Inducible Nitric-oxide Synthase Is Regulated by the Proteasome Degradation Pathway

Abstract

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis froml-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, andN-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited byN ω-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitorstrans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH4Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-κB inhibitor IκB-α, thus preventing activation of NF-κB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS. Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis froml-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, andN-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited byN ω-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitorstrans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH4Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-κB inhibitor IκB-α, thus preventing activation of NF-κB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS. nitric oxide nitric-oxide synthase inducible NOS N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal N-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norleucinal lipopolysaccharide NF-κB inhibitor human embryonic kidney human bladder transitional cell papilloma interleukin 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)- propane-1,3-diol HEK293 cell line stably expressing iNOS N ω-nitro-l-arginine methyl ester −9, iNOS splice variant with deletion of exons 8 and 9 Nitric oxide (NO),1 an important signaling and cytotoxic molecule, is synthesized froml-arginine by isoforms of nitric-oxide synthase (NOS) (1Palmer R.M. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9608) Google Scholar,2Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (819) Google Scholar). As a signaling molecule, NO is produced by two constitutive Ca2+-dependent isoforms, neuronal and endothelial NOS (or NOS1 and NOSIII, respectively). Ca2+-activated calmodulin binds to and transiently activates constitutive NOS dimers (2Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (819) Google Scholar, 3Abu-Soud H.M. Stuehr D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (407) Google Scholar). Because of the transient nature of elevated Ca2+ levels, the activity of NO produced is short-lived. As an agent of inflammation and cell-mediated immunity, NO is produced by a Ca2+-independent cytokine-inducible NOS (iNOS or NOSII) that is widely expressed in diverse cell types under transcriptional regulation by inflammatory mediators (4Xie Q.-W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1745) Google Scholar, 5Geller D.A. Lowenstein C.J. Shapiro R.A. Nussler A.K. Di Silvio M. Wang S.C. Nakayama D.K. Simmons R.L. Snyder S.H. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3491-3495Crossref PubMed Scopus (825) Google Scholar, 6Eissa N.T. Strauss A.J. Haggerty C.M. Choo E.K. Chu S.C. Moss J. J. Biol. Chem. 1996; 271: 27184-27187Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Calmodulin is tightly bound to iNOS even at basal Ca2+ levels, and therefore iNOS is notably distinguished from the constitutive isoforms by its prolonged production of a relatively large amount of NO (7Cho H.J. Xie Q.-W. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (565) Google Scholar). iNOS has been implicated in the pathogenesis of many diseases including Alzheimer's disease, pulmonary tuberculosis, asthma, lung cancer, transplant rejection, cerebral infarct, glaucoma, bacterial pneumonia, inflammatory bowel disease, arthritis, and septic shock (8Vodovotz Y. Lucia M.S. Flanders K.C. Chesler L. Xie Q.-W. Smith T.W. Weidner J. Mumford R. Webber R. Nathan C. Roberts A.B. Lippa C.F. Sporn M.B. J. Exp. Med. 1996; 184: 1425-1433Crossref PubMed Scopus (264) Google Scholar, 9Nathan C. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (849) Google Scholar, 10Guo F.H. Comhair S.A.A. Zheng S. Dweik R.A. Eissa N.T. Thomassen M.J. Calhoun W. Erzurum S.C. J. Immunol. 2000; 164: 5970-5980Crossref PubMed Scopus (237) Google Scholar). The activity of an enzyme can be controlled through the regulation of its synthesis, catalytic activity, or degradation. Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about its degradation. The 26S proteasome is a large multisubunit protease responsible for the selective degradation of a number of short-lived regulatory proteins, the activity of which must be tightly regulated, such as NF-κB, STAT1,fos/jun, and cyclins (11Ciechanover A. