Ubiquitination of inducible nitric oxide synthase is required for its degradation.
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 degrees 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.
Kolodziejski
Pawel J
PJ
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Musial
Aleksandra
A
Koo
Ja-Seok
JS
Eissa
N Tony
NT
D000818:Animals; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D016207:Cytokines; D011994:Recombinant Proteins; D025801:Ubiquitin; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease