From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0664
Received for publication, December 5, 2002, and in revised form, February 26, 2003
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ABSTRACT |
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Cystathionine The transsulfuration pathway converts homocysteine to cysteine
(Fig. 1), the limiting reagent in the
synthesis of the tripeptide, glutathione
(GSH).1 GSH is the most
abundant antioxidant in mammalian cells and plays an important role in
maintaining intracellular redox homeostasis and in cellular defense
against oxidative stress. Recent studies from our laboratory have
revealed that ~50% of the cysteine in the GSH pool in cultured human
liver cells is derived from homocysteine (1). Thus, the
transsulfuration pathway is a quantitatively significant contributor to
GSH biosynthesis and provides a direct link between homocysteine and
GSH-dependent redox homeostasis.
-synthase (CBS) catalyzes the
first of two steps in the transsulfuration pathway that converts
homocysteine to cysteine, a precursor of glutathione, a major
intracellular antioxidant. Tumor necrosis factor-
(TNF
), which is
known to enhance production of reactive oxygen species, increased CBS
activity and glutathione levels in HepG2 cells. Western blot analysis
revealed that the higher CBS activity correlated with cleavage of the
enzyme to a truncated form. This cleavage was suppressed by inhibitors of superoxide production or by transfection with an expression vector
for manganese superoxide dismutase. The commonly used proteasome inhibitors, MG132 and lactacystin but not
N-acetyl-Leu-Leu-norleucinal, suppressed the TNF
-induced
response. Targeted proteolysis of CBS was also observed in livers of
mice injected with lipopolysaccharide, which is known to induce TNF
.
Together, these data reveal a novel and previously unknown mechanism of
regulation for homocysteine-linked glutathione homeostasis in cells
challenged by oxidative stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Metabolic link between homocysteine and
glutathione-dependent redox homeostasis.
Elevated levels of homocysteine are an independent risk factor for atherosclerosis in the cerebral, coronary, and peripheral vasculature (2) and are correlated with neural tube defects and Alzheimer's disease (3, 4). However, despite the recent media and scientific attention to hyperhomocysteinemia-linked heart disease, our understanding of the regulation of homocysteine metabolism and its consequent effects on the downstream GSH biosynthetic pathway remains poor.
Cystathionine -synthase (CBS) catalyzes the first step in the
transsulfuration pathway, and its activity is redox-sensitive and
decreases ~2-fold under reductive conditions in vitro (5, 6). Our studies with the human hepatoma cell line, HepG2, have revealed
that oxidative stress induced by exogenous peroxides increases the flux
of homocysteine through transsulfuration and leads to enhanced GSH
synthesis (1).
Reactive oxygen species (ROS) have been implicated in various biological processes, including gene expression, cell growth, differentiation, proliferation, and apoptosis (7-9). It has been shown that the oxidative signaling pathways for intracellular and extracellular peroxides are different, at least in some systems (10). Thus, it is of significant interest to study the consequences of ROS generated via a signaling pathway on homocysteine-linked GSH homeostasis.
TNF is a polypeptide that induces a variety of cellular actions,
depending on its concentration and the type of cells where it acts
(11). As a ubiquitous pro-inflammatory cytokine, TNF
promotes cell
injury through several mechanisms, including the overproduction of
intracellular ROS that may damage critical cellular components such as
proteins, lipids, and DNA (12-14). However, the ability of TNF
to
kill cells appears to be restricted to tumor and virally infected
cells, because normal cells are generally insensitive to its toxic
effect (11). The molecular basis of TNF
-mediated cellular resistance
is not fully understood at present. The mechanism is probably
associated with enhancement of the intracellular antioxidant capacity
(13, 15-17).
The ability of TNF to stimulate endogenous ROS formation and enhance
GSH synthesis prompted us to examine the sensitivity of the flux
through CBS in HepG2 cells treated with TNF
. Additionally, mice were
employed as an animal model to study the effect of lipopolysaccharide (LPS)-induced ROS on the transsulfuration pathway. LPS is a component of the Gram-negative bacterial wall that triggers synthesis and release
of TNF
and stimulates production of ROS in experimental animals
(18-20). Our results reveal an unexpected post-translational mechanism
of activation of CBS induced by TNF
, namely, targeted proteolysis,
and implicates a role for O
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
HepG2 (from ATCC Repository) cells were grown
in MEM (Sigma) with 10% fetal bovine serum (FBS) and maintained at
37 °C, 5% CO2. When cells were 60-80% confluent, the
culture medium was changed to MEM lacking FBS and maintained under
these conditions for 24 h. Experiments were initiated with fresh
MEM lacking FBS and containing TNF (Promega), and cells were
harvested at the desired time. Unless specified otherwise, the
concentration of TNF
employed was 25 ng/ml. For measurement of
cystathionine, cells were preincubated for 1 h with 2.5 mM propargylglycine before treatment with TNF
.
