A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation.
Mol Cell Proteomics
; 19(12): 1968-1986, 2020 12.
Article
in En
| MEDLINE
| ID: mdl-32912968
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-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-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 IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-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 IκBα-loss was due to its sequestration along with IκBß into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα 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 IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB 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.
Key words
IkBa; IκB; Mallory-Denk-bodies; NF-kB; NF-κB; NMPP; PPIX; X-linked protoporphyria; ZnPP; ZnPPIX; affinity proteomics; erythropoietic protoporphyria; hepatotoxicity; immunoaffinity; inflammation; inflammatory response; knockouts*; label-free quantification; liver disease; liver inflammation; mass spectrometry; p62; protein aggregation; protein-protein interactions; proteomics.; α; ß
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
NF-kappa B
/
I-kappa B Proteins
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Protein Aggregates
/
Inflammation
/
Liver
Type of study:
Prognostic_studies
Limits:
Animals
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Humans
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Male
Language:
En
Journal:
Mol Cell Proteomics
Year:
2020
Document type:
Article