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2727-2730Crossref PubMed Scopus (366) Google Scholar, 12Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (921) Google Scholar, 13Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1267) Google Scholar). In this report, we identify the 26S proteasome as the major pathway responsible for human iNOS degradation. Human iNOS is targeted for degradation by the proteasome, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. Furthermore, we demonstrate that the proteasome pathway regulates human iNOS at both transcriptional and post-translational levels. Lactacystin, calpastatin, and taurocholic acid were obtained from Calbiochem-Novabiochem.N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (ALLN),N-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norleucinal (MG132),trans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, NH4Cl,N-α-p-tosyl-l-lysine chloromethyl ketone, N-p-tosyl-l-phenylalanine chloromethyl ketone, and lipopolysaccharide (LPS) were from Sigma. All other reagents were purchased from either Fisher or sources stated in the text. 1E8-B8 (Research and Diagnostic Antibodies) is a monoclonal antibody specific for the iNOS isoform and can detect both human and murine iNOS (8Vodovotz Y. Lucia M.S. Flanders K.C. Chesler L. Xie Q.-W. Smith T.W. Weidner J. Mumford R. Webber R. Nathan C. Roberts A.B. Lippa C.F. Sporn M.B. J. Exp. Med. 1996; 184: 1425-1433Crossref PubMed Scopus (264) Google Scholar, 14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar). The monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase was from Advanced Immunochemical. A goat polyclonal antibody (Santa Cruz Biotechnology) and a rabbit polyclonal antibody (New England Biolabs) were used in immunoblotting for the detection of IκB-α and phosphorylated IκB-α, respectively. Human embryonic kidney (HEK) 293, human bladder transitional cell papilloma (RT4), and murine macrophage RAW 264.7 cell lines were purchased from the American Type Culture Collection. The cells were cultured in phenol red-free improved minimal essential medium (HEK293), Dulbecco's modified Eagle's (RAW 264.7), or McCoy's media (RT4) supplemented with 2 mmglutamine and 10% heat-inactivated filtered (40 nm-filter) fetal bovine serum (Hyclone Laboratories, Inc.) at 37 °C in 5% CO2. Human iNOS cDNA, inserted into the expression vector pRc/CMV (Invitrogen) under the control of the cytomegalovirus promoter, was used. Cationic lipid-mediated transient transfection was done using LipofectAMINE and a transfection-enhancing Plus reagent (Life Technologies, Inc.) following the manufacturer's instructions. RT4 cells were incubated with or without a mixture of interleukin (IL)-1β (0.5 ng/ml), tumor necrosis factor-α (10 ng/ml), and IL-6 (200 units/ml) (6Eissa N.T. Strauss A.J. Haggerty C.M. Choo E.K. Chu S.C. Moss J. J. Biol. Chem. 1996; 271: 27184-27187Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Knour A. Krause S.W. Rehli M. Kreutz M. Andreesen R. J. Immunol. 1996; 157: 2109-2115PubMed Google Scholar). RAW 264.7 cells were incubated with or without 50 ng/ml LPS (4Xie Q.-W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1745) Google Scholar, 6Eissa N.T. Strauss A.J. Haggerty C.M. Choo E.K. Chu S.C. Moss J. J. Biol. Chem. 1996; 271: 27184-27187Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). After gently rinsing twice with phosphate-buffered saline, the cell layer was lysed on ice for 45 min in iNOS extraction buffer consisting of 40 mm bis-Tris propane buffer, pH 7.7, 150 mm NaCl, and 10% glycerol with 20 mm detergent sodium taurocholate and in the presence of protease inhibitors (phenylmethylsulfonyl fluoride (1 mm), pepstatin-A (10 μg/ml), leupeptin (10 μg/ml), aprotinin (10 μg/ml), phenanthroline (10 μg/ml), and benzamidine HCl (16 μg/ml)) (PharMingen). Lysates were centrifuged (16,000 ×g, 5 min, 4 °C) and supernatants were stored at −80 °C (14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar). In some experiments, to lyse cells and precipitate proteins immediately, 20% trichloroacetic acid or 1% boiling SDS was added directly to cells. Total protein concentrations were determined by using a bicinchoninic acid reagent (Pierce). Cell lysates (50 μg) were mixed with one-third volume of 4× Laemmli sample buffer (200 mm Tris-HCl, pH 6.8, 8% SDS, 0.004% bromphenol blue, 40% glycerol, and 400 mm dithiothreitol), boiled at 95 °C for 5 min, and then subjected to SDS/polyacrylamide gel electrophoresis. After SDS/polyacrylamide gel electrophoresis, immunoblotting was done with the 1E8-B8 antibody. An enhanced chemiluminescence system was used for detection (SuperSignal West Pico, Pierce). Images were acquired using a cooled charge-coupled device camera (Eagle Eye II Still Video System, Stratagene). NOS activity in the cell culture was determined by measuring the accumulation of nitrite in culture media. 100 μl of culture medium was mixed with 100 μl of Griess reagent for 10 min at room temperature, and absorbance at 543 nm was recorded in a microplate reader. Serial dilutions of sodium nitrite were used as standards (16Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (11108) Google Scholar). We investigated the degradation pathway for iNOS in the HEK293 cell line, an epithelial cell line that does not express any of the NOS genes and has been used extensively to study exogenously expressed iNOS (14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar, 17Xie Q.