Hsp90 inhibition renders iNOS aggregation and the clearance of iNOS aggregates by proteasomes requires SPSB2

Abstract

Inducible nitric oxide synthase (iNOS) plays important roles in cell injury and host defense. Our early study demonstrated that heat shock protein 90 (Hsp90) interacts with iNOS and this interaction enhances iNOS function. Recently, we reported that Hsp90 is also essential for iNOS gene transactivation. In the present study, we investigate the role of Hsp90 in controlling iNOS protein stability. In mouse macrophages, Hsp90 inhibition dissociated Hsp90 from iNOS and the latter subsequently formed aggregates. Aggregation deactivated iNOS. iNOS aggregates were cleared by the ubiquitin-proteasome system (UPS) inside cells. CHIP, an Hsp90-dependent E3 ligase, was previously implicated in iNOS turnover. However, CHIP knockdown had little effect on iNOS degradation in Hsp90-inhibited cells, indicating that other E3 ligases accounted for the clearance of iNOS aggregates. Further studies revealed that the SPRY domain-containing SOCS box protein 2 (SPSB2), an E3 ligase-recruiting protein, was essential for the ubiquitination of iNOS aggregates. SPSB2 knockdown or deleting the SPSB2-interacting domain on iNOS prevented the clearance of iNOS aggregates in Hsp90-inhibited cells. Thus, besides modulating iNOS function and gene transcription, Hsp90 is also essential for the protein stability of iNOS. Hsp90 blockade induces iNOS aggregation and SPSB2 is required for UPS degradation of iNOS aggregates.

Targeting SOCS Proteins to Control JAK-STAT Signalling in Disease

“…A well-characterised substrate of SPSB1, 2 and 4 is inducible nitric oxide synthase (iNOS), a key effector of the innate immune response. By promoting iNOS protein degradation, SPSB proteins prevent the accumulation of cytotoxic levels of nitric oxide that could damage ultimately host tissue [57,58]. E3 ubiquitin ligase: defined as a protein that recruits an E2 conjugation enzyme that has been loaded with ubiquitin, a small protein found in all eukaryotic organisms.…”

iNOS Interacts with Autophagy Receptor p62 and is Degraded by Autophagy in Macrophages

“…However, prolonged iNOS upregulation will generate high-level NO which may cause tissues damage, thus efficient degradation of iNOS is a critic mechanism for elimination of inflammation [32]. iNOS has been revealed to be degraded by UPS [33,34] and our data revealed for the first time that iNOS can also be degraded by autophagy, in a selective manner via the interaction with p62. Compared with the dramatic accumulation of iNOS when proteasome is inhibited [35], the increase of iNOS level is moderate during autophagy inhibition.…”

Involvement of Nitric Oxide in Protecting against Radical Species and Autoregulation of M1-Polarized Macrophages through Metabolic Remodeling

“…Calcium ions are the principle activators for NOS1 and NOS3 via binding to calmodulin, but NOS2, which contains bound calmodulin, is constitutively active. Accordingly, the production of •NO by NOS2 is mainly regulated through gene expression and the degradation of the NOS2 protein by proteasomes [ 104 , 105 ]. NF- κ B and IRF1 are the main transcriptional regulatory factors for the induction of NOS2 and, as a result, their inhibition suppresses NOS2 induction [ 106 ].…”