Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from L-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, and N-acetyl-L-leucinyl-Lleucinyl-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 by N -nitro-Larginine 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 inhibitors transepoxysuccinyl-L-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH 4 Cl 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-B inhibitor IB-␣, thus preventing activation of NF-B. 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 (NO),1 an important signaling and cytotoxic molecule, is synthesized from L-arginine by isoforms of nitricoxide synthase (NOS) (1, 2). As a signaling molecule, NO is produced by two constitutive Ca 2ϩ -dependent isoforms, neuronal and endothelial NOS (or NOS1 and NOSIII, respectively). Ca 2ϩ -activated calmodulin binds to and transiently activates constitutive NOS dimers (2, 3). Because of the transient nature of elevated Ca 2ϩ levels, the activity of NO produced is shortlived. 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 (4 -6). Calmodulin is tightly bound to iNOS even at basal Ca 2ϩ levels, and therefore iNOS is notably distinguished from the constitutive isoforms by its prolonged production of a relatively large amount of NO (7). 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, infl...

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.

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