Animal Experiments--
The procedures employed were approved by
the Animal Care Committee at the University of Nebraska-Lincoln.
Animals were housed and sacrificed in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals.
Male mice (BALB/c strain) weighing 30-50 g were divided into two
groups. The LPS-treated group received LPS (2.5 mg/kg, intraperitoneal)
and the control group received saline (1 ml, intraperitoneal). At the
desired time following injection, animals were anesthetized with an
intraperitoneal injection of Beuthanasia®-D Special containing 9.5 mg
of pentobarbital sodium and 1.25 mg of phenytoin sodium
(Schering-Plough Animal Health Corp., Bloomfield, NJ). The mice were
sacrificed by exsanguination, and the livers were rapidly removed,
freeze-clamped in liquid nitrogen, and stored at 70 °C until
further used.
Measurement of ROS-- ROS generation in cells was assessed using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes) as a probe. One hour prior to harvesting of cells, H2DCFDA (10 µg/ml) was added to culture dishes, followed by washing twice with PBS to remove excess probe. Cells were harvested by trypsinization and resuspended in 1 ml of PBS. The cell suspension (10 µl) was mixed with 990 µl, and DCF fluorescence was measured by a fluorometer (PerkinElmer Life Sciences LS50) with excitation at 490 nm and emission at 520 nm and a slit width of 5 nm for both excitation and emission. The fluorescence value was normalized by protein concentration.
Measurement of Intracellular Thiol Concentration-- The cells were washed twice with ice-cold PBS and collected by scraping and divided into two portions. The first (50 µl) was centrifuged at 2,000 × g for 5 min. The supernatant (40 µl) was discarded, and 90 µl of lysis buffer (containing 20 mM Hepes, 25 mM KCl, 0.5% Nonidet P-40, and 1 mM phenylmethanesulfonyl fluoride, pH 7.4) was added. The suspension was kept on ice for 10 min. Following centrifugation at 10,000 × g for 5 min, the supernatant was used to determine protein concentration by the Bradford reagent (Bio-Rad) using bovine serum albumin as the standard. The second aliquot of cells was deproteinized by mixing with an equal amount (50 µl) of fixing solution (16.8 g/liter metaphosphoric acid, 5 M NaCl, and 5 mM EDTA). After 10-min incubation at room temperature, the mixture was centrifuged at 10,000 × g for 3 min, and the supernatant was collected. For measurement of GSH levels in liver, the tissue was homogenized in liquid nitrogen. The homogenate was deproteinized as described above, and supernatant was collected. The supernatants from cell extract and liver homogenate were used to determine thiol compounds by high-performance liquid chromatography as described previously (21).
Measurement of CBS Activity--
The cells were washed twice
with ice-cold PBS and collected by scraping. KCl (150 µl of 1.12%
(w/v)) was added to tubes containing cells and mixed. The cells were
frozen in liquid nitrogen for 5 min and then thawed at room
temperature. After three such cycles, cell lysates were centrifuged at
15,000 × g for 10 min at 4 °C, and the supernatant
was collected. For measurement of CBS activity in liver, the liver
homogenate was mixed with 1.12% (w/v) KCl, and the supernatant was
collected. CBS activity in HepG2 and liver was measured according to
the method reported by Kashiwamata and Greenberg (22). Briefly, 72 µl
of supernatant was mixed with 3 µl of 1.2 mM pyridoxal
phosphate, 15 µl of 2.5 mM propargylglycine, and 10 µl
of 1 M Tris-HCl, pH 8.3. A blank was prepared with the same
components except that 72 µl of 1.12% KCl replaced the supernatant. The mixture was incubated at 37 °C for 15 min. Then, the mixture was
combined with 15 µl of 1.0 M serine and 30 µl of 0.75 M homocysteine and mixed. In some experiments, 0.38 mM AdoMet was added to the reaction mixture to test the
effect of this allosteric regulator of the full-length form of CBS. The
mixture was incubated at 37 °C for 30 min and terminated by adding
15 µl of 50% (v/v) trichloroacetic acid. The mixture was
centrifuged at 10,000 × g for 2 min. 100 µl of
supernatant was mixed with 800 µl of ninhydrin solution. The mixture
was boiled for 5 min and then cooled on ice for 2 min. The absorbance
of the solution was determined at 455 nm. The activity was calculated
using an extinction coefficient for cystathionine of 1155 M1cm
1, and 1 unit of activity
represents formation of 1 nmol of cystathione h
1
mg
1 of total protein at 37 °C.
Northern Blotting-- Northern blot analysis was performed as described previously (1). The bands were quantified by densitometry using Image software (National Institutes of Health). The band intensities were normalized versus 28 S rRNA.
