title: Regulation of inducible nitric-oxide synthase by the SPRY domain- and SOCS box-containing proteins.
abstract: Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan. nishiya@med.hokudai.ac.jp
authors.forename: Tadashi
authors.initials: T
authors.lastname: Nishiya
authors.forename: Kazuma
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Maekawa
authors.forename: Emi
authors.initials: E
authors.lastname: Kajita
authors.forename: Takahiro
authors.initials: T
authors.lastname: Horinouchi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujimuro
authors.forename: Kouetsu
authors.initials: K
authors.lastname: Ogasawara
authors.forename: Takashi
authors.initials: T
authors.lastname: Uehara
authors.forename: Soichi
authors.initials: S
authors.lastname: Miwa
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000502:physiology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D050826:Suppressor of Cytokine Signaling Proteins; D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D044767:Ubiquitin-Protein Ligases