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• W1967535331 abstract "Cystathionine औ-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-α (TNFα), 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 notN-acetyl-Leu-Leu-norleucinal, suppressed the TNFα-induced response. Targeted proteolysis of CBS was also observed in livers of mice injected with lipopolysaccharide, which is known to induce TNFα. Together, these data reveal a novel and previously unknown mechanism of regulation for homocysteine-linked glutathione homeostasis in cells challenged by oxidative stress. Cystathionine औ-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-α (TNFα), 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 notN-acetyl-Leu-Leu-norleucinal, suppressed the TNFα-induced response. Targeted proteolysis of CBS was also observed in livers of mice injected with lipopolysaccharide, which is known to induce TNFα. Together, these data reveal a novel and previously unknown mechanism of regulation for homocysteine-linked glutathione homeostasis in cells challenged by oxidative stress. glutathione cystathionine औ-synthase reactive oxygen species tumor necrosis factor-α lipopolysaccharide manganese superoxide dismutase dichlorodihydrofluorescein diacetate S-adenosylmethionine peroxynitrite minimal essential medium fetal bovine serum N-acetyl- Leu-Leu-norleucinal 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 ∼507 of the cysteine in the GSH pool in cultured human liver cells is derived from homocysteine (1Mosharov E. Cranford M.R. Banerjee R. Biochemistry. 2000; 39: 13005-13011Crossref PubMed Scopus (429) Google Scholar). Thus, the transsulfuration pathway is a quantitatively significant contributor to GSH biosynthesis and provides a direct link between homocysteine and GSH-dependent redox homeostasis. Elevated levels of homocysteine are an independent risk factor for atherosclerosis in the cerebral, coronary, and peripheral vasculature (2Refsum H. Ueland P.M. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1836) Google Scholar) and are correlated with neural tube defects and Alzheimer's disease (3Mills J.L. McPartlin J.M. Kirke P.N. Lee Y.J. Conle M.R. Weir D.G. Lancet. 1995; 345: 149-151Abstract PubMed Scopus (500) Google Scholar, 4Clarke R. Smith A.D. Jobst K.A. Refsum H. Sutton L. Ueland P.M. Arch. Neruol. 1998; 55: 1449-1455Crossref PubMed Scopus (1261) Google Scholar). 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 औ-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 (5Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar,6Taoka S. Lepore B.W. Kabil Ö. Ojha S. Ringe D. Banerjee R. Biochemistry. 2002; 41: 10454-10461Crossref PubMed Scopus (126) Google Scholar). 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 (1Mosharov E. Cranford M.R. Banerjee R. Biochemistry. 2000; 39: 13005-13011Crossref PubMed Scopus (429) Google Scholar). Reactive oxygen species (ROS) have been implicated in various biological processes, including gene expression, cell growth, differentiation, proliferation, and apoptosis (7Sen C.K. Packer L. FASEB J. 1996; 10: 709-720Crossref PubMed Scopus (1772) Google Scholar, 8Suzuki Y.J. Forman H.J. Sevanian A. Free Radic. Biol. Med. 1997; 22: 269-285Crossref PubMed Scopus (1255) Google Scholar, 9Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1006) Google Scholar). It has been shown that the oxidative signaling pathways for intracellular and extracellular peroxides are different, at least in some systems (10Pantopoulos K. Mueller S. Atzberger A. Ansorge W. Stremmel W. Hentze M.W. J. Biol. Chem. 1997; 272: 9802-9808Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Thus, it is of significant interest to study the consequences of ROS generated via a signaling pathway on homocysteine-linked GSH homeostasis. TNFα is a polypeptide that induces a variety of cellular actions, depending on its concentration and the type of cells where it acts (11Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (730) Google Scholar). As a ubiquitous pro-inflammatory cytokine, TNFα promotes cell injury through several mechanisms, including the overproduction of intracellular ROS that may damage critical cellular components such as proteins, lipids, and DNA (12Li Y.P. Schwartz R.J. Waddell I.D. Holloway B.R. Reid M.B. FASEB J. 1998; 12: 871-880Crossref PubMed Google Scholar, 13Morales A. Garcia-Ruiz C. Miranda M. Mari M. Colell A. Ardite E. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 30371-30379Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 14Liu Y. Tergaonkar V. Krishna S. Androphy E.J. J. Biol. Chem. 1999; 274: 24819-24827Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, the ability of TNFα to kill cells appears to be restricted to tumor and virally infected cells, because normal cells are generally insensitive to its toxic effect (11Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (730) Google Scholar). The molecular basis of TNFα-mediated cellular resistance is not fully understood at present. The mechanism is probably associated with enhancement of the intracellular antioxidant capacity (13Morales A. Garcia-Ruiz C. Miranda M. Mari M. Colell A. Ardite E. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 30371-30379Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 15Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (836) Google Scholar, 16Czaja M.J. Schilsky M.L. Xu Y. Schmiedeberg P. Compton A. Ridnour L. Oberley L.W. Am. J. Physiol. 1994; 266: G737-G744PubMed Google Scholar, 17Diehl A.M. Immunol. Rev. 2000; 174: 160-171Crossref PubMed Scopus (267) Google Scholar). The ability of TNFα 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 TNFα. 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 TNFα and stimulates production of ROS in experimental animals (18Sprong R.C. Winkelhuyzen-Janssen A.M. Aarsman C.J. van Oirschot J.F. van der Bruggen T. van Asbeck B.S. Am. J. Respir. Crit. Care Med. 1998; 157: 1283-1293Crossref PubMed Scopus (186) Google Scholar, 19Bautista A.P. Spitzer J.J. Am. J. Physiol. 1990; 259: G907-G912PubMed Google Scholar, 20Zurovsky Y. Gispaan I. Am. J. Kid. Dis. 1995; 25: 51-57Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Our results reveal an unexpected post-translational mechanism of activation of CBS induced by TNFα, namely, targeted proteolysis, and implicates a role for O2⨪ in this process. HepG2 (from ATCC Repository) cells were grown in MEM (Sigma) with 107 fetal bovine serum (FBS) and maintained at 37 °C, 57 CO2. When cells were 60–807 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 TNFα (Promega), and cells were harvested at the desired time. Unless specified otherwise, the concentration of TNFα employed was 25 ng/ml. For measurement of cystathionine, cells were preincubated for 1 h with 2.5 mm propargylglycine before treatment with TNFα. 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. 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 ॖg/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 ॖl) was mixed with 990 ॖl, 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. The cells were washed twice with ice-cold PBS and collected by scraping and divided into two portions. The first (50 ॖl) was centrifuged at 2,000 × g for 5 min. The supernatant (40 ॖl) was discarded, and 90 ॖl of lysis buffer (containing 20 mmHepes, 25 mm KCl, 0.57 Nonidet P-40, and 1 mmphenylmethanesulfonyl 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 ॖl) of fixing solution (16.8 g/liter metaphosphoric acid, 5 m NaCl, and 5 mmEDTA). 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 (21Reed D. Babson J. Beatty P. Brodie A. Ellis W. Potter D. Anal. Biochem. 1980; 106: 55-62Crossref PubMed Scopus (1119) Google Scholar). The cells were washed twice with ice-cold PBS and collected by scraping. KCl (150 ॖl of 1.127 (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 1.127 (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 (22Kashiwamata S. Greenberg D.M. Biochim. Biophys. Acta. 1970; 212: 488-500Crossref PubMed Scopus (104) Google Scholar). Briefly, 72 ॖl of supernatant was mixed with 3 ॖl of 1.2 mm pyridoxal phosphate, 15 ॖl of 2.5 mm propargylglycine, and 10 ॖl of 1 m Tris-HCl, pH 8.3. A blank was prepared with the same components except that 72 ॖl of 1.127 KCl replaced the supernatant. The mixture was incubated at 37 °C for 15 min. Then, the mixture was combined with 15 ॖl of 1.0 m serine and 30 ॖl of 0.75m 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 ॖl of 507 (v/v) trichloroacetic acid. The mixture was centrifuged at 10,000 × g for 2 min. 100 ॖl of supernatant was mixed with 800 ॖl 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 1155m−1cm−1, and 1 unit of activity represents formation of 1 nmol of cystathione h−1mg−1 of total protein at 37 °C. Northern blot analysis was performed as described previously (1Mosharov E. Cranford M.R. Banerjee R. Biochemistry. 2000; 39: 13005-13011Crossref PubMed Scopus (429) Google Scholar). The bands were quantified by densitometry using Image software (National Institutes of Health). The band intensities were normalized versus 28 S rRNA. Western blot analysis of CBS was performed as described previously (1Mosharov E. Cranford M.R. Banerjee R. Biochemistry. 2000; 39: 13005-13011Crossref PubMed Scopus (429) Google Scholar). 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 was performed using the GeneJammer reagent (Stratagene) according to the manufacturer's specifications. Briefly, HepG2 cells were transfected with 7 ॖg 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 TNFα (25 ng/ml) and harvested after 24 h. Data from experiments were expressed as means ± S.D. Statistical comparisons were made using the Student's paired t test. 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 (23Zhu H. Bannenberg G.L. Moldeus P. Shertzer H.G. Arch. Toxicol. 