Western Blotting-- Western blot analysis of CBS was performed as described previously (1). Purified polyclonal antibodies against CBS were used for detection of proteins. The membrane was exposed to ECL Hyperfilm (Amersham Biosciences) for 1-5 min, and the film was developed. To ensure equal loading, the membrane was stripped by incubation with 0.1 M lysine, pH 2.9, for 30 min and re-probed using a commercial antibody against actin (Sigma). The bands were quantified densitometrically.
Transfection--
Transfection was performed using the
GeneJammer reagent (Stratagene) according to the manufacturer's
specifications. Briefly, HepG2 cells were transfected with 7 µg of
pCR3-catalase (a gift from Dr. S. G. Rhee, National Institutes of
Health), pcDNA3-Mn-SOD (a gift from Dr. L. W. Oberley,
University of Iowa), or an empty vector, pcDNA3 (Invitrogen, CA).
48 h following transfection, the medium was replaced with fresh
MEM lacking FBS. 24 h later, the cells were treated with TNF
(25 ng/ml) and harvested after 24 h.
Statistical Analysis--
Data from experiments were expressed
as means ± S.D. Statistical comparisons were made using the
Student's paired t test.
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RESULTS |
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Effect of TNF on CBS--
HepG2 cells were incubated with
H2DCFDA that readily diffuses into cells and is trapped
after cleavage by intracellular esterases. Oxidation by intracellular
ROS results in the formation of a highly fluorescent product, DCF, from
H2DCF (23). Enhanced ROS production by cells stimulated by
TNF
(25 ng/ml) has been reported previously (13) and was confirmed
by the observation of a 2-fold increase in DCF fluorescence within
2 h reaching a maximal level in 8 h (not shown). Under these
conditions, morphological changes or evidence of cell death due to
TNF
exposure were not observed.
To monitor changes in the intracellular cystathionine concentration,
which is very low, propargylglycine was used to inhibit the next enzyme
in the pathway, -cystathionase. Addition of propargylglycine to the
culture medium results in a time-dependent increase in the
concentration of cystathionine. Addition of TNF
(25 ng/ml) to HepG2
cells increased cystathionine levels by ~76% after 24 h (Fig.
2A). Under these conditions, a
corresponding increase in GSH content was observed (Fig.
2B), which is in agreement with results reported previously
(13).
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Next, we determined whether increased formation of cystathionine
elicited by TNF was due to an increase in CBS activity. Addition of
TNF
increased CBS activity by ~50% of control values after
16 h of treatment (p < 0.01) and ~60% of
control values after 24 h (p < 0.01, Fig.
3A). The effect of TNF
on
CBS activity was found to be dose-dependent, and 10-25
ng/ml was sufficient to achieve a maximal effect (Fig.
3B).
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The possible influence of TNF on CBS mRNA levels was probed by
Northern blot analysis. As shown in Fig.
4A, TNF
did not alter the
amount of the CBS transcript, ruling out a role for this cytokine in
transcriptional regulation of CBS expression. We next performed Western
blot analysis to determine if TNF
increases CBS activity by
increasing enzyme levels. Under in vitro conditions, two
forms of CBS have been characterized and correspond to the full-length
(with 63-kDa subunits) and truncated forms (with 45-kDa subunits),
respectively (24-26). The truncated form of the enzyme is derived by
limited proteolytic cleavage of the full-length form, is dimeric rather
than tetrameric, and displays a 4-fold higher
kcat value than the full-length form assayed in
the absence of the allosteric activator, AdoMet (24). Although the
45-kDa truncated form of CBS has been reported in aged rat and human liver extracts (27), only the 63-kDa full-length form is detected in
fresh tissues and in HepG2 and other cell types.
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Surprisingly, administration of TNF was found to elicit targeted CBS
cleavage, which was clearly observed at 16 and 24 h following
exposure to TNF
(Fig. 4, B and C). Decrease in
the level of the full-length form was paralleled by an increase in the
level of the truncated form of CBS after 16 and 24 h of treatment. These results suggest that the TNF
-induced increase in CBS activity is due to targeted proteolysis of the full-length tetrameric enzyme to
the more active truncated dimeric form.
Response of CBS to AdoMet after Treatment of TNF--
To
further confirm that the increase in CBS activity by TNF
is due to
the production of the truncated form that has been characterized
previously in vitro (24-26), we determined the activity of
the enzyme in the presence and absence of AdoMet, which is an
allosteric activator of the full-length enzyme. Limited proteolysis of
CBS to generate the truncated form is accompanied by loss of the
C-terminal regulatory domain and a concomitant loss of sensitivity to
the allosteric regulator, AdoMet (26). In control cells that were not
exposed to TNF
, addition of AdoMet resulted in a 3-fold increase in
CBS activity (Fig. 5). In contrast, CBS
activity was stimulated only 1.7-fold by AdoMet in TNF
-treated
cells. These results are consistent with the formation of truncated CBS
lacking the AdoMet-responsive regulatory domain in TNF
-treated
cells. Residual activation that is observed under these conditions is due to the remaining full-length form and corresponds well with the
conversion of ~70% of the full-length form to the truncated one
observed by Western analysis (Fig. 4C).
