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

Area of Science:

• Molecular Biology
• Cancer Genetics
• Cellular Biology

Background:

• Loss-of-function mutations in KEAP1 (Kelch-like ECH-associated protein 1) promote cancer by stabilizing the NRF2 (Nuclear factor erythroid 2-related factor 2) transcription factor.
• KEAP1 mutations are frequently observed in various cancers, leading to altered cellular metabolism and oxidative stress responses.

Purpose of the Study:

• To characterize the molecular mechanisms of KEAP1 ANCHOR mutants, a class comprising 40% of identified KEAP1 mutations.
• To investigate the structural and functional consequences of ANCHOR mutations on NRF2 stabilization and cellular localization.

Main Methods:

• Immunoprecipitation and BioID to study protein-protein interactions.
• Molecular dynamics simulations and limited proteolysis to model protein structure.
• Confocal fluorescent imaging and transmission electron microscopy to visualize cellular structures.

Main Results:

• KEAP1 ANCHOR mutants bind and ubiquitylate NRF2 but fail to promote its degradation, leading to increased NRF2 levels.
• ANCHOR mutations stabilize KEAP1 residues interacting with NRF2, potentially via an intramolecular salt bridge.
• Mutant KEAP1 proteins form p62/SQSTM1-dependent, membraneless biomolecular condensates with NRF2 in live cells.

Conclusions:

• KEAP1 ANCHOR mutations create a unique mechanism for NRF2 stabilization through phase separation into biomolecular condensates.
• These condensates may represent an intermediate state in the cellular response to proteasomal degradation impairment and the initiation of autophagy.
• Understanding ANCHOR mutants provides insights into cancer progression and potential therapeutic targets.