Drosophila Keap1 Proteins Assemble Nuclear Condensates in Response to Oxidative Stress

Area of Science:

• Cellular biology
• Molecular mechanisms of oxidative stress response

Background:

• The Keap1-Nrf2 pathway regulates oxidative stress responses, with Keap1 (Kelch-like ECH-associated protein 1) controlling Nrf2 (NF-E2-related factor 2) degradation.
• While Keap1-Nrf2 interaction is cytoplasmic, Keap1 proteins are increasingly recognized for nuclear roles in transcription, though mechanisms are unclear.

Purpose of the Study:

• To investigate the nuclear behavior and function of the Keap1 ortholog in Drosophila (dKeap1) under oxidative stress.
• To elucidate the molecular mechanisms underlying dKeap1 nuclear localization and foci formation.

Main Methods:

• Treatment of Drosophila cells with oxidative stimuli.
• Fluorescence recovery after photobleaching (FRAP) to assess dKeap1 mobility.
• Analysis of dKeap1 domains (NTD, CTD, Kelch) for foci formation.
• In vitro condensate formation assays using CTD-YFP fusion proteins.

Main Results:

• Oxidative stress induced nuclear accumulation and stable foci formation of dKeap1.
• dKeap1 mobility was reduced within these nuclear foci.
• Both N-terminal (NTD) and C-terminal (CTD) domains were essential for foci formation, with intrinsically disordered regions in the CTD promoting condensate formation.
• The Kelch domain inhibited condensate formation, and its deletion caused cytoplasmic foci even without oxidative stress.

Conclusions:

• dKeap1 forms nuclear condensates upon oxidative stress, regulated by its NTD and CTD, suggesting a novel mechanism for nuclear function.
• The Kelch domain acts as a negative regulator of dKeap1 nuclear condensate formation.
• Findings provide new insights into Keap1's broader roles in oxidative response, development, and disease.