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.Ubiquitination and the subsequent proteasomal degradation of regulatory proteins represent a key mechanism for various cellular processes, including cell cycle control (1), apoptosis (2), cellular stress regulation (3), and antigen presentation (4). The ubiquitination of proteins is performed by a set of three enzymes: E1, E2, and E3 (5). E3 is a ubiquitin ligase that confers specificity to ubiquitination by recognizing target substrates and mediating the transfer of ubiquitin from E2 to the substrate. The E3 ubiquitin ligases are a large, diverse group of proteins, and many of them can act alone, but some RING-type E3 ubiquitin ligases, such as the ECS 3 (Elongin B/C-Cullin-5 (Cul5)-SOCS (suppressor of cytokine signaling)-box protein)-type E3 ubiquitin ligase, are found as components of much larger multi-protein complexes (6). The SOCS box is ϳ40 residues in length and contains a BC box and a Cul5 box, which binds Elongin C and Cul5, respectively (7). The SOCS box has been found in more than 50 proteins encompassing nine different families that are defined by the type of domain or motif they possess upstream of the SOCS box (6, 8). The SPRY domainand SOCS box-containing proteins, SPSB1 to SPSB4 (also known as SSB-1 to SSB-4), are characterized by a central SPRY domain essential for protein interacti...

The E3 Ubiquitin Ligase Neuregulin Receptor Degradation Protein 1 (Nrdp1) Promotes M2 Macrophage Polarization by Ubiquitinating and Activating Transcription Factor CCAAT/Enhancer-binding Protein β (C/EBPβ)

“…In addition, macrophages from mutant-Nrdp1 (lacking E3 ubiquitin ligase) transgenic mice also showed similar impaired expression of iNOS after LPS stimulation (data not shown), suggesting that Nrdp1 inhibits iNOS expression in an E3 ubiquitin ligase-independent manner. iNOS protein level in cells can be up-regulated by increased transcription of iNOS gene or the decreased ubiquitinationand proteasome-mediated degradation of iNOS protein (22,23). A variety of transcription factors, such as nuclear factor-B (NF-B), activating protein-1 (AP-1), C/EBP, C/EBP␤, octamerbinding transcription factor-1 (Oct-1), have been proved to bind with the iNOS promoter and enhance the transcription of the iNOS gene.…”

“…A variety of signals can regulate Arg1 expression in macrophages. Among them, IL-4 is by far the most potent cytokine that polarizes macrophages to AAM (22). As the Arg1 level was significantly higher in Nrdp1-TG macrophages than that in WT macrophages, we further explored whether Nrdp1 influences the M2 macrophage polarization triggered by IL-4.…”

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…SPSB1, 2 and 4 are similar both in amino acid sequence and in function. We, and others, have recently shown that inducible nitric oxide synthase (iNOS/NOS2) is a physiological target of the mammalian SPSB1 and SPSB2 proteins, suggesting a role for these proteins in regulating the innate immune response (70)(71)(72). SPSB1, 2 and 4 interact via their SPRY domain with a DINNN motif in the N-terminal region of iNOS and through the SOCS box-associated CRL ubiquitinate iNOS causing it to be degraded by the proteasome.…”

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-β Signaling Pathway Targeting the Type II Receptor

“…Recently, SPSB1 and SPSB2 have been shown to interact with inducible nitric-oxide synthase (iNos), targeting it for ubiquitination and proteasomal degradation and implicating the SPSB proteins in regulating the host response to infection (37)(38)(39). SPSB1 has also been reported to interact with c-Met to enhance the hepatocyte growth factor-induced Erk-Elk-1-Sre pathway by an unknown mechanism (27), whereas SPSB1, -2, and -4 have been reported to bind to the intracellular protein prostate apoptosis response 4 (Par4) (28).…”