Tumor Necrosis Factor-alpha -induced Targeted Proteolysis of Cystathionine beta -Synthase Modulates Redox Homeostasis*

Cheng-Gang Zou and Ruma BanerjeeDagger

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystathionine beta -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-alpha (TNFalpha ), 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 TNFalpha -induced response. Targeted proteolysis of CBS was also observed in livers of mice injected with lipopolysaccharide, which is known to induce TNFalpha . 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

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.


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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 beta -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.

TNFalpha 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, TNFalpha 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 TNFalpha 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 TNFalpha -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 TNFalpha 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 TNFalpha . 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 TNFalpha and stimulates production of ROS in experimental animals (18-20). Our results reveal an unexpected post-translational mechanism of activation of CBS induced by TNFalpha , namely, targeted proteolysis, and implicates a role for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in this process.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TNFalpha (Promega), and cells were harvested at the desired time. Unless specified otherwise, the concentration of TNFalpha employed was 25 ng/ml. For measurement of cystathionine, cells were preincubated for 1 h with 2.5 mM propargylglycine before treatment with TNFalpha .

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, 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 M-1cm-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 TNFalpha (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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of TNFalpha 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 TNFalpha (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 TNFalpha 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, gamma -cystathionase. Addition of propargylglycine to the culture medium results in a time-dependent increase in the concentration of cystathionine. Addition of TNFalpha (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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Fig. 2.   Effect of TNFalpha on cystathionine and GSH. HepG2 Cells were incubated with 25 ng/ml TNFalpha in the presence (A) or absence of 2.5 mM proparglyglycine (B). Cellular cystathionine (A) and GSH (B) concentrations were determined by high performance liquid chromatography as described under "Experimental Procedures." Empty bars represent values without treatment of TNFalpha ; solid bars represent values with treatment of TNFalpha . *, p < 0.05 versus control (without TNFalpha ) at each time point. Results are the means ± S.D. of four experiments.

Next, we determined whether increased formation of cystathionine elicited by TNFalpha was due to an increase in CBS activity. Addition of TNFalpha 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 TNFalpha 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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Fig. 3.   Time and dose dependence of the TNFalpha effect on CBS activity. A, HepG2 cells were incubated with 25 ng/ml TNFalpha for varying times; B, HepG2 Cells were incubated with varying TNFalpha concentrations for 24 h. CBS activity was determined as described under "Experimental Procedures." A, empty bars represent values without treatment of TNFalpha , and solid bars represent values with treatment of TNFalpha . *, p < 0.05 versus control (without TNFalpha ) at each time. In B: *, p < 0.05 versus control (without TNFalpha ). Results are the means ± S.D. of three experiments.

The possible influence of TNFalpha on CBS mRNA levels was probed by Northern blot analysis. As shown in Fig. 4A, TNFalpha 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 TNFalpha 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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Fig. 4.   Effect of TNFalpha on CBS mRNA and protein levels. HepG2 cells were incubated with 25 ng/ml TNFalpha . A, Northern blot analysis. Equal loading of samples was ensured by comparing the 28 S rRNA in each lane. The blot is representative of five independent experiments. B, Western blot analysis of CBS. Total protein in cell extracts were separated on 10% SDS-PAGE. CBS and actin were detected using specific antibodies as described under "Experimental Procedures." The blot is typical of five experiments. C, graphic representation of truncated CBS quantitated by spot densitometry of the Western blots and normalized to actin levels, used as an equal loading control.

Surprisingly, administration of TNFalpha was found to elicit targeted CBS cleavage, which was clearly observed at 16 and 24 h following exposure to TNFalpha (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 TNFalpha -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 TNFalpha -- To further confirm that the increase in CBS activity by TNFalpha 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 TNFalpha , 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 TNFalpha -treated cells. These results are consistent with the formation of truncated CBS lacking the AdoMet-responsive regulatory domain in TNFalpha -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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Fig. 5.   Response of CBS to AdoMet after treatment with TNFalpha . HepG2 Cells were incubated with 25 ng/ml TNFalpha . CBS activity was determined in the presence (solid bars) and absence (empty bars) of 0.38 mM AdoMet. *, p < 0.05 versus control (without AdoMet) at each time. Results are the means ± S.D. of three experiments.

Role of ROS in Cleavage of CBS Induced by TNFalpha -- 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 TNFalpha -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 TNFalpha , suggesting a role for mitochondrial generation of ROS in TNFalpha -mediated regulation of CBS.


