PCR gene fusion technique (16) as follows. To produce MLP-GFP (COOH-terminal GFP-tagged M-LP), two separate PCRs to amplify the M-LP
and GFP genes were performed: the M-LP gene was amplified using the
primer set exMLPS2 and exMG1A2 and first-strand cDNA synthesized
using the total RNAs from the kidneys of 9-month-old mice as the
template and the GFP gene was amplified using the primer set exMG2S and
exGFPA and pEGFP as the template. Then, the two PCR products were mixed
with the primer set exMLPS2 and exGFPA and extended by the PCR to form
the chimeric gene. Likewise, to produce GFP-MLP
(NH2-terminal GFP-tagged M-LP), separate PCRs for GFP and
M-LP gene amplification were performed with the two primer sets exGFPS
and exGM1A and exGM2S and exMLPA, respectively. Then, the two PCR
products were mixed with the primer set exGFPS and exMLPA, extended by
the PCR and each PCR-amplified chimeric gene was cloned into the
EcoRI/BamHI site of pcDNA3.1 (the resulting vectors were designated pcDNA3.1/MLP-GFP and pcDNA3.1/GFP-MLP, respectively).
Expression Vectors for M-LP
Variants--
NH2-terminal truncated Cells and Transfections--
COS-7 cells were maintained in
Dulbecco's modified Eagle's medium containing 1 mM
L-glutamine, 50 units/ml penicillin, 50 µg/ml
streptomycin, and 10% (v/v) fetal calf serum (Invitrogen) and
transiently transfected using LipofectAMINE Plus reagent (Invitrogen), according to the manufacturer's instructions.
Confocal Microscopy--
Cells were plated in 3.5-cm
poly-D-lysine-coated glass-bottomed dishes (MatTek,
Ashland, MA) and 24 h after transfection, fluorescent images were
analyzed by a laser scanning confocal microscope LSM-GB200 (Olympus,
Tokyo, Japan). An argon laser at 488 nm was used for excitation and the
fluorescent signals emitted by GFP and DsRED2 were detected using a
535-nm band-pass filter and a 570-nm long pass filter, respectively.
Generation of Anti-M-LP Antibodies--
The cDNA encoding
NH2-terminal amino acids 1-103 or COOH-terminal amino
acids 105-194 of M-LP were cloned in-frame with glutathione S-transferase into the pGEX-6P-1 vector (Amersham
Biosciences, Tokyo, Japan), introduced into Escherichia coli
INV Enzyme Assays--
The activities of enzymes involved in ROS
metabolism in COS-7 cells transfected with pcDNA3.1/MLP and
pcDNA3.1 as a control were determined. Three days after
transfection, culture media and cell lysates were recovered and their
enzymatic activities were determined. The cell lysates used for the
measurement of SOD and Gpx activities were prepared by mixing
them with 250 mM Tris-HCl (pH 7.5) containing 500 µM phenylmethylsulfonyl fluoride and 1 µg/ml aprotinin
and subjecting them to three freeze-thaw cycles, after which the SOD
and Gpx activities were assayed using a SOD assay kit-WST (Dojin
Molecular Technologies, Kumamoto, Japan) and a glutathione peroxidase
assay kit (Cayman, Ann Arbor, MI), respectively. To investigate the
effects of the anti-M-LP antibody on the SOD activities, cell lysates
were incubated with antibody serially diluted with 25 mM
Tris-HCl (pH 7.4) containing 8% (w/v) sodium chloride and 0.2% (w/v)
potassium chloride at 37 °C for 1 h and the remaining
activities were measured.
Cell lysates were prepared for the measurement of catalase (CAT)
activity as follows. The cells were washed with phosphate-buffered saline (pH 7.4), suspended in phosphate-buffered saline containing 5 mM EDTA, 0.01% (w/v) digitonin, and 0.25% (w/v) sodium
cholate and sonicated 30 s for 8-10 times until the solution was
cleared. The sonicated sample was centrifuged at 12,000 × g for 30 min at 4 °C to remove cell debris and the
catalase activity of the supernatant was measured using a BIOXYTECH
Catalase-520 kit (OxisResearch, Portland, OR). Protein
concentrations were determined using a QuantiPro BCA assay kit (Sigma).
The data shown are mean ± S.E. Differences between means were
determined using Student's t test and those at
p < 0.05 were considered significant.
