Ubiquitination of inducible nitric oxide synthase is required for its degradation

Abstract

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.

Regulation of inducible nitric oxide synthase by aggresome formation

“…iNOS is expressed in vivo in response to cytokines and inflammatory mediators (18)(19)(20). Therefore, we sought to examine the subcellular localization of cytokine-induced iNOS.…”

Regulation of inducible nitric oxide synthase by rapid cellular turnover and cotranslational down-regulation by dimerization inhibitors

“…HEK 293 cells are human embryonic kidney epithelial cells that do not contain any of the NOS genes, and they have been previously used for the characterization of human iNOS (13)(14)(15). We used a HEK 293 cell line that stably expresses human iNOS (9,10). Pulse-chase analysis revealed that human iNOS in these cells has a half-life of Ϸ2.0 Ϯ 0.1 h (Fig.…”

“…Although much is known about factors affecting the synthesis and catalytic activity of iNOS, little is known about mechanisms regulating its degradation. Recently, the ubiquitin-proteasome pathway has been identified as the major pathway responsible for iNOS degradation (9,10). However, the cellular regulatory mechanisms governing the targeting of iNOS for degradation have yet to be studied.…”

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions

“…The theoretical molecular mass for human iNOS protein is 131 kDa, and the 9 kDa mass difference could be explained either by alternative splicing or differences in glycosylation. Recent work 45,46 suggested that a novel mechanism involving iNOS ubiquitination and proteasomic proteolysis could also play a role in the regulation of iNOS activity. As previously proposed by Zhao et al 21 and Coers et al, 47 the small molecular weight bands revealed by Western blot could be considered as proteolytic degradation products.…”