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Role of ROS in Cleavage of CBS Induced by TNF--
To gain
insight into the role of intracellular ROS in regulating the cleavage
of CBS, cells were treated with specific inhibitors of ROS production
and various antioxidants or ROS scavengers. Among the inhibitors of ROS
production, apocynin has been shown to inhibit the function of NADPH
oxidase, which produces ROS in the plasma membrane (28). In contrast,
rotenone and thenoyltrifluoroacetone are inhibitors of complexes
I and II, respectively, in the mitochondrial electron transport chain
(29). As shown in Fig. 6A,
apocynin (0.3 mM) was ineffective in suppressing
TNF
-mediated proteolysis of CBS suggesting that this pathway for ROS
formation is not involved in signaling CBS cleavage. In contrast, the
combination of rotenone (5 µM) and
thenoyltrifluoroacetone (5 µM) substantially inhibited cleavage of CBS induced by TNF
, suggesting a role for mitochondrial generation of ROS in TNF
-mediated regulation of CBS.
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O (30, 31). Pretreatment of HepG2 cells with Tiron (5 mM), a cell-permeable scavenger of O
. In
contrast, the effect of TNF
on CBS was unaffected by GSH ethyl ester, a cell-permeable scavenger of H2O2 (33).
It has been shown that TNF
induces the production of O
) (34). To examine the role of ONOO
on
TNF
-induced CBS cleavage, HepG2 cells were pretreated with uric
acid, a scavenger of peroxynitrite (32). As shown in Fig. 6A, uric acid (1 mM) did not attenuate the
TNF
-dependent effect on CBS. In addition, treatment of
HepG2 cells with 25 mM dimethyl sulfoxide, a hydroxyl
radical scavenger (35), did not abolish TNF
-mediated cleavage of
CBS. These results specifically implicate O
-induced cleavage of CBS and are consistent with
the previous report that exogenous H2O2, which
enhances flux of homocysteine through the transsulfuration pathway,
does not induce targeted proteolysis of CBS (1).
Superoxide dismutase (SOD) catalyzes dismutation of O-induced CBS cleavage was further confirmed by
transient transfection of HepG2 cells with an expression plasmid for
Mn-SOD. After incubation with TNF
for 24 h, CBS was detected by
Western blotting. As shown in Fig. 6B, transfection with
Mn-SOD inhibited TNF
-induced cleavage of CBS. In contrast,
transfection with an empty vector or an expression plasmid for catalase
(data not shown), did not affect CBS cleavage induced by TNF
.
Effect of Proteasome Inhibitors on Cleavage of CBS Induced by
TNF--
In general, TNF
induces degradation of cellular
proteins by activating one of two principal pathways: cytosolic
calcium-activated proteases (calpains) and the ubiquitin-proteasome
pathway (36). Although ubiquitin-directed degradation in proteasomes
generally leads to complete degradation in most cases, in a few cases,
limited proteolysis is observed (37). We found that ALLN, an inhibitor of calpains I and II as well as an inhibitor of the proteasome (38),
did not block cleavage of CBS induced by TNF
(Fig.
7). In contrast, lactacystin and MG132,
which are widely used as inhibitors of the 20 S and 26 S proteasomes
(36), respectively, effectively inhibited cleavage of CBS induced by
TNF
, implicating a role for the proteasome in proteolytic activation
of CBS.
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GSH Level and CBS Activity in Livers of Mice Treated with
LPS--
We were interested in determining whether our unexpected
observation of targeted proteolysis of CBS in cultured cells could be
extended to an animal model. Administration of the pro-inflammatory agent LPS has been shown to stimulate production of ROS, such as
O, and host susceptibility to LPS appears to
be correlated with the levels of circulating TNF
that develop in
response to LPS (39). Injection of mice with LPS resulted in increased
CBS activity and GSH concentration in livers within 4h (Fig.
8, A and B).
Western blot analysis revealed that the increase in CBS activity was
accompanied by increased conversion of full-length CBS to the truncated
form (Fig. 8C).