Measurement of mRNA Levels--
Total RNA from mouse kidneys
was prepared as described in our previous report (1) and total RNA was
extracted from COS-7 cells using Sepasol-RNA I Super (Nacalai Tesque,
Kyoto, Japan), according to the manufacturer's instructions. The
mRNA levels were determined by quantitative real-time PCR analysis
with a LightCycler Instrument (Roche Molecular Diagnostics) using the QuantiTect SYBR Green PCR kit (Qiagen, Chatsworth, CA) as previously described (17). The primer sequences and product size for each gene are
summarized in Table II. The amount of
mRNA was normalized to the internal control,
glyceraldehyde-3-phosphate dehydrogenase. All PCR assays were performed
in triplicate and the intra-assay variability was <7%.
M-LP Localizes to Peroxisomes--
A peroxisomal localization of
M-LP was considered highly likely in view of the significant homology
with both the amino acid sequences (30.4% identity, 66.7% similarity)
and hydrophilicity profile of the peroxisomal membrane protein Mpv17.
Accordingly, colocalization analysis of GFP-tagged M-LP and a
peroxisomal marker protein was performed. First, the effectiveness of
DsRED2-PTS1 as a peroxisomal marker was tested. Punctate fluorescence
signals were observed in cells expressing DsRED2-PTS1 and they
colocalized completely with the signals because of GFP-PTS1 (Fig.
1A), which has been used as a
peroxisomal marker in previous studies (18, 19), whereas diffuse
cytosolic fluorescence was observed in control cells expressing DsRED2.
These results demonstrate that DsRed2-PTS1, as well as GFP-PTS1, is an
effective marker for peroxisomal protein. Next, cells were
co-transfected with DsRED2-PTS1 and either MLP-GFP or GFP-MLP and their
intracellular distributions were examined (Fig. 1B).
Punctate green fluorescence because of MLP-GFP was observed in
MLP-GFP-expressing cells and was superimposable on the red fluorescence
because of DsRED2-PTS1, demonstrating clearly that M-LP was targeted to
peroxisomes. However, GFP-MLP was detected in peroxisomes and the
cytoplasm. These results could be interpreted as meaning that the mPTS
exists around the NH2-terminal and the interaction between
the mPTS and its receptor(s) was hindered by tagging GFP onto the
NH2 terminus of M-LP.
The Transmembrane Segment 1 (TMS1) and NH2-terminal
Half of the Loop between TMS1 and TMS2 Are Required for Peroxisomal
Membrane Targeting--
To assess the mPTS of M-LP more closely, we
constructed several NH2- and COOH-terminal deletion
mutants, as shown in Fig. 2A,
and examined their intracellular localizations (Fig. 1C). From the putative topology of M-LP derived from its primary structure, three TMSs, TMS1-(16-34), TMS2-(92-110), and TMS3-(151-168)
have been predicted (2).
Although a great deal of information about peroxisomal matrix protein
import has been obtained, our understanding of how peroxisomal membrane
proteins are targeted and integrated into peroxisomal membranes is
still poor. The sequences involved in targeting or insertion of
peroxisomal membrane proteins described so far are as follows: in
Pichia pastoris Pas2p (20) and human (21) and Hansenula polymorpha (22) Pex3p, positively charged amino
acids at the NH2-terminal region were shown to be involved
in targeting or insertion; in Saccharomyces cerevisiae
Pex15p, the COOH-terminal luminal tail was required for targeting (23);
in rat PMP70, an internal region that roughly corresponded to the TMS3
was necessary for targeting and insertion (24); in S. cerevisiae PMP47, TMS2 plus an adjacent cytoplasmic oriented
sequence, a matrix facing basic cluster, and an anchoring TMS were
sufficient for targeting (25); in human PMP34, the loop region between
TMS4 and TMS5 and three transmembrane segments were required for
targeting and insertion (26); and in rat PMP22, the
NH2-terminal region and more than one TMS were required for
targeting and insertion (27). Although the amino acid sequences of M-LP
and PMP22 are very similar (25.0% identity, 72.1% similarity), those
in the NH2-terminal regions are quite different and the
mPTS identified in PMP22 was not found in M-LP. Honsyo and Fujiki (26)
pointed out that no conclusive consensus sequence had been observed,
but the feature common to most of these proteins was the presence of
positively charged amino acids located in the flanking region of a TMS.
