At the forefront of NF-κB research, a collaborative review authored by Professors Alexander Hoffmann and Genhong Cheng from UCLA alongside Nobel laureate David Baltimore from Caltech offers an all-encompassing synthesis of NF-κB’s multifaceted mechanisms and therapeutic potentials. Published in the open-access journal Immunity & Inflammation on September 4, 2025, this authoritative review dissects NF-κB’s canonical and non-canonical activation pathways, the nuanced layers of transcriptional regulation, and the clinical implications of targeting this pathway in diverse diseases.

The canonical NF-κB signaling pathway is predominantly activated by external stimuli such as microbial infection or inflammatory cues. Upon engagement of pattern recognition receptors like Toll-like receptors (TLRs), or cytokine receptors such as TNFR1, and antigen receptors including T cell receptors (TCR) and B cell receptors (BCR), a cascade ensues that culminates in the assembly and activation of the inhibitor of κB kinase (IKK) complex. This complex phosphorylates the inhibitory protein IκBα, marking it for degradation and thereby liberating NF-κB dimers to translocate into the nucleus where they drive transcription of target genes. This pathway is tightly modulated by sophisticated negative feedback loops through proteins like IκB and A20, ensuring balanced immune responses. Dysregulation here can precipitate severe conditions, such as cytokine storms triggered by hyperactive TLR4 signaling or tumorigenesis linked to chronic IKKβ activation.

In juxtaposition, the non-canonical NF-κB pathway unfolds with markedly slower kinetics and principally governs adaptive immune functions including lymphoid organ development and B cell survival. Activated by a limited cohort of tumor necrosis factor receptor superfamily members, this axis hinges on the NF-κB-inducing kinase (NIK) to drive processing of the p100 precursor into p52, shaping a distinct NF-κB dimer composition. The non-canonical route is intricately regulated, with aberrations frequently implicated in malignancies and autoimmune pathologies. Persistent NIK stabilization is a hallmark of several B cell lymphomas, while sustained BAFF signaling prolongs autoreactive B cell lifespan in systemic lupus erythematosus, illustrating the clinical significance of this pathway’s homeostasis.

Importantly, these seemingly discrete signaling routes intersect and engage in molecular cross-talk, with NIK influencing canonical IKK complexes and canonical NF-κB activity inducing expression of components like p100 and A20, creating a highly interconnected regulatory network. This integration ensures that NF-κB responses are finely tuned to cellular context and stimulus type, harmonizing immune activation and developmental processes in a tightly controlled manner.

Transcriptional regulation by NF-κB is exceedingly dynamic and context-specific. The functional outcomes depend heavily on the composition of NF-κB dimers—combinations of RelA, RelB, c-Rel, p50, and p52 subunits—which differ in DNA-binding specificity and interactions with chromatin remodelers and co-regulators. Furthermore, various post-translational modifications on NF-κB subunits provide an additional regulatory dimension, enabling rapid, reversible control of transcriptional activity. This complexity allows NF-κB to exert differential effects on gene expression, sometimes exhibiting opposing functions in inflammation and cell survival, underscoring the pathway’s duality in health and disease.

The pathological spectrum influenced by NF-κB is broad, encompassing chronic inflammatory disorders, oncogenesis, neurodegeneration, metabolic syndromes, cardiovascular diseases, and autoimmunity. Hoffmann and colleagues provide a detailed review of therapeutic modalities targeting NF-κB signaling, ranging from small-molecule inhibitors to biologics that dampen upstream receptor activation or kinase activity. Despite significant progress, these interventions are constrained by side effects such as immunosuppression, development of drug resistance, inadvertent promotion of tumorigenesis, and toxicity. Such challenges highlight the imperative for next-generation strategies with improved precision.

Emerging therapeutic avenues aimed at selectively modulating NF-κB subunits or harnessing novel technologies like proteolysis-targeting chimeras (PROTACs), gene editing tools, nanomedicine delivery systems, and combinatorial immunotherapies represent promising directions for overcoming existing limitations. Tailored approaches that consider the context-dependent nature of NF-κB signaling could revolutionize treatment paradigms in inflammatory and neoplastic diseases by maximizing efficacy while minimizing adverse outcomes.

Looking ahead, the authors emphasize the necessity of integrating cutting-edge technologies including multi-omics analytics, high-resolution imaging, and artificial intelligence-driven data interpretation to dissect NF-κB’s spatiotemporal regulation at molecular and systemic scales. Advancements in these areas will facilitate unprecedented insights into how NF-κB orchestrates complex cellular responses in vivo, paving the way for rational and personalized therapeutic interventions.

