p62-Dependent Phase Separation of Patient-Derived KEAP1 Mutations and NRF2

Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1...

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Published in:	Molecular and cellular biology Vol. 38; no. 22
Main Authors:	Cloer, E. W., Siesser, P. F., Cousins, E. M., Goldfarb, D., Mowrey, D. D., Harrison, J. S., Weir, S. J., Dokholyan, N. V., Major, M. B.
Format:	Journal Article
Language:	English
Published:	United States Taylor & Francis 01-11-2018 American Society for Microbiology
Subjects:	autophagy biomolecular condensates clusters hypomorphs KEAP1 liquid-liquid phase separation lung cancer membraneless organelles mutations NRF2 oxidative stress p62/SQSTM1 Spotlight lung cancer membraneless organelles autophagy mutations NRF2 liquid-liquid phase separation KEAP1 p62/SQSTM1 hypomorphs clusters oxidative stress biomolecular condensates
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Summary:	Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Citation Cloer EW, Siesser PF, Cousins EM, Goldfarb D, Mowrey DD, Harrison JS, Weir SJ, Dokholyan NV, Major MB. 2018. p62-dependent phase separation of patient-derived KEAP1 mutations and NRF2. Mol Cell Biol 38:e00644-17. https://doi.org/10.1128/MCB.00644-17.
ISSN:	1098-5549 0270-7306 1098-5549
DOI:	10.1128/MCB.00644-17