-W. Leung M. Fuortes M. Sassa S. Nathan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4891-4896Crossref PubMed Scopus (79) Google Scholar). We had previously produced an HEK293 cell line with stable expression of human iNOS (14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar), henceforth referred to as HEK293-iNOS. We tested the effects of the potent yet nonspecific proteasome inhibitors, ALLN and MG132, and the highly specific proteasome inhibitor lactacystin on iNOS expression in this cell line (13Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1267) Google Scholar, 18Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1508) Google Scholar). Cells were incubated with proteasome inhibitors or the corresponding vehicle for 20 h and then harvested by lysis on ice (see “Experimental Procedures”) (14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar). Lactacystin, ALLN, and MG132 blocked human iNOS degradation leading to its accumulation, which could be detected by the immunoblotting of cell lysates with a specific antibody for iNOS (Fig. 1 A). In some experiments, to lyse cells and precipitate proteins immediately, 20% trichloroacetic acid (Fig. 1 B) or boiling SDS-containing buffer (Fig. 1 C) was added directly to the cells. In all cases, inhibition of proteasomal activity with lactacystin led to iNOS accumulation, indicating that the effect of proteasomal inhibition on iNOS was not the result of an in vitro effect during cell lysis. To further characterize human iNOS degradation via the proteasome pathway, we examined the dose and time dependence of the effect of lactacystin on iNOS expression. Incubation of HEK293-iNOS cells for 20 h with concentrations of lactacystin resulted in a corresponding in iNOS accumulation In experiments, of HEK293-iNOS cells with as little as 2 lactacystin for resulted in a time-dependent accumulation of iNOS in HEK293-iNOS cells in iNOS expression could be detected as as 8 h after lactacystin and to further the time of the for iNOS accumulation after proteasomal inhibition with HEK293 cells stably expressing human iNOS were incubated in the presence of vehicle or 2 lactacystin for The cell lysates (50 μg) were by using an antibody. The data of in of the iNOS in A expressed as the in cells with cells with vehicle In to its in the degradation of regulatory proteins, the 26S proteasome has been implicated in the of and proteins S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (921) Google Scholar). In this we investigated the proteasomal degradation of human iNOS on NO production by iNOS. We tested the effect of lactacystin on iNOS expressed in HEK293-iNOS cells incubated in the presence or of the NOS N ω-nitro-l-arginine methyl ester Biol. 1992; Google Scholar). Incubation of the cells in the presence of resulted in the inhibition of NO synthesis by iNOS as by measuring nitrite accumulation in culture media not Incubation of the cells with 10 lactacystin for 20 h blocked iNOS degradation in the presence or of were obtained when the specific iNOS (50 was used of not M. J.S. 1995; Google Scholar). These data that the of iNOS to NO is not for its by the proteasome degradation all NOS isoforms, for the synthesis of NO human iNOS is in its (2Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (819) Google Scholar, 17Xie Q.-W. Leung M. Fuortes M. Sassa S. Nathan C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4891-4896Crossref PubMed Scopus (79) Google Scholar). We previously a splice variant of human iNOS that lacks exons 8 and 9 a for and NO synthesis (14Eissa N.T. Yuan J. Haggerty C.M. Choo E.K. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7625-7630Crossref PubMed Scopus (76) Google Scholar). dimers produce In we in human iNOS as a for and in is a mutant of human iNOS in which is by Although its to dimerize, bind the and an of iNOS N.T. Haggerty C.M. W. Moss J. J. Cell Biol. PubMed Scopus Google Scholar). To these iNOS via the proteasome pathway, we HEK293 cells with human iNOS, or and examined the effect of proteasome to the effect on iNOS, lactacystin blocked the degradation of and These that the of human iNOS by the proteasome degradation pathway is of the capacity of iNOS to dimerize or bind the substrate. To the of other that be in iNOS the effect of various degradation inhibitors was After a in the presence of the specific the cells were lysed by boiling in SDS-containing In contrast to proteasomal inhibitors, the calpain inhibitor 5 the inhibitor chloromethyl ketone, 100 or the inhibitor chloromethyl ketone, 50 did not lead to the accumulation of iNOS A). were obtained using the lysosomal protease inhibitorstrans-epoxysuccinyl-l-leucylamido-4-guanidino (50 and leupeptin (50 the protease inhibitor pepstatin-A and the lysosomal inhibitors or NH4Cl (Fig. and was a not in iNOS detection with lysosomal inhibitors Because of the in some of the iNOS in a for the lysosomal pathway in human iNOS degradation be As a lactacystin was used during the and resulted in a accumulation of iNOS, suggesting that the proteasome pathway a primary in human iNOS degradation. NF-κB is a major in the inducible expression of iNOS Q.