Proteomics

Dataset Information

47

Patient-derived KEAP1 superbinder mutants stabilize KEAP1 structure and sequester NRF2 in p62-dependent clusters


ABSTRACT: Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a pro-survival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a previous study of KEAP1 mutations observed in lung cancer, we classified 40% of the mutations as ‘superbinders’ (superbinders). These mutants bind and ubiquitylate NRF2 but do not promote NRF2 degradation. Here, we further investigated the molecular mechanism(s) driving the superbinder phenotype. BioID-based quantitative proteomic analysis of the R320Q and R470C superbinder mutations revealed increased co-complexed NRF2 without significant alteration to other KEAP1-associated proteins, including CUL3, VCP, and several ubiquitin receptors within the proteasome lid. Dynamic simulation modeling and limited proteolysis analyses suggest that superbinder mutations stabilize residues in KEAP1 that contact NRF2. In cells, KEAP1 R320Q and R470C mutants co-localize with NRF2, p62/SQSTM1 and polyubiquitin in spherical clusters that rapidly fuse and dissolve; KEAP1-NRF2 localization to these clusters requires p62. Expression of R320Q and R470C in lung cancer cells provided resistance to the reactive oxygen species-inducing drug bleomycin. We present a model wherein superbinder mutations alter the conformational dynamics of the KEAP1-NRF2 complex to alter the cycling of KEAP1 between open and closed conformations, thus inhibiting NRF2 degradation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo sapiens  

TISSUE(S): Cell Culture

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Dennis Goldfarb  

LAB HEAD: Ben Major

PROVIDER: PXD008411 | Pride | 2018-11-07

REPOSITORIES: Pride

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Publications

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

Cloer E W EW   Siesser P F PF   Cousins E M EM   Goldfarb D D   Mowrey D D DD   Harrison J S JS   Weir S J SJ   Dokholyan N V NV   Major M B MB  

Molecular and cellular biology 20181029 22


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  ...[more]

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