A Critical Role for CHIP in the Aggresome Pathway

Abstract

Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed "physiologic aggresome," is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIPmediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTR⌬F508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

The physiologic aggresome mediates cellular inactivation of iNOS

“…Similar to what was previously described for aggresomes, the iNOS aggresome in NHBE cells was formed at the MTOC, surrounded the centrosome, and was distinct from other perinuclear structures such as the Golgi (supporting information (SI) Fig. S1) (7)(8)(9).…”

“…Recent work extends these observations to functional proteins such as iNOS (8,9). The functional significance of the latter observation is revealed by this study.…”

“…Recent evidence suggests that aggresome formation is also a physiologic mechanism to regulate certain cellular proteins. The latter process, termed ''physiologic aggresome,'' has been described in connection to cellular regulation of iNOS (8,9). We have recently shown that iNOS is expressed initially as a cytosolic protein but is eventually targeted to a perinuclear location, identified by our data as an aggresome.…”

“…We have previously shown that cytokine-induced iNOS is sequestered in a perinuclear aggresome in various human and murine cell lines (8,9). Airway bronchial epithelial cells represent a major source of iNOS in the lung (17,20).…”

“…We have previously shown that iNOS exists in 2 cellular compartments, the first being a diffuse cytosolic location and the second being a perinuclear compartment characterized as an iNOS aggresome, whose formation is accelerated by proteasomal inhibitors such as MG132 (8,9). Using live cell imaging and DAR, we monitored the subcellular NO production over various time points in HEK293-iNOS-GFP cells in which iNOS aggresome formation was accelerated by the proteasome inhibitor MG132 (Fig.…”