Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from L-arginine in response to inflammatory mediators. We have previously shown that iNOS is degraded through the 26S proteasome. Targeting of proteins for proteasomal degradation may or may not require their covalent linkage to multiubiquitin chains (ubiquitination). In addition, ubiquitination of a protein can serve functions other than signaling proteolysis. In this context, it is not known whether iNOS is subject to ubiquitination or whether ubiquitination is required for its degradation. In this study, we show that iNOS, expressed in HEK293 cells or induced in primary bronchial epithelial cells, A549 cells, or murine macrophages, is subject to ubiquitination. To investigate whether iNOS ubiquitination is required for its degradation, HEK293T cells were cotransfected with plasmids containing cDNAs of human iNOS and of the dominant negative ubiquitin mutant K48R. Disruption of ubiquitination by K48R ubiquitin resulted in inhibition of iNOS degradation. ts20 is a mutant cell line that contains a thermolabile ubiquitin-activating enzyme (E1) that is inactivated at elevated temperature, preventing ubiquitination. Incubation of ts20 cells, stably expressing human iNOS, at the nonpermissive temperature (40°C) resulted in inhibition of iNOS degradation and marked accumulation of iNOS. These studies indicate that iNOS is subject to ubiquitination and that ubiquitination is required for its degradation.

Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of many disorders. iNOS is notably distinguished from constitutive NOSs by its production of large amounts of NO for a prolonged period; hence, it was termed the high-output NOS. Understanding how cells regulate iNOS is a prerequisite for strategies aimed at modulating NO synthesis. iNOS is thought to be regulated primarily at the transcriptional level in response to cytokines and inflammatory mediators. In this study, we report a posttranslational regulatory mechanism for control of iNOS expression through a rapid cellular rate of turnover. Unexpectedly, iNOS cellular half-life was found to be relatively short. In primary bronchial epithelial cells, iNOS half-life was 1.6 ؎ 0.3 h. A similar half-life was found for iNOS in several cell lines. This fast rate of turnover is in sharp contrast to that reported for the constitutive NOS isoforms. iNOS half-life was not affected by intracellular depletion of tetrahydrobiopterin, a critical cofactor required for iNOS activity. Further, iNOS monomers and dimers had a similar half-life. Importantly, we discovered a previously unrecognized cotranslational down-regulation mechanism by which the newly discovered pyrimidineimidazole-based allosteric dimerization inhibitors of iNOS lead to reduced iNOS expression. This study provides insights into the cellular posttranslational mechanisms of iNOS and has important implications for design of selective iNOS inhibitors and their use in therapeutic strategies.degradation ͉ proteasome ͉ half-life N itric oxide (NO) is an important signaling and cytotoxic molecule that is synthesized from L-arginine by isoforms of nitric oxide synthase (NOS) (1-3). As a signaling molecule, NO is produced by two constitutive calcium (Ca 2ϩ )-dependent isoforms, neuronal NOS and endothelial NOS (or NOSI and NOSIII, respectively). Ca 2ϩ -activated calmodulin binds to and transiently activates constitutive NOS dimers (3). Because of the transient nature of elevated Ca 2ϩ levels, these isoforms produce small amounts of NO for short periods of time. As an agent of inflammation and cell-mediated immunity, NO is produced by a Ca 2ϩ -independent cytokine-inducible NOS (iNOS or NOSII) that is widely expressed in diverse cell types under transcriptional regulation by inflammatory mediators (2, 4). Calmodulin is tightly bound to iNOS even at basal Ca 2ϩ levels; therefore, iNOS is notably distinguished from the constitutive isoforms by its prolonged production of a relatively large amount of NO (5). iNOS has been implicated in the pathogenesis of many diseases including Alzheimer's disease, tuberculosis, asthma, transplant rejection, stroke, glaucoma, inflammatory bowel disease, arthritis, and septic shock (6, 7). Such wide implication has produced a corresponding intense interest in understanding the regulation of NO synthesis by iNOS with the goal of developing therapeutic strategies aimed at selective modulation of iNOS activity (8). Understan...

Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation. Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined, and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer. Dimerization of iNOS represents a potentially critical target for therapeutic intervention. In this study, we show that intracellular iNOS forms dimers that are ''undisruptable'' by boiling, denaturants, or reducing agents. Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. UD dimers do not form in Escherichia coli-expressed iNOS and could not be assembled in vitro, which suggests that an in vivo cellular process is required for their formation. iNOS UD dimers are not affected by intracellular depletion of H4B. However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers. This study reveals insight into the mechanisms of in vivo iNOS dimer formation. UD dimers represent a class of iNOS dimers that had not been suspected. This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activity in vivo.

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