-W. Y. Nathan C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Chu S.C. Moss J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is a that is by a of and cytokines S. M. Immunol. 1998; PubMed Scopus Google Scholar). NF-κB through a tightly of and proteasomal of a of inhibitor the of which is proteasomal inhibitors the degradation of phosphorylated and IκB-α, thus NF-κB activation M. Y. Immunol. 2000; PubMed Scopus Google Scholar). of the proteasome pathway were to with the induction of iNOS in by blocking NF-κB activation S. L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). We that the proteasome regulates iNOS on two at the transcriptional by IκB-α and thus NF-κB, and at the post-translational by iNOS To this we used a cytokine mixture of (0.5 ng/ml), tumor necrosis factor-α (10 ng/ml), and IL-6 (200 units/ml) to induce iNOS expression in RT4 cells (6Eissa N.T. Strauss A.J. Haggerty C.M. Choo E.K. Chu S.C. Moss J. J. Biol. Chem. 1996; 271: 27184-27187Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Knour A. Krause S.W. Rehli M. Kreutz M. Andreesen R. J. Immunol. 1996; 157: 2109-2115PubMed Google Scholar). The of 40 lactacystin 1 h prior to stimulation prevented iNOS induction A). The effect on iNOS induction was by the inhibition of IκB-α as by of IκB-α The in IκB-α with proteasomal inhibition was by inhibition of the degradation of phosphorylated IκB-α, as by immunoblotting cell lysates with an antibody C) Science. 2000; PubMed Scopus Google Scholar). We that in cells by cytokines to produce iNOS, iNOS is proteasomal inhibitors its degradation. To this RT4 cells were incubated for h in the presence of the cytokine mixture used by a with medium without cytokines and then in the presence of lactacystin for 20 The of cells with lactacystin led to a accumulation of iNOS (Fig. to that in with HEK293 cells expressing iNOS. is a effect of proteasomal inhibition on human iNOS. to the of prior on human iNOS degradation. The that iNOS in RT4 cells is through the proteasome pathway that the of the proteasome pathway in iNOS degradation is not to HEK293 cells, is to be a of To the to murine cells, we tested the effect of proteasomal inhibition on murine iNOS degradation. The murine macrophage cell line RAW 264.7 was by LPS (50 to induce iNOS expression (4Xie Q.-W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1745) Google Scholar, 6Eissa N.T. Strauss A.J. Haggerty C.M. Choo E.K. Chu S.C. Moss J. J. Biol. Chem. 1996; 271: 27184-27187Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 48 h the cells were incubated with lactacystin (40 for 20 in to for RT4 cells. Lactacystin blocked murine iNOS leading to its accumulation as detected by and by iNOS activity, by measuring nitrite accumulation in culture media (Fig. 9 These that iNOS to the proteasome in both human and murine cells, and of the of iNOS by transfection (HEK293), cytokines (RT4), or LPS (RAW has been that all of the proteasome pathway must be as a to been of proteins that by of Moreover, as has been with of a protein is to that its degradation must through a R. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google J. M. Roberts Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In is not the iNOS after proteasomal inhibition is and the of iNOS is for its degradation. of proteins on to be by R. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. Roberts Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of iNOS were in SDS/polyacrylamide gel after proteasomal inhibition (see and The of these to be has that cells control the of in through control protein degradation. the regulation of iNOS degradation cells control the of NO synthesis during inflammation and has been in of iNOS degradation to the of of NO by Y. C. J. Xie Q.-W. Nathan C. J. Exp. Med. 1993; PubMed Scopus Google Scholar). the of neuronal NOS has been to the of the enzyme by a the proteasome S. S. Y. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). this study was C. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google that in human cells, iNOS via the proteasome the of iNOS degradation to be an for NO the of proteins for degradation is highly selective (11Ciechanover A. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2727-2730Crossref PubMed Scopus (366) Google Scholar, 12Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (921) Google Scholar, 13Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1267) Google Scholar). the specific pathway for iNOS study the for such study is to the of R. a and a through the and the We for of the