Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1968-1985 |
| Number of pages | 18 |
| Journal | Molecular and Cellular Proteomics |
| Volume | 19 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 1 2020 |
Bibliographical note
Funding Information:Her, UCSF Liver Cell & Tissue Biology Core Facility (supported by NIDDK Grant P30DK26743) for hepatocyte isolation. We are most grateful to Dr. D. M. Bissell (UCSF) for valuable discussions and his critical review of our manuscript. We also gratefully acknowledge Prof. P. Ortiz de Montellano (UCSF) for valuable discussions of porphyrin chemistry, and Dr. G. Knudsen (UCSF) for her helpful comments on our IAP/MS methodology and critical review of our manuscript and proteomic data. We also sincerely thank Profs. T. Yanagawa, M. Komatsu, H. Zhu, N. Mizushima, R. Scheckman, and R. J. Youle for valuable cell-lines. Y. Liu is most grateful to Dr. D. Larsen, UCSF Nikon Imaging Center for her training in confocal immunofluorescence microscopy, and Drs. M. Laurance, UCSF HDF Comprehensive Cancer Center and A. Pico, The Gladstone Institute Bioinformatics Facility, for training on pathway analyses and visualization. Published as a BioRxiv preprint (March 21st, 2019): doi: https://doi.org/10.1101/ 585497. supported by NIH Grants GM44037 (M.A.C.), DK26506 (M.A. C.), GM25515 (P.O.M.), GM097057 (D.H.K.), DK087984 (J.J. C.), and CIHR grant #81189 (P.A.G.). We also acknowledge the UCSF Biomedical Mass Spectrometry and Proteomics Resource Center (Prof. A. L. Burlingame, Director) supported by the Adelson Medical Research Foundation.
Funding Information:
supported by NIH Grants GM44037 (M.A.C.), DK26506 (M.A. C.), GM25515 (P.O.M.), GM097057 (D.H.K.), DK087984 (J.J. C.), and CIHR grant #81189 (P.A.G.). We also acknowledge the UCSF Biomedical Mass Spectrometry and Proteomics Resource Center (Prof. A. L. Burlingame, Director) supported by the Adelson Medical Research Foundation.
Funding Information:
Acknowledgments—We gratefully acknowledge Mr. Chris Her, UCSF Liver Cell & Tissue Biology Core Facility (supported by NIDDK Grant P30DK26743) for hepatocyte isolation. We are most grateful to Dr. D. M. Bissell (UCSF) for valuable discussions and his critical review of our manuscript. We also gratefully acknowledge Prof. P. Ortiz de Montellano (UCSF) for valuable discussions of porphyrin chemistry, and Dr. G.
Publisher Copyright:
© 2020 Liu et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.