From this point of view, M-LP has two clusters of positively charged amino acids in the loop between TMS1 and TMS2 (the first cluster at
amino acid positions 37, 39, 46, 49, and 50; the second at positions
68, 72, 77, 80, and 85). Our results demonstrate that the presence of
the NH2-terminal half of the loop containing the first
cluster and adjacent TMS1 were sufficient for M-LP to function as an
mPTS. Therefore, it can be said that M-LP satisfies the law these
authors suggested.
SOD Activity Is Elevated in COS-7 Cells Transfected with
M-LP--
The high sequence and membrane topological homologies of
M-LP and Mpv17 have led to speculation that M-LP, as well
as Mpv17, functions as an ROS scavenger. Accordingly, we determined the activities of enzymes involved in ROS metabolism in COS-7 cells transfected with pcDNA3.1/MLP and pcDNA3.1 as a control. As
shown in Table III, there was no
significant difference between the Gpx or CAT activities of these
cells. However, the SOD activity of the cell lysates prepared from
COS-7 cells transfected with M-LP was significantly higher than that of
the control cells. The following two mechanisms may be responsible for
the elevation of SOD activity: activation of SOD at the protein level
and an increase in the amount of SOD enzyme expressed. However, in view
of our finding that preincubation with an anti-M-LP antibody did not
influence the SOD activity (data not shown), it seems unlikely that the SOD activity is regulated by an interaction with M-LP at the protein level.
Manganese-SOD (SOD2) mRNA Is Increased in COS-7 Cells
Transfected with M-LP--
Three SOD isoforms, a cytosolic,
copper-zinc SOD (SOD1), a mitochondrial, manganese SOD
(SOD2), and an extracellular SOD (SOD3) have been detected
in mammalian cells (27). The mRNA levels of these three SODs,
cellular Gpx (Gpx1), Gpx3, and CAT in COS-7 cells transfected with M-LP
were examined using quantitative real-time reverse transcriptase-PCR
(Fig. 3). Whereas the expression levels of five genes (SOD1, SOD3, Gpx1,
Gpx3, and CAT) were unchanged, an increase in SOD2
mRNA compared with the control cell level was observed, suggesting
that the increase in SOD activity in the M-LP expressing cells was due
mainly to up-regulation of the SOD2 gene. Recently, Wagner
et al. (15) performed the reverse experiment,
i.e. examination of the activities and mRNA levels of
antioxidant enzymes in Mpv17 null cells, and found that the absence of
the Mpv17 protein reduced expression of all three SOD isoforms and Gpx3
and concomitantly increased The Expression Patterns of SOD2 and M-LP Genes in Mouse Kidney
during Development and Aging Are Similar--
In our previous study,
we used comparative reverse transcriptase-PCR and showed that M-LP gene
expression changed age-dependently (2). Our finding that
M-LP affected expression of the SOD2 gene awoke our interest
in the expression patterns of the SOD2 gene during
development and aging. Therefore, the levels of M-LP and SOD2 mRNAs
in the kidneys of 3-day- to 15-month-old mice were measured using
quantitative real-time PCR. As shown in Fig.
4A, the amount of M-LP
expressed increased steadily during development, reached its highest
levels in adulthood and decreased gradually with aging. This
observation agreed quite well with our previous results (2). As
expected, the expression pattern of the SOD2 gene was very
similar to that of M-LP (Fig. 4B), suggesting that M-LP
participates in the regulation of SOD2 gene expression.
Age-dependent changes in SOD2 activity or its gene
expression have been reported in several mammalian tissues. However,
the expression patterns vary depending on species and/or tissues. For
instance, SOD2 activity was observed to be lower in human skin
fibroblast cell lines derived from fetal skin than those from postnatal
skin, although no postnatal age-dependent differences were
observed (29). Three-month-old rats had higher hepatic SOD2 activities
than 24-month-old rats, whereas there was no significant age-related
difference in the SOD2 activities of the lungs (30). In our previous
study, we showed that the M-LP gene is expressed mainly in the kidney
and spleen. The different expression patterns in various tissues may be
attributable to the participation of a tissue-specific molecule like
M-LP in the regulation of SOD2.
