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The transcription regulators of the NF-κB family have emerged as a critical factor affecting the function of various adult tissues. The NF-κB family transcription factors are homo- and heterodimers made up of five monomers (p50, p52, RelA, cRel and RelB). The family is distinguished by sequence homology in their DNA binding and dimerization domains, which enables them to bind similar DNA response elements and participate in similar biological programs through transcriptional activation and repression of hundreds of genes. Even though the family members are closely related in terms of sequence and function, they all display distinct activities. In this review, we discuss the sequence characteristics, protein and DNA interactions, and pathogenic involvement of one member of family, NF-κB/p52, relative to that of other members. We pinpoint the small sequence variations within the conserved region that are mostly responsible for its distinct functional properties.

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB–inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB–inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.

NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components--first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.

Metabolic disorders such as insulin resistance and hypertension are potential risk factors for aging and neurodegenerative diseases. These conditions are reversed in Chromogranin A (CgA) knockout (CgA-KO) mice. CgA is known to be associated with protein aggregates in the brains of neurodegenerative diseases including Alzheimer’s disease (AD). Here, we investigated the role of CgA in Tau pathogenesis in AD and corticobasal degeneration (CBD). CgA ablation in Tauopathy mice (PS19) (CgA-KO/PS19) reduced pathological Tau aggregation and spreading, extended lifespan, and improved cognitive function. Transcriptomic and metabolite analysis of mouse cortices revealed elevated alpha-1-adrenergic receptors (Adra1) expression and high Epinephrine (EPI) levels in PS19 mice compared to WT mice, mirroring observations in AD and CBD patients. CgA depletion in PS19 mice lowered cortical EPI levels and the expression of Adra1 back to normal. Treatment of WT hippocampal organotypic slice cultures with EPI or Adra1 agonist promoted, while an Adra1 antagonist inhibited Tau hyperphosphorylation and formation of neurofibrillary tangles, which is unaltered upon CgA depletion. These findings demonstrate the involvement of CgA in Tau pathogenesis and highlight the interplay between the EPI-Adra1 signaling pathway and CgA in Tauopathy. Chromogranin A (CgA) ablation in Tauopathy mice reduces toxic Tau buildup, reverses cognitive decline and improves lifespan. Mechanistically, the deletion of CgA normalizes the adrenergic signaling axis in the brain.

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of IκBα by IKK2/β, a highly conserved prototypical Ser/Thr kinase in vertebrates, is critical for canonical NF-κB activation. Here, we report that human IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG +1 (DLG in IKK1/α and 2) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its IκBα substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of IκBα at S32 or S36, but rather directs phosphorylation of IκBα at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to IκBα in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-κB activation.

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM—RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5′-GGGGT G ACCCC-3′) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5’-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease. NEIL2, a mammalian DNA repair enzyme, has been reported to suppress infection induced inflammation. Here, the authors characterize its role in modulating the severity of SARS-CoV-2 infection.

Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.

Nuclear factor kappa B (NFκB) is a transcription factor that controls inflammation and cell survival. In clinical histology, elevated NFκB activity is a hallmark of poor prognosis in inflammatory disease and cancer, and may be the result of a combination of diverse micro-environmental constituents. While previous quantitative studies of NFκB focused on its signaling dynamics in single cells, we address here how multiple stimuli may combine to control tissue level NFκB activity. We present a novel, simplified model of NFκB (SiMoN) that functions as an NFκB activity calculator. We demonstrate its utility by exploring how type I and type II interferons modulate NFκB activity in macrophages. Whereas, type I IFNs potentiate NFκB activity by inhibiting translation of IκBα and by elevating viral RNA sensor (RIG-I) expression, type II IFN amplifies NFκB activity by increasing the degradation of free IκB through transcriptional induction of proteasomal cap components (PA28). Both cross-regulatory mechanisms amplify NFκB activation in response to weaker (viral) inducers, while responses to stronger (bacterial or cytokine) inducers remain largely unaffected. Our work demonstrates how the NFκB calculator can reveal distinct mechanisms of crosstalk on NFκB activity in interferon-containing microenvironments.