Drosophila Keap1 Forms Nuclear Condensates Under Oxidative Stress

Study Background and Research Question

The Keap1-Nrf2 signaling axis is a central regulator of cellular responses to oxidative and xenobiotic stress. In mammals and Drosophila, Keap1 binds Nrf2 in the cytoplasm, promoting its ubiquitin-dependent degradation under resting conditions. Upon oxidative insult, this interaction is disrupted, allowing Nrf2 to accumulate in the nucleus and activate transcription of antioxidant genes. While the cytoplasmic regulatory circuit is well-established, accumulating evidence suggests that Keap1 family proteins, including the Drosophila ortholog dKeap1, also enter the nucleus, bind chromatin, and directly modulate gene expression. However, the mechanisms underlying this nuclear function—and whether they are linked to phase separation or condensate biology—have remained unclear. The central research question addressed by Ji et al. (Antioxidants 2026, 15, 134) is: how does dKeap1 respond to oxidative stress at the nuclear level, and what structural domains mediate its functional assembly within the nucleus?

Key Innovation from the Reference Study

This study provides the first direct demonstration that dKeap1 forms stable nuclear condensates in vivo in response to oxidative stress. Importantly, these condensates exhibit properties associated with biomolecular phase separation, implicating a new mechanism for Keap1-dependent transcriptional regulation. Furthermore, detailed domain mapping reveals that both N-terminal and C-terminal domains of dKeap1 are essential for condensate formation, while the Kelch domain acts as a negative regulator. The identification of two intrinsically disordered regions (IDRs) in the CTD and their sufficiency to drive in vitro condensate formation highlight a modular architecture that likely governs stress-responsive nuclear assembly.

Methods and Experimental Design Insights

The authors employed a multifaceted approach to dissect dKeap1 nuclear dynamics and the requirements for condensate formation:

• Genetic Models: Drosophila stocks expressing wild-type and mutant forms of dKeap1, including domain deletions and fluorescent fusion constructs, enabled precise structure-function analysis.
• Live-Cell Imaging: Fluorescence microscopy and FRAP (fluorescence recovery after photobleaching) were used to monitor dKeap1 subcellular localization, mobility, and condensate dynamics in response to oxidative treatments.
• In Vitro Phase Separation Assays: Recombinant dKeap1 domains fused to YFP were expressed and purified, allowing for direct testing of condensate formation under controlled conditions. This in vitro work required precise removal of fusion tags, a step often facilitated by high-specificity proteases such as HRV 3C-based reagents.
• Domain Mapping: Systematic deletions and domain swaps delineated the contributions of the NTD, CTD, and Kelch domains to condensate assembly and subcellular distribution.

Notably, the methodology integrates both in vivo and in vitro systems, leveraging the strengths of Drosophila genetics and recombinant protein biochemistry.

Core Findings and Why They Matter

• dKeap1 assembles into nuclear condensates following oxidative stress: Upon treatment, dKeap1 accumulates in the nucleus and forms discrete, stable foci with reduced internal mobility (as shown by FRAP). This supports a model where stress-responsive nuclear bodies are central to Keap1 function (reference study).
• The CTD harbors IDRs sufficient for phase separation: Two IDRs within the C-terminal domain (CTD) were necessary and sufficient to drive condensate formation in vitro, consistent with well-established principles of phase separation in nuclear organization.
• Kelch domain suppresses condensate assembly: Deletion of the Kelch domain led to inappropriate cytoplasmic condensates, suggesting that intramolecular regulation balances dKeap1 localization and assembly.
• Both NTD and CTD are required for in vivo nuclear condensate formation: Loss of either domain abrogated nuclear foci, indicating a coordinated structural requirement for stress-induced nuclear assembly.

These findings extend the canonical view of Keap1 from cytoplasmic Nrf2 regulation to a direct, modular role in nuclear organization, phase separation, and chromatin control. This has significant implications for understanding how oxidative stress modulates transcription and development.

Comparison with Existing Internal Articles

Several recent thought-leadership articles have contextualized the intersection of protein purification and nuclear condensate research. For example, "Precision Cleavage: Redefining Fusion Protein Purification" discusses how high-specificity proteases like PreScission Protease (an HRV 3C fusion protein) enable the generation of native proteins for condensate studies. This is directly relevant to the workflows in the Ji et al. study, where recombinant dKeap1 domains were analyzed for phase separation properties. Similarly, "Nuclear Condensate Assembly by Drosophila Keap1 in Oxidative Stress" provides an accessible overview of the core findings, emphasizing the structural determinants of condensate formation and their implications for chromatin regulation.

What sets the reference study apart is its mechanistic dissection of domain contributions, the demonstration of both in vivo and in vitro condensate formation, and the direct link to oxidative stress response, thereby advancing the field beyond descriptive localization studies. The use of precise protein cleavage and purification methods, as highlighted in internal resources, is critical for these mechanistic insights.

Limitations and Transferability

• Species specificity: While Drosophila models provide genetic tractability, the extent to which dKeap1 nuclear condensate mechanisms are conserved in mammalian systems remains to be validated.
• In vitro–in vivo extrapolation: Although CTD-YFP fusion proteins recapitulate condensate formation in vitro, the physiological relevance of these assemblies depends on the nuclear context and potential co-factors present in intact cells.
• Domain interaction complexity: The study maps broad requirements for the NTD, CTD, and Kelch domains, but the fine-grained molecular interactions and post-translational modifications that govern dKeap1 assembly await further structural characterization.

Thus, while the findings offer a compelling mechanistic advance, careful translation to mammalian or disease models is warranted.

Protocol Parameters

• Fusion protein tag removal: For biochemical analysis of dKeap1 domains, use site-specific cleavage at the Gln-Gly bond within engineered HRV 3C recognition sites, enabling recovery of untagged domains for phase separation assays.
• Protein purification workflow: Maintain low temperature (4°C) during protease cleavage and purification steps to preserve the structural integrity of IDRs and prevent non-specific aggregation, as recommended for low temperature protease activity.
• Fluorescent labeling: Employ YFP or similar tags for live-cell imaging and in vitro condensate visualization, ensuring tag removal for subsequent biophysical characterization if necessary.

Research Support Resources

To replicate or extend studies involving fusion protein tag cleavage and characterization of condensate-forming domains, researchers can utilize PreScission Protease (PSP) (SKU K1101), a recombinant HRV 3C protease fused to GST. This enzyme provides high specificity for cleavage at the Gln-Gly bond and is optimized for low temperature protease activity, facilitating recovery of native proteins suitable for phase separation and chromatin studies. For further workflow guidance and case studies, see "Precision in Protein Purification: PreScission Protease". APExBIO’s PSP supports rigorous protein purification workflows required for advanced nuclear condensate research, but workflow optimization should be tailored to the biophysical requirements of the target protein system.