ROS are generated constantly during oxidative metabolism and cause
cellular damage by reacting with proteins, lipids, DNA, and
carbohydrates. SOD is considered to be one of the most important constituents of the first line of defense against ROS production by
virtue of its ability to convert highly reactive superoxide radicals to
hydrogen peroxide and molecular oxygen. Our data demonstrated that, of
the three SODs, only the SOD2 gene was up-regulated by the
expression of M-LP. The biological importance of SOD2 has been shown in
studies on knockout mice (31, 32): homozygous mutant mice died less
than 3 weeks after birth, probably because of severe impairment of
mitochondrial function because of the elevated level of ROS (28).
Analysis of the 5'-flanking region resulted in the identification of
several regulatory sequences, including Sp1, NF-
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-glutamyl transpeptidase
enzyme activity and mRNA expression level were higher, whereas
plasma glutathione peroxidase (Gpx3) and superoxide dismutase (SOD)
activities were lower, in Mpv17 null cells than normal cells (15).
These results strongly suggest that the Mpv17 protein is involved in
enzymatic antioxidant defense systems. The aims of this study
were first, to confirm the peroxisomal localization of M-LP and
characterize the mPTS of this new protein and second, to investigate
the activities and expression of antioxidant enzymes to determine
whether there is a connection between M-LP and ROS metabolism.
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Synthetic oligonucleotide primers used for construction of expression
vectors
15MLP-GFP
(NH2-terminal amino acids 1-15 are deleted from M-LP),
34MLP-GFP (NH2-terminal amino acids 1-34 are deleted),
and
91MLP-GFP (NH2-terminal amino acids 1-91 are
deleted) were generated by the PCR using the primer pair sets 16MGS and
exGFPA, 35MGS and exGFPA, or 92MGS and exGFPA, respectively, and
pcDNA3.1/MLP-GFP as the template. The PCR products were cloned into
the EcoRI/BamHI site of pcDNA3.1.
COOH-terminal truncation variants 34MLP-GFP (comprising
NH2-terminal amino acids 1-34), 55MLP-GFP (comprising
NH2-terminal amino acids 1-55), 91MLP-GFP (comprising
NH2-terminal amino acids 1-91), and an NH2-
and COOH-terminal truncation variant 16/55MLP-GFP (comprising amino
acids 16-55) were generated as follows. The GFP gene was PCR-amplified
using the primer set exGFPS and exGFPA and pEGFP as the template and the PCR product was cloned into the EcoRI/BamHI
sites of pcDNA3.1 (the resulting vector was designated
pcDNA3.1/GFP). DNA fragments encoding amino acids 1-34, 1-55,
1-91, and 16-55 of M-LP were PCR amplified using the primer sets 91MS
and 34MA, 91MS and 55MA, 91MS and 91MA, or 15DMS and 55MA,
respectively, and pcDNA3.1/GFP-MLP as the template. The PCR
products were cloned into the KpnI/HindIII sites
of pcDNA3.1/GFP, which contained the coding sequence of GFP behind
the KpnI/HindIII site.
F' (Invitrogen), and induced using standard procedures. The
glutathione S-transferase fusion proteins were solubilized
from inclusion bodies using 8 M urea and affinity purified
using glutathione-Sepharose-4B (Amersham Biosciences). For antibody
production, the glutathione S-transferase fusion proteins
were emulsified with adjuvant (TiterMax Gold; CytRx, Atlanta, GA),
injected intramuscularly into Japanese white rabbits three times and
the immunoglobulin G fractions were purified using a protein A column
(Hitrap Protein A; Amersham Biosciences).
Primers for quantitative real-time PCR analyses
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Fig. 1.
Colocalization analysis of GFP-tagged M-LP
with DsRED2-PTS1, a fluorescent marker of peroxisomes.
COS-7 cells were cotransfected with GFP-PTS1 and DsRED2-PTS1
(A), GFP-tagged M-LP and DsRED2-PTS1 (B), or
GFP-tagged deletion mutants of M-LP and DsRED2-PTS1 (C),
respectively. The fluorescent images because of GFP
(green) and DsRED (red) were analyzed by a
laser scanning confocal microscope.