1994; 68: 582-587Crossref PubMed Scopus (249) Google Scholar). Enhanced ROS production by cells stimulated by TNFα (25 ng/ml) has been reported previously (13Morales A. Garcia-Ruiz C. Miranda M. Mari M. Colell A. Ardite E. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 30371-30379Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) 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 TNFα 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, γ-cystathionase. Addition of propargylglycine to the culture medium results in a time-dependent increase in the concentration of cystathionine. Addition of TNFα (25 ng/ml) to HepG2 cells increased cystathionine levels by ∼767 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 (13Morales A. Garcia-Ruiz C. Miranda M. Mari M. Colell A. Ardite E. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 30371-30379Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Next, we determined whether increased formation of cystathionine elicited by TNFα was due to an increase in CBS activity. Addition of TNFα increased CBS activity by ∼507 of control values after 16 h of treatment (p < 0.01) and ∼607 of control values after 24 h (p < 0.01, Fig.3A). The effect of TNFα on CBS activity was found to be dose-dependent, and 10–25 ng/ml was sufficient to achieve a maximal effect (Fig.3B). The possible influence of TNFα on CBS mRNA levels was probed by Northern blot analysis. As shown in Fig.4A, TNFα 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 TNFα 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 (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar, 25Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar, 26Kery V. Poneleit L. Kraus J. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (139) Google Scholar). 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 higherkcat value than the full-length form assayed in the absence of the allosteric activator, AdoMet (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar). Although the 45-kDa truncated form of CBS has been reported in aged rat and human liver extracts (27Skovby F. Kraus J.P. Rosenberg L.E. J. Biol. Chem. 1984; 259: 588-593Abstract Full Text PDF PubMed Google Scholar), only the 63-kDa full-length form is detected in fresh tissues and in HepG2 and other cell types. Surprisingly, administration of TNFα was found to elicit targeted CBS cleavage, which was clearly observed at 16 and 24 h following exposure to TNFα (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 TNFα-induced increase in CBS activity is due to targeted proteolysis of the full-length tetrameric enzyme to the more active truncated dimeric form. To further confirm that the increase in CBS activity by TNFα is due to the production of the truncated form that has been characterized previously in vitro (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar, 25Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar, 26Kery V. Poneleit L. Kraus J. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (139) Google Scholar), 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 (26Kery V. Poneleit L. Kraus J. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (139) Google Scholar). In control cells that were not exposed to TNFα, 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 TNFα-treated cells. These results are consistent with the formation of truncated CBS lacking the AdoMet-responsive regulatory domain in TNFα-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 ∼707 of the full-length form to the truncated one observed by Western analysis (Fig. 4C). 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 (28Beswick R.A. Dorrance A.M. Leite R. Webb R.C. Hypertension. 2001; 38: 1107-1111Crossref PubMed Scopus (306) Google Scholar). In contrast, rotenone and thenoyltrifluoroacetone are inhibitors of complexes I and II, respectively, in the mitochondrial electron transport chain (29Corda S. Laplace C. Vicaut E. Duranteau J. Am. J. Respir. 2001; 24: 762-768Google Scholar). As shown in Fig. 6A, apocynin (0.3 mm) was ineffective in suppressing TNFα-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 ॖm) and thenoyltrifluoroacetone (5 ॖm) substantially inhibited cleavage of CBS induced by TNFα, suggesting a role for mitochondrial generation of ROS in TNFα-mediated regulation of CBS. O2⨪ is one of the primary ROS produced in cells treated with TNFα (30Meier B. Radeke H.H. Selle S. Younes M. Sies H. Resch K. Habermehl G.G. Biochem. J. 1989; 263: 539-545Crossref PubMed Scopus (571) Google Scholar, 31Hennet T. Richter C. Peterhans E. Biochem. J. 1993; 289: 587-592Crossref PubMed Scopus (261) Google Scholar). Pretreatment of HepG2 cells with Tiron (5 mm), a cell-permeable scavenger of O2⨪ (32Li C.Q. Trudel L.J. Wogan G.N. Chem. Res. Toxicol. 