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IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments. We want to thank the Bigas’ and Espinosa’s lab members for constructive discussions and suggestions and technical support. This work has been funded by Instituto de Salud Carlos III FEDER (PI19/0013), Agencia Estatal de Investigación, Spain (PID2019-104695RB-I00) to A.B., NIH/GM085490 to G.G., BIO2017-83650-P to JVF and Generalitat de Catalunya 2017SGR135. LM is a predoctoral fellow of 2015FI-B00806 and 2016FI-B1 00110 and MF has financial support by the Universitat de Vic-Universitat Central de Catalunya PhD fellowships program. The authors thankfully acknowledge the computer resources at Pirineus and the technical support provided by the Spanish Supercomputer Network (BCV-2020-1-0001).

IκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for IκB homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied.We here show that serine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBα association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-IκBα binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding and impaired goblet cell differentiation, comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.

IκB kinase 2/β (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2’s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors.SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a ‘Cys-Cys motif’ for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.

LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.

The human IκB Kinase (IKK) is a multisubunit protein complex of two kinases and one scaffolding subunit that controls induction of transcription factor NF-κB activity. IKK behaves as an entity of aberrantly high apparent molecular weight in solution. Recent X-ray crystallographic and cryo-electron microscopy structures of individual catalytic subunits (IKK1/IKKα and IKK2/IKKβ) reveal that they are both stably folded dimeric proteins that engage in extensive homo-oligomerization through unique surfaces that are required for activation of their respective catalytic activities. The NEMO/IKKγ subunit is a predominantly coiled coil protein that is required for activation of IKK through the canonical NF-κB signaling pathway. Here we report size-exclusion chromatography, multi-angle light scattering, analytical centrifugation, and thermal denaturation analyses of full-length human recombinant NEMO as well as deletion and disease-linked variants. We observe that NEMO is predominantly a dimer in solution, although by virtue of its modular coiled coil regions NEMO exhibits complicated solution dynamics involving portions that are mutually antagonistic toward homodimerization. This behavior causes NEMO to behave as a significantly larger sized particle in solution. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and provides the complex-bound IKK2 further propensity toward homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO subunit dimers which helps clarify its role in diseases and in IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers.

The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5′-GGGRNNNYCC-3′ (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.

The NF-κB (Nuclear Factor kappa B) transcription factor plays crucial roles in the regulation of numerous biological processes including development of the immune system, inflammation, and innate and adaptive immune responses. Control over the immune cell functions of NF-κB results from signaling through one of two different routes: the canonical and noncanonical NF-κB signaling pathways. Present at the end of both pathways are the proteins NF-κB, IκB, and the IκB kinase (IKK). These proteins work together to deliver the myriad outcomes that influence context-dependent transcriptional control in immune cells. In the present chapter, we review the structural information available on NF-κB, IκB, and IKK, the critical terminal components of the NF-κB signaling, in relation to their physiological function.

The NF-κB (Nuclear Factor kappa B) transcription factor plays crucial roles in the regulation of numerous biological processes including development of the immune system, inflammation, and innate and adaptive immune responses. Control over the immune cell functions of NF-κB results from signaling through one of two different routes: the canonical and noncanonical NF-κB signaling pathways. Present at the end of both pathways are the proteins NF-κB, IκB, and the IκB kinase (IKK). These proteins work together to deliver the myriad outcomes that influence context-dependent transcriptional control in immune cells. In the present chapter, we review the structural information available on NF-κB, IκB, and IKK, the critical terminal components of the NF-κB signaling, in relation to their physiological function.

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

The nuclear factor-κB (NF-κB) signalling pathway participates in a multitude of biological processes, which imply the requirement of a complex and precise regulation. IκB (for Inhibitor of kappaB) proteins, which bind and retain NF-κB dimers in the cytoplasm, are the main contributors to negative regulation of NF-κB under non-stimulation conditions. Nevertheless, increasing evidences indicate that IκB proteins exert specific nuclear roles that directly contribute to the control of gene transcription. In particular, hypophosphorylated IκBβ can bind the promoter region of TNFα leading to persistent gene transcription in macrophages and contributing to the regulation of the inflammatory response. Recently, we demonstrated that phosphorylated and SUMOylated IκBα reside in the nucleus of the cells where it binds to chromatin leading to specific transcriptional repression. Mechanistically, IκBα associates and regulates Polycomb Repressor Complex activity, a function that is evolutionary conserved from flies to mammals, as indicate the homeotic phenotype of Drosophila mutants. Here we discuss the implications of chromatin-bound IκBα function in the context of tumorigenesis.

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation., M.M. is an investigator of the Sara Borrell program (CD09/00421). E.L.A. is a recipient of a predoctoral fellowship from the Department of Education, Universities and Research of the Basque Government (BFI-2011), and V.R. was a recipient of FIMIM predoctoral fellowship. L.E. is an investigator at the Carlos III program. This work was supported by a grant from the Instituto de Salud Carlos III (PI041890), Fundación Mutua Madrileña, AGAUR (SGR23), and RTICCS/FEDER (RD06/0020/0098 and RD12/0036/0054). M.D. was funded by the Ministerio de Ciencia e Innovación (BFU2009-09074 and MEC-CONSOLIDER CSD2007-00023), Generalitat Valenciana (PROMETEO 2008/134), and a grant from European Union Research (UE-HEALH-F2-2008-201666). J.V-F. was funded by a MICINN grant (CTQ2008-00755) and the EC VPH (no. FP7-2007-IST-223920).