Nuclear Condensate Formation by Drosophila Keap1 in Oxidative Stress

Study Background and Research Question

The Keap1-Nrf2 signaling axis is a well-established mechanism through which cells respond to oxidative and xenobiotic stress, orchestrating the transcription of antioxidant and detoxification genes. In the canonical model, Keap1 (Kelch-like ECH-associated protein 1) binds Nrf2 (NF-E2–related factor 2) in the cytoplasm, promoting Nrf2's proteasomal degradation. Upon oxidative challenge, this interaction is disrupted, allowing Nrf2 to translocate to the nucleus and activate protective gene expression programs. Despite extensive research into Keap1-Nrf2 cytoplasmic dynamics, the nuclear functions of Keap1 family proteins—including their roles in chromatin regulation and development—remain incompletely understood. The referenced study (Ji et al., 2026) addresses the key question: How does Drosophila Keap1 (dKeap1) function in the nucleus during oxidative stress, and what mechanisms underlie its potential to form nuclear condensates?

Key Innovation from the Reference Study

The principal innovation of this work lies in demonstrating that dKeap1, beyond its well-known cytoplasmic activities, relocates to the nucleus and assembles into stable nuclear condensates upon oxidative stress. These condensates are characterized as biomolecular assemblies—reminiscent of liquid–liquid phase-separated (LLPS) compartments—that may serve to reorganize nuclear microenvironments and modulate chromatin-based gene regulation. Importantly, the study delineates structural requirements for condensate formation, identifying both the N-terminal and C-terminal domains as essential, and illuminating the role of dKeap1’s C-terminal intrinsically disordered regions (IDRs) in driving phase separation. This nuclear condensation activity suggests a new paradigm for Keap1 function in stress adaptation and developmental gene regulation.

Methods and Experimental Design Insights

To dissect the nuclear role of dKeap1, the authors employed a combination of in vivo and in vitro approaches:

• Fluorescence Live Imaging: Drosophila tissues subjected to oxidative stress were imaged to track the subcellular localization of dKeap1, with particular attention to the formation of nuclear foci.
• Fluorescence Recovery After Photobleaching (FRAP): FRAP experiments quantified the mobility of dKeap1 within nuclear foci, providing evidence for stable, less dynamic condensates post-stress exposure.
• Domain Deletion Mutagenesis: Systematic truncations of dKeap1 were performed to identify domains required for nuclear condensate assembly. Constructs lacking either the N-terminal or C-terminal domains failed to form nuclear foci.
• In Vitro Phase Separation Assays: Purified dKeap1 C-terminal domain fused to YFP was tested for spontaneous condensate formation, confirming the phase separation capability of the IDR-containing region.
• Functional Rescue and Localization Studies: Deletion of the Kelch domain led to aberrant cytoplasmic condensate formation, implicating this domain as a negative regulator of phase separation.

This multifaceted strategy allowed the authors to link structural features of dKeap1 directly to its nuclear condensate-forming capacity under oxidative conditions (reference).

Core Findings and Why They Matter

Among the most significant findings, the study shows that:

• dKeap1 accumulates in the nucleus and forms stable nuclear foci in response to oxidative stress. These foci exhibit reduced molecular mobility, consistent with the properties of biomolecular condensates.
• Both N- and C-terminal domains are necessary for foci formation, while the intrinsically disordered regions within the C-terminal domain actively promote condensate assembly, as demonstrated in vitro.
• The Kelch domain suppresses aberrant condensate formation, as its removal triggers cytoplasmic foci even in unstressed cells.

These findings provide a mechanistic explanation for how dKeap1 might regulate nuclear processes, such as chromatin organization and gene expression, in response to cellular stress. The direct demonstration of LLPS-driven condensate formation by a redox sensor protein extends the understanding of nuclear organization and gene regulatory logic during stress adaptation. This work also aligns with broader evidence that phase separation is a common mechanism for controlling nuclear biochemical environments (Ji et al., 2026).

Comparison with Existing Internal Articles and Advances in Protein Purification

The reference study's insights into nuclear condensate assembly are complemented by advances in protein purification technologies that enable the study of such dynamic processes. For example, one internal article highlights how the use of PreScission Protease (PSP), a highly specific HRV 3C protease, supports gentle and precise fusion protein tag cleavage, preserving protein integrity during sample preparation for condensate and chromatin research. This is especially valuable in studies like Ji et al., where maintaining the native state of recombinant proteins is critical for in vitro phase separation assays. Other internal resources (see here) discuss practical optimization of PSP workflows in protein purification and their application in the study of nuclear protein complexes. Both sources emphasize the necessity of minimizing off-target cleavage and maintaining low temperature protease activity—parameters directly relevant to phase separation and LLPS research. By combining mechanistic discoveries about dKeap1 with methodological advances in protein purification enzyme technology, researchers can more faithfully model and interrogate nuclear condensate biology.

Limitations and Transferability

While the study robustly demonstrates that dKeap1 forms nuclear condensates in response to oxidative stress in Drosophila systems, several limitations should be acknowledged:

• Model Specificity: The findings are specific to Drosophila, and while mammalian Keap1 shares homologous domains, direct evidence for similar condensate behavior in mammalian cells is still lacking.
• Functional Consequences: The study focuses on condensate formation and structural requirements but does not fully resolve the downstream gene regulatory or chromatin remodeling activities of these condensates.
• Transferability to Other Stressors: The role of dKeap1 condensates in contexts beyond oxidative stress, or in response to different cellular cues, remains to be systematically explored.

Nevertheless, the general principles of LLPS-driven nuclear organization, and the structural determinants identified, are likely to inform future research across species and systems.

Protocol Parameters

• Oxidative stress induction: Apply oxidizing agents (e.g., paraquat) at established concentrations to Drosophila tissues or cells to trigger dKeap1 nuclear translocation.
• Live imaging: Use fluorescence microscopy with GFP/YFP-tagged dKeap1 constructs; observe nuclear foci formation over time post-stress.
• FRAP analysis: Photobleach a defined region within nuclear condensates and monitor fluorescence recovery to assess protein mobility.
• Mutagenesis: Generate truncation or domain-swap constructs to map regions essential for condensate assembly.
• In vitro phase separation: Purify recombinant dKeap1 C-terminal domain (often as a GST or YFP fusion) using affinity chromatography; assess condensate formation under low temperature and appropriate buffer conditions.

Research Support Resources

For studies requiring high-specificity fusion protein tag cleavage—such as preparation of recombinant protein domains for LLPS and condensate assays—researchers can employ PreScission Protease (PSP) (SKU K1101). This HRV 3C protease, supplied by APExBIO, is optimized for low temperature protease activity and precise GST fusion protein cleavage at the Gln-Gly bond, minimizing off-target effects and preserving native protein structure. Such attributes are especially useful in workflows investigating the phase behavior of proteins like dKeap1, where maintaining protein integrity is critical. For further optimization and mechanistic details, see the internal review on PreScission Protease applications in chromatin and condensate research.