Echoing the vision of Professor David Baltimore, who sadly passed away shortly after this publication, the translation of foundational NF-κB research into precision medicine holds promise for tailored combinatorial therapies that address individual patient heterogeneity. This personalized approach aims to harness the full therapeutic potential of NF-κB modulation while mitigating risks, aspiring to transform patient outcomes across a spectrum of immune-related and malignant diseases.

This comprehensive review not only honors the legacy of Prof. Baltimore but sets a new standard in our understanding of NF-κB’s centrality to immunology and beyond. It serves as a critical resource for researchers and clinicians seeking to unravel the intricate biology of this master regulator and to innovate effective therapeutic strategies that can alleviate human suffering caused by NF-κB dysregulation.

Subject of Research: Not applicable

Article Title: NF-κB: Master Regulator of Cellular Responses in Health and Disease

News Publication Date: 4-Sep-2025

References:
DOI: 10.1007/s44466-025-00014-0

Image Credits:
Prof. Alexander Hoffmann and Prof. Genhong Cheng from the University of California, U.S.

Keywords:
Immunology; Signal transduction; NF kappa B pathway; Inflammation; Immune response; Autoimmune disorders; Cancer research; Gene regulation; Drug development

NF-κB transcription factors form dimers from five key subunits—RelA (p65), RelB, c-Rel, NF-κB1 (p50/p105), and NF-κB2 (p52/p100)—allowing functional versatility in gene regulation. The dynamic interplay between these subunits and AR proteins orchestrates the fine-tuning of downstream transcription, a process now known to be heavily influenced by the ankyrin repeat domains acting as versatile protein–protein interaction modules. This structural motif appears instrumental in mediating not only inhibitory control but also chromatin remodeling and transcriptional specificity.

The IκB family, long recognized for its inhibitory regulation of NF-κB, is itself a fertile ground of AR domain-containing proteins. These encompass precursor proteins like p100 (IκBδ) and p105 (IκBγ), classical cytoplasmic inhibitors—IκBα, IκBβ, and IκBε—and the more recently appreciated nuclear IκBs, including Bcl-3, IκBζ, IκBNS, and IκBη. Each harbors six to eight ankyrin repeats that directly engage NF-κB dimers, thereby orchestrating nuanced regulatory outcomes. Intriguingly, these interactions transcend mere sequestration, as nuclear IκBs participate actively in transcriptional complexes to either repress or promote gene expression.

Among nuclear IκBs, IκBζ forms a transcriptionally active complex with p50 and p52 NF-κB subunits on specific target genes such as Lcn2, employing a critical aspartate residue within its first ankyrin repeat for the interaction. The nuanced recognition of specific NF-κB subunits highlights the precision of AR-mediated binding, suggesting architectural adaptability encoded within these motifs. Bcl-3 further exemplifies the multifaceted nature of these interactions, stabilizing p50 homodimers on DNA and preventing their ubiquitination, thereby modulating inflammatory gene expression. Structural studies reveal that Bcl-3 extensively contacts ARs 1, 6, and 7 of p50, underscoring the spatial specificity inherent in AR domain engagements.

IκBη extends this paradigm, utilizing its eight ankyrin repeats to bind p50, a process integral to its nuclear localization and function. This emphasizes that ARs not only mediate protein–protein interactions but can also influence subcellular distribution, offering a dual regulatory axis in transcriptional control. Collectively, these insights redefine nuclear IκBs from passive inhibitors to active transcriptional co-regulators, intricately sculpting NF-κB-driven gene expression.

Beyond classical NF-κB regulators, the oncogenic AR protein p28GANK shines as a compelling antagonist of NF-κB activity. Overexpressed in hepatocellular carcinoma, p28GANK contains seven ankyrin repeats structurally reminiscent of IκBs and exerts profound effects on NF-κB RelA (p65) subunit activity. Contrasting mechanistic reports converge on its ability to bind RelA via these repeats, suppressing its transcriptional activity through different molecular routes. One pathway involves modulation of RelA acetylation levels by recruiting the deacetylase SIRT1, dampening transcription without affecting nuclear translocation or DNA binding. Alternatively, other evidence indicates p28GANK enforces cytoplasmic retention of RelA by exporting it through a CRM-1-dependent pathway, effectively sequestering NF-κB from chromatin. This duality underscores the functional versatility provided by the ankyrin repeat scaffold in modulating key oncogenic signaling molecules.