2002; 15: 527-535Crossref PubMed Scopus (51) Google Scholar), effectively blocked the cleavage of CBS mediated by TNFα. In contrast, the effect of TNFα on CBS was unaffected by GSH ethyl ester, a cell-permeable scavenger of H2O2 (33Ishibashi M. Akazawa S. Sakamaki H. Matsumoto K. Yamasaki H. Yamaguchi Y. Goto S. Urata Y. Kondo T. Nagataki S. Free Radic. Biol. Med. 1997; 22: 447-454Crossref PubMed Scopus (67) Google Scholar). It has been shown that TNFα induces the production of O2⨪ as well as NO, resulting in the formation of peroxynitrite (ONOO−) (34Phelps D.T. Ferro T.J. Higgins P.J. Shankar R. Parker D.M. Johnson A. Am. J. Physiol. 1995; 269: L551-L559PubMed Google Scholar). To examine the role of ONOO− on TNFα-induced CBS cleavage, HepG2 cells were pretreated with uric acid, a scavenger of peroxynitrite (32Li C.Q. Trudel L.J. Wogan G.N. Chem. Res. Toxicol. 2002; 15: 527-535Crossref PubMed Scopus (51) Google Scholar). As shown in Fig.6A, uric acid (1 mm) did not attenuate the TNFα-dependent effect on CBS. In addition, treatment of HepG2 cells with 25 mm dimethyl sulfoxide, a hydroxyl radical scavenger (35Haddad J.J. Land S.C. FEBS Lett. 2001; 505: 269-274Crossref PubMed Scopus (225) Google Scholar), did not abolish TNFα-mediated cleavage of CBS. These results specifically implicate O2⨪ as the ROS that is involved in TNFα-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 (1Mosharov E. Cranford M.R. Banerjee R. Biochemistry. 2000; 39: 13005-13011Crossref PubMed Scopus (429) Google Scholar). Superoxide dismutase (SOD) catalyzes dismutation of O2⨪ to H2O2 and O2. The involvement of O2⨪ in TNFα-induced CBS cleavage was further confirmed by transient transfection of HepG2 cells with an expression plasmid for Mn-SOD. After incubation with TNFα for 24 h, CBS was detected by Western blotting. As shown in Fig. 6B, transfection with Mn-SOD inhibited TNFα-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 TNFα. In general, TNFα induces degradation of cellular proteins by activating one of two principal pathways: cytosolic calcium-activated proteases (calpains) and the ubiquitin-proteasome pathway (36Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although ubiquitin-directed degradation in proteasomes generally leads to complete degradation in most cases, in a few cases, limited proteolysis is observed (37Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar). We found that ALLN, an inhibitor of calpains I and II as well as an inhibitor of the proteasome (38Wu H.-M. Chi K.-H. Lin W.-W. FEBS Lett. 2002; 526: 101-105Crossref PubMed Scopus (78) Google Scholar), did not block cleavage of CBS induced by TNFα (Fig.7). In contrast, lactacystin and MG132, which are widely used as inhibitors of the 20 S and 26 S proteasomes (36Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), respectively, effectively inhibited cleavage of CBS induced by TNFα, implicating a role for the proteasome in proteolytic activation of CBS. 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 O2⨪, in the liver (19Bautista A.P. Spitzer J.J. Am. J. Physiol. 1990; 259: G907-G912PubMed Google Scholar) and kidney (20Zurovsky Y. Gispaan I. Am. J. Kid. Dis. 1995; 25: 51-57Abstract Full Text PDF PubMed Scopus (36) Google Scholar) of mice. LPS also induces production of TNFα, and host susceptibility to LPS appears to be correlated with the levels of circulating TNFα that develop in response to LPS (39Dinges M.M. Schlievert P.M. Infect. Immun. 2001; 69: 7169-7172Crossref PubMed Scopus (46) Google Scholar). 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). Two forms of CBS have been characterized under in vitroconditions, a full-length and a truncated form, which exist in the tetrameric and dimeric oligomerization states, respectively (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar). 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 (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar, 25Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar, 26Kery V. Poneleit L. Kraus J. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (139) Google Scholar). 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 TNFα-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 (24Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (83) Google Scholar, 26Kery V. Poneleit L. Kraus J. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (139) Google Scholar). 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 (6Taoka S. Lepore B.W. Kabil Ö. Ojha S. Ringe D. Banerjee R. Biochemistry. 2002; 41: 10454-10461Crossre" @default.
• W1967535331 created "2016-06-24" @default.
• W1967535331 creator A5013907641 @default.
• W1967535331 creator A5018201691 @default.
• W1967535331 date "2003-05-01" @default.
• W1967535331 modified "2023-09-28" @default.
• W1967535331 title "Tumor Necrosis Factor-α-induced Targeted Proteolysis of Cystathionine औ-Synthase Modulates Redox Homeostasis" @default.
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