Drosophila Keap1 Nuclear Condensates in Oxidative Stress Response

Study Background and Research Question

The Keap1-Nrf2 pathway is a cornerstone of cellular defense, orchestrating transcriptional responses to oxidative and xenobiotic stress. Under non-stress conditions, Keap1 (Kelch-like ECH-associated protein 1) acts as a cytoplasmic repressor, targeting Nrf2 for ubiquitin-mediated degradation. Upon oxidative challenge, this interaction is disrupted, allowing Nrf2 to translocate to the nucleus and activate genes responsible for antioxidant and detoxification functions. While the cytoplasmic dynamics of Keap1-Nrf2 signaling are well characterized, recent studies hint at additional nuclear roles for Keap1 family proteins, particularly in developmental transcription programs and chromatin regulation. However, the molecular mechanisms underpinning Keap1's nuclear functions remain poorly understood. The current study (Antioxidants 2026, 15, 134) addresses this gap by investigating how the Drosophila Keap1 ortholog (dKeap1) responds to oxidative stress at the nuclear level.

Key Innovation from the Reference Study

This work demonstrates for the first time that dKeap1 assembles into stable, phase-separated nuclear condensates following oxidative stress. Unlike its canonical role in the cytoplasm, dKeap1's nuclear function appears to depend on the orchestrated activity of distinct protein domains and the formation of biomolecular condensates—membraneless compartments implicated in organizing nuclear processes. The discovery that both the N-terminal and C-terminal domains of dKeap1 are necessary for nuclear condensate assembly, and that the Kelch domain acts as a negative regulator of this process, provides a new paradigm for understanding how oxidative stress modulates nuclear architecture and transcriptional regulation in metazoans.

Methods and Experimental Design Insights

The research combined in vivo and in vitro approaches to dissect the structural requirements for dKeap1 condensate formation and their response to oxidative cues:

• Live Cell Imaging: Drosophila tissues and cultured cells expressing tagged dKeap1 were subjected to oxidative stress. High-resolution fluorescence microscopy tracked dKeap1 localization and condensate formation dynamics.
• Domain Mapping and Mutational Analysis: Deletion constructs targeting the N-terminal, Kelch, and C-terminal domains, as well as intrinsic disorder regions (IDRs), were generated to define regions essential for condensate formation.
• FRAP (Fluorescence Recovery After Photobleaching): This technique assessed the mobility and stability of dKeap1 within nuclear condensates, revealing reduced protein mobility indicative of a phase-separated state.
• In Vitro Condensate Assays: Purified dKeap1 domain fusion proteins were tested for their intrinsic ability to phase separate under controlled conditions, confirming that the C-terminal domain and its IDRs drive condensate formation.

The study also examined the effects of domain deletions on both nuclear and cytoplasmic distribution, showing that removal of the Kelch domain leads to abnormal cytoplasmic foci even in the absence of stress.

Core Findings and Why They Matter

The central findings are as follows (reference study):

• Oxidative stress induces dKeap1 nuclear accumulation and condensate assembly. Gradual formation of stable nuclear foci is observed in response to oxidative challenge.
• Reduced mobility of dKeap1 in nuclear condensates. FRAP analyses confirmed that dKeap1 becomes less dynamic within these foci, consistent with liquid–liquid phase separation (LLPS) behavior.
• Domain-specific requirements: Both N-terminal (NTD) and C-terminal (CTD) domains are essential for condensate formation, while the Kelch domain suppresses aberrant condensate assembly in the cytoplasm.
• Intrinsic disorder and phase separation: Two intrinsically disordered regions (IDRs) within the CTD are necessary and sufficient for condensate formation in vitro, highlighting the importance of protein disorder in nuclear organization.

These insights represent a significant advance in our mechanistic understanding of how stress-responsive factors like dKeap1 may organize nuclear microenvironments to modulate gene expression. By demonstrating that Keap1 nuclear function is intimately tied to phase separation and specific domain architecture, the study bridges the gap between stress signaling and chromatin biology, with implications for both development and disease processes.

Comparison with Existing Internal Articles

The reference study's focus on nuclear condensates and domain-driven assembly mechanisms aligns closely with recent discussions in the scientific literature regarding the intersection of protein purification technologies and condensate biology. Internal articles such as "Harnessing Precision Proteolysis: PreScission Protease in Translational Protein Research" emphasize the necessity of maintaining protein native structure and interactions during isolation, particularly for studies probing condensate formation or chromatin association. The use of highly specific protein purification enzymes, including PreScission Protease (PSP), ensures that recombinant proteins used in such studies retain their functional domains and intrinsic disorder regions essential for phase separation. Further, the article "PreScission Protease: Precision Tag Cleavage for Nuclear Protein Research" provides troubleshooting strategies tailored for nuclear protein workflows, echoing the challenges encountered in the experimental design of the present study.

Limitations and Transferability

While the findings provide compelling evidence for dKeap1 nuclear condensate assembly in Drosophila, several limitations should be recognized:

• Species specificity: The study uses the Drosophila ortholog, and although mammalian Keap1 shares structural and functional features, direct extrapolation to human systems will require further validation.
• In vitro versus in vivo context: While IDR-driven condensate formation is recapitulated in vitro, the full repertoire of interacting partners and post-translational modifications present in vivo may modulate condensate dynamics.
• Functional readouts: The precise impact of dKeap1 condensate formation on transcriptional output and developmental gene regulation warrants deeper investigation.

Nevertheless, the mechanistic framework established here is readily transferable to broader studies of nuclear phase separation, chromatin organization, and stress-responsive transcriptional control.

Protocol Parameters

• Oxidative stress induction: Optimize exposure conditions to promote robust dKeap1 nuclear accumulation without compromising cell viability.
• Fusion protein tag cleavage: Employ a highly specific protease to remove affinity tags, preserving native protein conformation and post-translational modifications critical for condensate studies (see below for practical resources).
• Low temperature protease activity: Conduct cleavage reactions at 4°C to maintain protein stability and prevent unwanted aggregation, particularly for proteins with significant intrinsic disorder.
• Imaging and FRAP: Use high-sensitivity fluorescence microscopy and validate condensate formation using photobleaching assays to confirm phase separation properties.

Research Support Resources

For researchers aiming to replicate or extend these findings, the choice of protein purification enzyme is critical for preserving functional domains necessary for condensate assembly. PreScission Protease (PSP) (SKU K1101) provides HRV 3C protease specificity and efficient GST fusion protein cleavage at low temperatures, supporting workflows that require retention of native structure and activity. According to the internal article, PSP is especially suitable for sensitive nuclear protein and condensate studies, enabling precise removal of fusion tags and facilitating downstream analyses. Aliquot storage and optimized buffer conditions are recommended to maximize enzyme activity and reproducibility. For detailed guidance on integrating PSP into nuclear protein workflows, see the resources linked above.