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Fig. 6.   Effect of inhibitors of ROS production and scavengers of ROS on TNFalpha -induced cleavage of CBS. A, HepG2 cells were preincubated with apocynin (0.3 mM); rotenone/trifluoracetone (Rot/TTFA, 5 µM each); Tiron (5 mM); GSH ethyl ester (GSH, 5 mM); uric acid (1 mM); and dimethyl sulfoxide (DMSO, 25 mM) before treatment with 25 ng/ml TNFalpha for 16 h. B, HepG2 cells were transfected with 7 µg of pcDNA3-Mn-SOD or an empty vector, pcDNA3, for 24 h. After treatment with TNFalpha for 24 h, CBS and actin were detected by Western blot analysis. The blot is representative of three independent experiments.

O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is one of the primary ROS produced in cells treated with TNFalpha (30, 31). Pretreatment of HepG2 cells with Tiron (5 mM), a cell-permeable scavenger of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (32), effectively blocked the cleavage of CBS mediated by TNFalpha . In contrast, the effect of TNFalpha on CBS was unaffected by GSH ethyl ester, a cell-permeable scavenger of H2O2 (33). It has been shown that TNFalpha induces the production of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> as well as NO, resulting in the formation of peroxynitrite (ONOO-) (34). To examine the role of ONOO- on TNFalpha -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 TNFalpha -dependent effect on CBS. In addition, treatment of HepG2 cells with 25 mM dimethyl sulfoxide, a hydroxyl radical scavenger (35), did not abolish TNFalpha -mediated cleavage of CBS. These results specifically implicate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> as the ROS that is involved in TNFalpha -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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> to H2O2 and O2. The involvement of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in TNFalpha -induced CBS cleavage was further confirmed by transient transfection of HepG2 cells with an expression plasmid for Mn-SOD. After incubation with TNFalpha for 24 h, CBS was detected by Western blotting. As shown in Fig. 6B, transfection with Mn-SOD inhibited TNFalpha -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 TNFalpha .

Effect of Proteasome Inhibitors on Cleavage of CBS Induced by TNFalpha -- In general, TNFalpha 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 TNFalpha (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 TNFalpha , implicating a role for the proteasome in proteolytic activation of CBS.


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Fig. 7.   Effect of calpain and proteasome inhibitors on TNFalpha -induced cleavage of CBS. HepG2 cells were preincubated with ALLN (calpain I inhibitor, 20 µM), lactacystin (20 S proteasome inhibitor, 4 µM), or MG132 (calpain and proteasome inhibitor, 10 µM) for 1 h prior to treatment with 25 ng/ml TNFalpha for 16 h. Changes in CBS were monitored by Western blot analysis, and actin was employed as an equal loading control.

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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, in the liver (19) and kidney (20) of mice. LPS also induces production of TNFalpha , and host susceptibility to LPS appears to be correlated with the levels of circulating TNFalpha 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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Fig. 8.   Effect of LPS administration to mice on GSH concentration and CBS. Changes in GSH level (A) and CBS activity (B). *, p < 0.05 versus control (without AdoMet) at each time. Results are the means ± S.D. of three experiments. C, Western blot analysis of mouse liver extracts using CBS antibodies. The blot is representative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TNFalpha -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 TNFalpha on CBS activity appears to be post-translational, because changes in mRNA and protein levels are not observed (Fig. 4). Rather, TNFalpha -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 TNFalpha 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 NFkappa B 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 TNFalpha -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 TNFalpha to effect targeted proteolysis is currently unknown, ROS induced by TNFalpha may serve as second messengers in cell signaling (49). It has been shown that TNFalpha -mediated activation of NF-kappa B is associated with the production of ROS and is antiapoptotic (50). TNFalpha 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 TNFalpha -induced cleavage of CBS (Fig. 6). In contrast, the NADPH oxidase inhibitor, apocynin, failed to inhibit TNFalpha -induced cleavage of CBS. These results support the view that the mitochondrial respiratory chain is the major source of TNFalpha -induced ROS (53) that is involved in the signaling pathway leading to cleavage of CBS.

O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is one of the primary ROS produced in cells induced by TNFalpha (30, 31). Once O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is generated in response to TNFalpha , 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> scavengers, Tiron or Mn-SOD, scavengers of H2O2 and ONOO- did not abolish cleavage of CBS by TNFalpha (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 TNFalpha . 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 TNFalpha -induced cleavage of CBS, and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> formation appears to be important in this process.

Although the physiological significance of regulation of CBS by TNFalpha 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 TNFalpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: GSH, glutathione; CBS, cystathionine beta -synthase; ROS, reactive oxygen species; TNFalpha , tumor necrosis factor-alpha ; 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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