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DISCUSSION |
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Two forms of CBS have been characterized under in vitro conditions, a full-length and a truncated form, which exist in the tetrameric and dimeric oligomerization states, respectively (24). The full-length enzyme is a modular protein in which the N-terminal heme domain is followed by a catalytic core and a C-terminal regulatory domain. Deletion of the latter incurs loss of sensitivity to the allosteric activator, AdoMet (24-26). The basal activity associated with the full-length enzyme is enhanced 2- to 3-fold in the presence of AdoMet. In contrast, the kcat for the truncated form is 4-fold higher than the basal activity exhibited by the full-length form but is not responsive to AdoMet. Thus, the effective change in kcat accompanying truncation of CBS is a 1.5- to 2-fold increase over the full-length form in the presence of physiological concentrations of AdoMet. Although the kinetic and spectroscopic properties of the two forms have been studied quite extensively with purified recombinant enzyme, the physiological relevance, if any, of the truncated form is unknown.
In this study, we have made the unexpected observation that a
TNF-induced increase in the flux of homocysteine through the transsulfuration pathway is accompanied by an increase in CBS activity
that is correlated with targeted proteolysis of the enzyme to generate
a truncated form. The concomitant loss of sensitivity of CBS activity
to AdoMet is consistent with the loss of the C-terminal regulatory
domain and generation of the form that has been characterized quite
extensively under in vitro conditions (24, 26). Targeted proteolysis leading to loss of ~18 kDa from the N terminus is incompatible with retention of enzyme activity, because it would destroy the active site as revealed by the structure of the protein (6,
40). An 18-kDa fragment was not detected in these experiments, which
could be due to its instability or the absence of antigenic epitopes in
this fragment. An 18-kDa fragment is similarly not seen during in
vitro limited proteolysis of purified human CBS, which leads to
formation of a 45-kDa band.
The regulation exerted by TNF on CBS activity appears to be
post-translational, because changes in mRNA and protein levels are
not observed (Fig. 4). Rather, TNF
-induced cleavage of CBS results
in the disappearance of the full-length enzyme in HepG2 cells and in
livers of mice treated with LPS with the concomitant appearance of the
truncated form (Figs. 4 and 8). Although our observations with HepG2
cells and mouse livers are qualitatively similar, the time course for
the appearance of the truncated form is different. This is not
surprising due to the differences in the experimental systems in
addition to the fact that TNF
is not the only cytokine induced by
LPS. It has been shown that LPS also induces the production of other
inflammatory cytokines, such as interleukin-1, which may produce ROS
(41).
The activity of CBS is modulated by several different mechanisms. These include: (i) allosteric regulation by AdoMet (24-26) or by Ca2+/calmodulin (42), which increase CBS activity in the absence of protein synthesis; (ii) transcriptional regulation by hormones, such as glucocorticoid and insulin, which stimulates and inhibit CBS gene expression, respectively (43); or (iii) limited proteolysis of the full-length tetrameric form to the truncated dimeric form induced in vitro (27). To our knowledge, regulation by limited proteolysis has not been observed in vivo. Full-length CBS is the predominant form detected in fresh tissue, and limited proteolysis is observed when fresh extracts of human or rat liver are incubated at 4 °C for 7 days or treated with trypsin (27). Interestingly, a pathogenic amber mutation of CBS at codon 409 (W409X) has been reported in a homocystinuric patient (44). However, the predicted ~45-kDa truncated enzyme is not seen due to nonsense-mediated decay of the mutant mRNA precluding translation (45).
The pathway leading to cleavage of CBS is unclear and could, in
principle, involve either the proteasome or a protease. Although the
proteasome in most cases leads to complete degradation of target
proteins, few exceptions are known in which the outcome is limited
proteolysis. For example, the first step in the proteolytic activation
of NFB from its precursor protein p105 to yield p50 is catalyzed by
the proteasome (37, 46). More recently, limited proteolysis of a
membrane-tethered SPT23 transcription factor by the proteasome has been
reported (47).
Among the inhibitors that we tested in this study, MG132 and
lactacystin have been widely used to inhibit the proteasome. However,
their specificity is not absolute, and MG132 and lactacystin have been
reported to inhibit calpain (36) and cathepsin A (48), respectively.
Thus, the inhibition studies do not allow us to unequivocally
distinguish between the involvement of proteasomal versus
nonproteasomal proteases in the TNF-induced cleavage of CBS. Lack of
inhibition by ALLN could result from differences in its kinetics and/or
efficacy, because our observations are made after a significant lag (17 h) following inhibitor addition.
Although the signaling mechanism utilized by TNF to effect targeted
proteolysis is currently unknown, ROS induced by TNF
may serve as
second messengers in cell signaling (49). It has been shown that
TNF
-mediated activation of NF-
B is associated with the
production of ROS and is antiapoptotic (50). TNF
induces generation
of ROS at two sites: the mitochondrial respiratory chain (51) and the
plasma membrane-associated NADPH oxidases (52). Pretreatment of HepG2
cells with rotenone and thenoyltrifluoroacetone, which are inhibitors
of complex I and II of mitochondrial electron transport chain, blocked
TNF
-induced cleavage of CBS (Fig. 6). In contrast, the NADPH oxidase
inhibitor, apocynin, failed to inhibit TNF
-induced cleavage of CBS.