The ASPP family, encompassing ASPP1, ASPP2, and the inhibitory iASPP, further exemplify AR-domain-mediated regulation at the interface of apoptosis and inflammation. Characterized by their C-terminal proline-rich region, four ankyrin repeats, and SH3 domain, these proteins utilize their ANK-SH3 composite to engage the p65 subunit of NF-κB. Through these interactions, ASPP2 can interface with NF-κB pathways, integrating apoptotic control with inflammatory signaling, a nexus vital for cellular fate decisions in stress and disease contexts. The inhibitory iASPP likewise binds p65, indicating that modulation by AR proteins spans activation to suppression within NF-κB-driven transcription.

Intriguingly, the NF-κB family itself is autoregulatory through its ankyrin repeats. The ubiquitin ligase KPC1 targets the AR domain of NF-κB precursor p105, enhancing its ubiquitination and limiting proteasomal processing into p50. This regulatory mechanism influences the balance of NF-κB dimers and downstream gene expression, impacting tumor suppressor expression and immune cell recruitment. The capacity of AR domains within NF-κB proteins to attract ubiquitin ligases reflects a sophisticated self-modulatory feedback controlling signaling amplitude and duration.

Turning attention to Notch signaling, the Notch intracellular domain (NICD) features its own cluster of seven ankyrin repeats essential for transcriptional activation. NICD interacts directly with the transcription factor RBPJ via its AR domain, initiating expression of key downstream genes such as Dll4, establishing positive feedback loops that underpin cell fate determination during development and angiogenesis. This interaction is finely modulated by another AR domain-containing protein, GIT1, which competes with NICD for RBPJ binding, inhibiting the Dll4-Notch1 axis in stalk cells. Such competition preserves cellular heterogeneity and supports angiogenic sprouting, highlighting AR domains as dynamic modules regulating signal flux beyond simple activation.

Notably, the gene NRARP, itself a Notch target, encodes a protein comprising three ankyrin repeats that extend the NICD ankyrin repeat stack upon forming a tripartite complex with NICD1 and RBPJ. This extension acts as a negative feedback loop, tempering Notch signaling output and illustrating how AR domain architecture can shape transcription factor complex conformation and function. This mechanistic insight into NRARP’s role completes a feedback circuit integral for fine-tuning vascular development.

This emerging paradigm underscores ankyrin repeats as modular units of regulation transcending canonical structural roles. Their presence across diverse proteins—ranging from classical inhibitors like IκBs to oncoproteins like p28GANK, and signaling mediators like NICD and NRARP—demonstrates a conserved evolutionary strategy to exploit repeat motifs for dynamic protein interactions, subcellular localization, and transcriptional control. Such versatility grants AR-containing proteins the ability to govern multiple signaling pathways simultaneously, making them prime candidates for therapeutic targeting in inflammation, cancer, and developmental disorders.

Given the ubiquity and functional diversity of ankyrin repeats, future research will undoubtedly uncover novel AR-containing players and mechanisms in epigenetic and transcriptional regulation. Structural biology combined with systems-level analysis of AR-mediated interactomes promises to reveal comprehensive networks that govern cellular identity and response, providing unprecedented opportunities to manipulate these pathways in disease intervention. The exquisite specificity and adaptability of AR domains offer templates for designing small molecules or biologics that modulate protein–protein interactions currently deemed undruggable.

As our understanding expands, the convergent roles of ankyrin repeat proteins in both NF-κB and Notch signaling pathways underscore the integrative nature of cellular signaling hubs. They act not only as structural motifs but as finely tuned regulatory elements that determine the specificity, timing, and magnitude of transcriptional responses. This knowledge pivots ankyrin repeats from peripheral structural components to central regulatory nodes with broad impact on health and disease.

In summary, the intricate dance of ankyrin repeat-containing proteins in modulating transcription factors like NF-κB and NICD reveals a landscape of complex protein interaction networks vital for cellular regulation. Their modulation of gene expression networks implicates these AR modules as keystones in the balance between homeostasis and pathology. Unlocking their mechanistic secrets heralds a new chapter in molecular biology, where precise control over these repeat domains might pave the way for novel therapies across a spectrum of inflammatory, oncogenic, and developmental diseases.

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Subject of Research: Ankyrin repeat-containing proteins and their roles in epigenetic and transcriptional regulation.

Article Title: The role of ankyrin repeat-containing proteins in epigenetic and transcriptional regulation.

Article References: Wu, M., Zhao, Y., Yang, J. et al. The role of ankyrin repeat-containing proteins in epigenetic and transcriptional regulation. Cell Death Discov. 11, 232 (2025). https://doi.org/10.1038/s41420-025-02519-4

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41420-025-02519-4