Liu Y, Trnka MJ, Guan S, Kwon D, Kim DH, Chen JJ, Greer PA, Burlingame AL, Correia MA

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-?B activation. However, the precise mechanism that links protein aggregation to NF-?B-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with ...

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MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an I?B?-loss with consequent NF-?B activation. Four known mechanisms of I?B?-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 I?B?-loss was due to its sequestration along with I?B? into insoluble aggregates, thereby releasing NF-?B. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-?B subunit p65, which stably interacts with I?B? under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with I?B? under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with I?B? 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 I?B?-nuclear import. The concurrent aggregation of I?B?, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of I?B? and its consequent binding and termination of NF-?B 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.

Date: Dec. 01, 2019

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