These results support the view that the mitochondrial respiratory chain
is the major source of TNF
-induced ROS (53) that is involved in the
signaling pathway leading to cleavage of CBS.
O (30, 31). Once O
,
H2O2 and ONOO
may be produced in
subsequent reactions. ·OH is generated in turn by reduction of
H2O2 or via ONOO
(54). However,
in contrast to the O
did not abolish cleavage of CBS by TNF
(Fig. 6).
The lack of involvement of H2O2 and
ONOO
was further confirmed by the observation that
Me2SO, a scavenger of ·OH, did not affect the
cleavage of CBS by TNF
. In addition, we have previously reported
that exogenous H2O2, which enhances flux of
homocysteine through the transsulfuration pathway, does not induce
targeted proteolysis of CBS (1). Together, these results imply
selectivity of the ROS involved in TNF
-induced cleavage of CBS, and
O
Although the physiological significance of regulation of CBS by TNF
is not clear, it is likely to be related to redox homeostasis and
oxidant defense. CBS catalyzes the first step in cysteine biosynthesis
from homocysteine via the transsulfuration pathway and is a
quantitatively significant contributor to the GSH pool in liver (Fig.
1). The ability of mammalian cells to preserve intracellular functions
during oxidative challenge critically depends on the use and
replenishment of protective antioxidant systems. Previous studies have
revealed that cellular resistance or sensitivity to TNF
is
associated with increased or decreased SOD levels, respectively (15,
55). Treatment of rat astrocytes with LPS has been shown to increase
transcription of glucose-6-phosphate dehydrogenase (56) and may be
important in preventing GSH depletion and protecting astrocytes against
nitric oxide-mediated cell injury. Recent studies from our laboratory
have indicated that exogenous peroxides result in increased flux of
homocysteine through the transsulfuration pathway and enhanced GSH
synthesis in HepG2 cells, although the mechanism of this regulation is
not known (1). In contrast, antioxidants exert an opposing effect (57).
Thus, it is clear that the transsulfuration pathway plays an important role in the regulation of intracellular redox balance. The redox sensitivity of the transsulfuration pathway can be rationalized as an
autocorrective response that leads to increased GSH synthesis in cells
challenged by oxidative stress. Our studies reveal a novel mechanism
for redox regulation based on targeted proteolysis of a key enzyme in
the transsulfuration pathway, CBS.
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ACKNOWLEDGEMENTS |
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We thank Drs. L. W. Oberley (University of Iowa) and S. G. Rhee (National Institutes of Health) for the generous gifts of the expression plasmids for Mn-SOD and catalase, respectively.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK64959 and by the American Heart Association.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.
An Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 402-472-2941; Fax:
402-472-7842; E-mail: rbanerjee1@unl.edu.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M212376200
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ABBREVIATIONS |
---|
The abbreviations used are:
GSH, glutathione;
CBS, cystathionine -synthase;
ROS, reactive oxygen species;
TNF
, tumor necrosis factor-
;
LPS, lipopolysaccharide;
Mn-SOD, manganese
superoxide dismutase;
H2DCFDA, dichlorodihydrofluorescein diacetate;
AdoMet, S-adenosylmethionine;
ONOO
, peroxynitrite;
MEM, minimal essential medium;
FBS, fetal bovine
serum;
ALLN, N-acetyl-
Leu-Leu-norleucinal.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mosharov, E., Cranford, M. R., and Banerjee, R. (2000) Biochemistry 39, 13005-13011[CrossRef][Medline] [Order article via Infotrieve] |
2. | Refsum, H., Ueland, P. M., Nygard, O., and Vollset, S. E. (1998) Annu. Rev. Med. 49, 31-62[CrossRef][Medline] [Order article via Infotrieve] |
3. | Mills, J. L., McPartlin, J. M., Kirke, P. N., Lee, Y. J., Conle, M. R., and Weir, D. G. (1995) Lancet 345, 149-151[CrossRef][Medline] [Order article via Infotrieve] |
4. | Clarke, R., Smith, A. D., Jobst, K. A., Refsum, H., Sutton, L., and Ueland, P. M. (1998) Arch. Neruol. 55, 1449-1455[CrossRef] |
5. |
Taoka, S.,
Ohja, S.,
Shan, X.,
Kruger, W. D.,
and Banerjee, R.