15MLP-GFP, the mutant lacking the
NH2-terminal tail consisting of the first 15 amino acids,
was targeted properly to peroxisomes, as revealed by its colocalization
with DsRED2-PTS1. However, the signals from cells expressing
34MLP-GFP, which lacks the NH2-terminal tail and TMS1,
did not correspond to peroxisomes, although several punctate signals
were observed. In the case of
91MLP-GFP, which lacks the
NH2-terminal tail, TMS1, and the loop between TMS1 and
TMS2, overall diffuse cytosolic fluorescence was observed. Next, in the
cases of mutants truncated at the COOH-terminal region, 91MLP-GFP
comprising residues 1-91, which include the NH2-terminal
tail, TMS1, and the loop between TMS1 and TMS2, and 55MLP-GFP
comprising residues 1-55, which include the NH2-terminal tail, TMS1, and half of the loop between TMS1 and TMS2, were located exclusively in peroxisomes, whereas 34MLP-GFP comprising residues 1-34, which include the NH2-terminal tail and TMS1, was
partially localized in peroxisomes. These results demonstrate that TMS1 and the NH2-terminal half of the loop between TMS1 and TMS2
are required for the correct peroxisomal localization of M-LP. To confirm this hypothesis, 16/55MLP-GFP, comprising residues 16-55, which include TMS1 and the NH2-terminal half of the loop
between TMS1 and TMS2, was constructed and its intracellular
localization was examined. As shown in Fig. 1C, the signals
were localized exclusively in peroxisomes, suggesting that TMS1 and the
NH2-terminal half of the loop between TMS1 and TMS2 are
sufficient to function as an mPTS.
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Fig. 2.
Schematic presentation of deletion mutants
and the peroxisome targeting signal of M-LP. A,
localization to peroxisomes is indicated by: +, localized; ±,
partially localized; , not localized. B, positively
charged amino acids in the loop between TMS1 and TMS2 are indicated by
+. The region required for peroxisomal membrane targeting is indicated
by a bar.
Enzyme activities in COS-7 cells transfected with pcDNA3.1 and
pcDNA3.1/MLP
-glutamyl transpeptidase expression.
Therefore, M-LP seems to have the same function as the Mpv17 protein
from the point of view that its action leads to a reduction in ROS
production.
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Fig. 3.
The mRNA levels of enzymes involved in
ROS metabolism in COS-7 cells transfected with M-LP. The mRNA
levels of SOD1, SOD2, SOD3, Gpx1, Gpx3, and CAT in M-LP expressing
cells (n = 4) were determined by quantitative real-time
PCR analysis. The results are expressed as ratios relative to the value
for the control cells transfected with pcDNA3.1 (n = 4). The increase in the SOD2 mRNA level was statistically
significant (*, p = 0.028).
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Fig. 4.
Age-dependent expression of M-LP
(A) and SOD2 (B) mRNAs in mouse
kidney detected by quantitative real-time PCR analysis. The
results are expressed as percentages of the maximum mean value.
D, day; W, week; M, month.
B, CCAAT-enhancer
binding protein, and an antioxidant-response element (33). Of the
antioxidant enzymes, only SOD2 is induced by various stimuli, such as
tumor necrosis factor-
(34), interleukin-1 (35), and X-irradiation
(36) that are known to produce ROS or induce intracellular ROS
generation. The mechanism responsible for the induction of SOD2 under
these conditions has yet to be elucidated, however, it has been
proposed that tumor necrosis factor-
and interleukin-1 regulate SOD2
expression by activating the transcription factor NF-
B (37, 38).
Thus, further investigation is needed to clarify the relationship
between M-LP and these factors and the role(s) of M-LP in ROS metabolism.
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ACKNOWLEDGEMENTS |
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We thank F. Nakamura for secretarial assistance. We also thank S. Ikeda and K. Ikeda for heartfelt encouragement.
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FOOTNOTES |
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* This work was supported in part by Japan Society for the Promotion of Science Grants-in-aid 12670390 (to R. I.), 12357003, 13877069 (to T. Y.), and 12307011 (to K. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-776-61-3111; Fax: 81-776-61-8108; E-mail: ireiko@fmsrsa.fukui-med.ac.jp.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M210886200
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ABBREVIATIONS |
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The abbreviations used are: M-LP, Mpv17-like protein; GFP, green fluorescent protein; ROS, reactive oxygen species; SOD, superoxide dismutase; PTS, peroxisome targeting signal; mPTS, peroxisome targeting signal of membrane protein; Gpx, glutathione peroxidase; CAT, catalase; TMS, transmembrane segment.
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