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

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.