(1998)
J. Biol. Chem.
273,
25179-25184 |
6. | Taoka, S., Lepore, B. W., Kabil, Ö., Ojha, S., Ringe, D., and Banerjee, R. (2002) Biochemistry 41, 10454-10461[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Sen, C. K.,
and Packer, L.
(1996)
FASEB J.
10,
709-720 |
8. | Suzuki, Y. J., Forman, H. J., and Sevanian, A. (1997) Free Radic. Biol. Med. 22, 269-285[CrossRef][Medline] [Order article via Infotrieve] |
9. | Finkel, T. (1998) Curr. Opin. Cell Biol. 10, 248-253[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Pantopoulos, K.,
Mueller, S.,
Atzberger, A.,
Ansorge, W.,
Stremmel, W.,
and Hentze, M. W.
(1997)
J. Biol. Chem.
272,
9802-9808 |
11. | Beutler, B., and Cerami, A. (1988) Annu. Rev. Biochem. 57, 505-518[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Li, Y. P.,
Schwartz, R. J.,
Waddell, I. D.,
Holloway, B. R.,
and Reid, M. B.
(1998)
FASEB J.
12,
871-880 |
13. |
Morales, A.,
Garcia-Ruiz, C.,
Miranda, M.,
Mari, M.,
Colell, A.,
Ardite, E.,
and Fernandez-Checa, J. C.
(1997)
J. Biol. Chem.
272,
30371-30379 |
14. |
Liu, Y.,
Tergaonkar, V.,
Krishna, S.,
and Androphy, E. J.
(1999)
J. Biol. Chem.
274,
24819-24827 |
15. | Wong, G. H., and Goeddel, D. V. (1988) Science 242, 941-944[Medline] [Order article via Infotrieve] |
16. | Czaja, M. J., Schilsky, M. L., Xu, Y., Schmiedeberg, P., Compton, A., Ridnour, L., and Oberley, L. W. (1994) Am. J. Physiol. 266, G737-G744[Medline] [Order article via Infotrieve] |
17. | Diehl, A. M. (2000) Immunol. Rev. 174, 160-171[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sprong, R. C.,
Winkelhuyzen-Janssen, A. M.,
Aarsman, C. J.,
van Oirschot, J. F.,
van der Bruggen, T.,
and van Asbeck, B. S.
(1998)
Am. J. Respir. Crit. Care Med.
157,
1283-1293 |
19. | Bautista, A. P., and Spitzer, J. J. (1990) Am. J. Physiol. 259, G907-G912[Medline] [Order article via Infotrieve] |
20. | Zurovsky, Y., and Gispaan, I. (1995) Am. J. Kid. Dis. 25, 51-57[Medline] [Order article via Infotrieve] |
21. | Reed, D., Babson, J., Beatty, P., Brodie, A., Ellis, W., and Potter, D. (1980) Anal. Biochem. 106, 55-62[Medline] [Order article via Infotrieve] |
22. | Kashiwamata, S., and Greenberg, D. M. (1970) Biochim. Biophys. Acta 212, 488-500[Medline] [Order article via Infotrieve] |
23. | Zhu, H., Bannenberg, G. L., Moldeus, P., and Shertzer, H. G. (1994) Arch. Toxicol. 68, 582-587[CrossRef][Medline] [Order article via Infotrieve] |
24. | Taoka, S., Widjaja, L., and Banerjee, R. (1999) Biochemistry 38, 13155-13161[CrossRef][Medline] [Order article via Infotrieve] |
25. | Shan, X., and Kruger, W. D. (1998) Nat. Genet. 19, 91-93[Medline] [Order article via Infotrieve] |
26. | Kery, V., Poneleit, L., and Kraus, J. (1998) Arch. Biochem. Biophys. 355, 222-232[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Skovby, F.,
Kraus, J. P.,
and Rosenberg, L. E.
(1984)
J. Biol. Chem.
259,
588-593 |
28. |
Beswick, R. A.,
Dorrance, A. M.,
Leite, R.,
and Webb, R. C.
(2001)
Hypertension
38,
1107-1111 |
29. | Corda, S., Laplace, C., Vicaut, E., and Duranteau, J. (2001) Am. J. Respir. 24, 762-768 |
30. | Meier, B., Radeke, H. H., Selle, S., Younes, M., Sies, H., Resch, K., and Habermehl, G. G. (1989) Biochem. J. 263, 539-545[Medline] [Order article via Infotrieve] |
31. | Hennet, T., Richter, C., and Peterhans, E. (1993) Biochem. J. 289, 587-592[Medline] [Order article via Infotrieve] |
32. | Li, C. Q., Trudel, L. J., and Wogan, G. N. (2002) Chem. Res. Toxicol. 15, 527-535[CrossRef][Medline] [Order article via Infotrieve] |
33. | Ishibashi, M., Akazawa, S., Sakamaki, H., Matsumoto, K., Yamasaki, H., Yamaguchi, Y., Goto, S., Urata, Y., Kondo, T., and Nagataki, S. (1997) Free Radic. Biol. Med. 22, 447-454[CrossRef][Medline] [Order article via Infotrieve] |
34. | Phelps, D. T., Ferro, T. J., Higgins, P. J., Shankar, R., Parker, D. M., and Johnson, A. (1995) Am. J. Physiol. 269, L551-L559[Medline] [Order article via Infotrieve] |
35. | Haddad, J. J., and Land, S. C. (2001) FEBS Lett. 505, 269-274[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Mallampalli, R. K.,
Ryan, A. J.,
Salome, R. G.,
and Jackowski, S.
