Drosophila Keap1 Forms Nuclear Condensates Under Oxidative Stress

Study Background and Research Question

The Keap1-Nrf2 signaling axis is a cornerstone of the cellular response to oxidative and xenobiotic stresses, orchestrating the transcription of antioxidant and detoxification genes. Disruptions in this pathway are implicated in a wide spectrum of human diseases, notably cancer and neurodegenerative disorders. Traditionally, Keap1 functions in the cytoplasm, targeting Nrf2 for degradation and maintaining cellular homeostasis. However, emerging evidence indicates that both Keap1 and Nrf2 can localize to the nucleus and directly modulate gene expression. The precise molecular mechanisms and the significance of nuclear Keap1, particularly in developmental gene regulation and chromatin organization, remain incompletely understood. The present study (Antioxidants 2026, 15, 134) addresses a key question: how does Drosophila Keap1 (dKeap1) behave in the nucleus under oxidative stress, and what are the structural requirements for its nuclear functions?

Key Innovation from the Reference Study

The central innovation of this work is the demonstration that dKeap1 assembles into stable nuclear condensates in response to oxidative stimuli, a process dependent on specific protein domains. This finding establishes a new paradigm for Keap1 family proteins as dynamic regulators of nuclear architecture, acting not only through canonical stress signaling but also via the formation of nonmembranous nuclear compartments. The study provides mechanistic insights by mapping the domain requirements and identifying intrinsically disordered regions (IDRs) that facilitate phase separation—a feature increasingly recognized as fundamental to nuclear organization and gene regulation.

Methods and Experimental Design Insights

The authors employed a combination of live-cell fluorescence imaging, genetic manipulation, in vitro phase separation assays, and FRAP (fluorescence recovery after photobleaching) analysis to dissect dKeap1 behavior in Drosophila cells. Oxidative stress was induced chemically, and the subcellular localization and dynamics of dKeap1 fused to fluorescent tags (e.g., YFP) were tracked over time. To probe the molecular determinants of condensate formation, a series of dKeap1 truncation and domain deletion mutants were engineered. These included constructs lacking the N-terminal domain (NTD), the C-terminal domain (CTD), or the Kelch domain, as well as isolated IDRs fused to fluorophores. In vitro assays assessed the propensity of these proteins to self-associate and form droplets under defined conditions. The requirement for specific domains in condensate assembly was correlated with in vivo nuclear behavior post-oxidative challenge.

Protocol Parameters

• Oxidative stress induction: Drosophila cells were exposed to defined concentrations of oxidants (e.g., hydrogen peroxide) for controlled time intervals to elicit nuclear translocation and condensate formation by dKeap1.
• Fluorescent fusion constructs: dKeap1 and its variants were fused to YFP or other tags for live imaging of subcellular localization.
• Domain mapping: Systematic deletion mutants (ΔKelch, ΔNTD, ΔCTD, and isolated IDRs) were generated to pinpoint structural elements essential for phase separation.
• FRAP analysis: Mobility of dKeap1 within nuclear foci was quantified to distinguish liquid-like from gel-like condensate properties.
• In vitro phase separation: Recombinant fusion proteins were expressed and purified, then subjected to droplet formation assays under physiological buffer conditions to recapitulate in vivo condensate assembly.

Core Findings and Why They Matter

The study's major findings are as follows:

• Nuclear accumulation and foci assembly: Upon oxidative stress, dKeap1 rapidly accumulates in the nucleus and organizes into stable nuclear foci. These structures exhibit reduced protein mobility, indicative of biomolecular condensates.
• Requirement for NTD and CTD: Both the N-terminal and C-terminal domains are necessary for condensate formation. Deletion of either domain abrogates nuclear foci assembly, underscoring their cooperative roles.
• Role of IDRs: Two intrinsically disordered regions within the CTD are sufficient to drive condensate formation in vitro, confirming that phase separation is encoded at the sequence level.
• Kelch domain as a negative regulator: Deletion of the Kelch domain leads to spontaneous cytoplasmic condensates, even in the absence of stress, and enhances phase separation in vitro. Thus, the Kelch domain functions as a suppressor of condensate assembly, fine-tuning the nuclear versus cytoplasmic localization and activity of dKeap1.

These findings position dKeap1 as a regulator of nuclear organization through phase separation, providing a mechanistic link between oxidative signaling and chromatin-associated gene regulation. The results also highlight the importance of IDRs in scaffolding nuclear condensates, a theme shared with other chromatin regulators and transcriptional machinery (reference study).

Comparison with Existing Internal Articles

While the reference study focuses on the mechanistic biology of nuclear condensates, internal resources such as PreScission Protease: Precision Fusion Protein Tag Cleavage explore the technical challenges associated with studying such protein assemblies. For example, purification of recombinant proteins containing IDRs or prone to phase separation often necessitates highly specific tag removal to avoid off-target effects and preserve native structure. Internal articles highlight how PreScission Protease (PSP), leveraging HRV 3C protease specificity, enables precise fusion protein tag cleavage at the Gln-Gly bond under low temperature—conditions that help stabilize sensitive protein domains. These workflow considerations are directly relevant when working with dKeap1 constructs, as improper tag removal or proteolysis can disrupt IDR-mediated condensation properties, potentially confounding mechanistic studies (see discussion on enzyme selection for challenging recombinant proteins).

Limitations and Transferability

Despite its mechanistic depth, the study is limited to Drosophila systems and may not fully recapitulate the diversity of Keap1 functionalities in mammals. The in vitro assays, while informative, rely on recombinant fusion proteins that may not entirely capture posttranslational modifications or interactome complexity in vivo. Additionally, the study does not directly interrogate the functional consequences of condensate assembly on specific gene expression outcomes or developmental processes. Thus, while the domain logic and role of IDRs are likely conserved, direct extrapolation to human Keap1-Nrf2 biology requires further validation.

Why this cross-domain matters, maturity, and limitations

The formation of biomolecular condensates by dKeap1 situates this protein at the interface between stress response signaling and nuclear organization, with broad implications for developmental biology and disease. However, translation of these findings to therapeutic or clinical applications is premature. The current work establishes foundational cell biological principles, but the maturity of the field will depend on future studies linking condensate dynamics to organismal physiology and pathology.

Research Support Resources

For researchers aiming to dissect the molecular mechanisms of protein phase separation or to purify IDR-rich fusion proteins for similar studies, the selection of a highly specific and low-temperature compatible protein purification enzyme is critical. PreScission Protease (PSP) (SKU K1101) offers HRV 3C protease-based cleavage precision, supporting efficient GST fusion protein cleavage at 4°C and minimizing unwanted proteolysis of sensitive domains, as discussed in multiple workflow articles. This reagent can be integrated into workflows requiring tag removal from dKeap1 or related constructs, ensuring high-fidelity recovery of native protein for downstream condensate biology research.