A Novel Mechanism for NF-kB-activation via IkB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-kB activation. However, the precise mechanism that links protein aggregation to NF-kB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IkBa-loss with consequent NF-kB activation. Four known mechanisms of IkBa-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IkBa-loss was due to its sequestration along with IkBb into insoluble aggregates, thereby releasing NF-kB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-kB subunit p65, which stably interacts with IkBa under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IkBa under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IkBa after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IkBa-nuclear import. The concurrent aggregation of IkBa, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IkBa and its consequent binding and termination of NF-kB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.