(2000)
J. Biol. Chem.
275,
9699-9708 |
37. | Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785[Medline] [Order article via Infotrieve] |
38. | Wu, H.-M., Chi, K.-H., and Lin, W.-W. (2002) FEBS Lett. 526, 101-105[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Dinges, M. M.,
and Schlievert, P. M.
(2001)
Infect. Immun.
69,
7169-7172 |
40. |
Meier, M.,
Janosik, M.,
Kery, V.,
Kraus, J. P.,
and Burkhard, P.
(2001)
EMBO J.
20,
3910-3916 |
41. |
Hsu, H. Y.,
and Wen, M. H.
(2002)
J. Biol. Chem.
277,
22131-22139 |
42. |
Eto, K.,
Ogasawara, M.,
Umemura, K.,
Nagai, Y.,
and Kimura, H.
(2002)
J. Neurosci.
22,
3386-3391 |
43. |
Ratnam, S.,
Maclean, K. N.,
Jacobs, R. L.,
Brosnan, M. E.,
Kraus, J. P.,
and Brosnan, J. T.
(2002)
J. Biol. Chem.
277,
42912-42918 |
44. | Kraus, J. P., Janosik, M., Kozich, V., Mandell, R., Shih, V., Sperandeo, M. P., Sebastio, G., de Franchis, R., Andria, G., Kluijtmans, L. A., Blom, H., Boers, G. H., Gordon, R. B., Kamoun, P., Tsai, M. Y., Kruger, W. D., Koch, H. G., Ohura, T., and Gaustadnes, M. (1999) Hum. Mutat. 13, 362-375[CrossRef][Medline] [Order article via Infotrieve] |
45. | Janosik, M., Oliveriusova, J., Janosikova, B., Sokolova, J., Kraus, E., Kraus, J. P., and Kozich, V. (2001) Am. J. Hum. Genet. 68, 1506-1513[CrossRef][Medline] [Order article via Infotrieve] |
46. | Lin, L., DeMartino, G. N., and Greene, W. C. (1998) Cell 92, 819-828[Medline] [Order article via Infotrieve] |
47. | Rape, M., Hoppe, T., Gorr, I., Kalocay, M., Richly, H., and Jentsch, S. (2001) Cell 107, 667-677[Medline] [Order article via Infotrieve] |
48. | Ostrowska, H., Wojcik, C., Wilk, S., Omura, S., Kozlowski, L., Stoklosa, T., Worowski, K., and Radziwon, P. (2000) Int. J. Biochem. Cell Biol. 32, 747-757[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Chandel, N. S.,
Schumacker, P. T.,
and Arch, R. H.
(2001)
J. Biol. Chem.
276,
42728-42736 |
50. |
Deshpande, S. S.,
Angkeow, P.,
Huang, J.,
Ozaki, M.,
and Irani, K.
(2000)
FASEB J.
14,
1705-1714 |
51. | Goossens, V., Grooten, J., De Vos, K., and Fiers, W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8115-8119[Abstract] |
52. | Thannickal, V. J., and Fanburg, B. L. (2000) Am. J. Physiol. 279, L1005-L1028 |
53. |
Goossens, V.,
Grooten, J.,
and Fiers, W.
(1996)
J. Biol. Chem.
271,
192-196 |
54. | Jacobson, M. D. (1996) Trends Biochem. Sci. 21, 83-86[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Manna, S. K.,
Zhang, H. J.,
Yan, T.,
Oberley, L. W.,
and Aggarwal, B. B.
(1998)
J. Biol. Chem.
273,
13245-13254 |
56. | Garcia-Nogales, P., Almeida, A., Fernandez, E., Medina, J. M., and Bolanos, J. P. (1999) J. Neurochem. 72, 1750-1758[CrossRef][Medline] [Order article via Infotrieve] |
57. | Vitvitsky, V., Mosharov, E., Tritt, M., Ataullakhanov, F., and Banerjee, R. (2003) Redox Rep. 8, 57-63[CrossRef][Medline] [Order article via Infotrieve] |