HDX-MS Analysis of Catalytic Activation of IKK2 in the IκB Kinase Complex.

The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub4) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.

Chromogranin A Deficiency Attenuates Tauopathy by Altering Epinephrine-Alpha-Adrenergic Receptor Signaling.

Metabolic disorders such as insulin resistance and hypertension are potential risk factors for aging and neurodegenerative diseases. These conditions are reversed in Chromogranin A knockout (CgA-KO) mice. This study investigates the role of CgA in Alzheimer's disease (AD) and corticobasal degeneration (CBD). CgA ablation in tauopathy mice (hTau) (CgA-KO/hTau) exhibited reduced tau aggregation, spreading, extended lifespan, and improved cognitive function. Transcriptomic and metabolite analysis of mouse cortices revealed altered alpha1-adrenergic receptors (Adra1) and high epinephrine (EPI) levels in hTau mice compared to WT mice, mirroring observations in AD and CBD patients. CgA-KO/hTau mice exhibited a reversal of EPI levels in the cortex and the expression of Adra1, nearly returning them to WT levels. Treatment of hippocampal slices with EPI or Adra1 agonist intensified, while an Adra1 antagonist inhibited tau hyperphosphorylation and aggregation. These findings highlight the interplay between the EPI-Adra signaling system and CgA in tauopathy.

Phosphorylation-induced flexibility of proto-oncogenic Bcl3 regulates transcriptional activation by NF-κB p52 homodimer.

The overexpression of proto-oncogene Bcl3 is observed in various cancers. Bcl3 is extensively phosphorylated and associates with homodimers of NF-κB p50 and p52 to regulate transcription. Through cellular and biochemical assays, we observed that phospho-mimetic Glu substitution at Ser366 in addition to previously studied Ser33, 114 and 446 is necessary to switch Bcl3 from an IκB-like inhibitor to a transcriptional activator. To study interactive features of p52 and Bcl3, and phosphorylation- mediated changes in Bcl3 that regulate DNA-binding by p52, we performed HDX-MS of both Bcl3 and p52 within various complexes. Nature of interactions within Bcl3:(p52:p52) complex in presence and absence of DNA, differential flexibility of Bcl3, and allosteric changes in Bcl3 upon phospho-modifications revealed why a facile accommodation of DNA requires phosphorylation. The inhibitory nature of unphosphorylated Bcl3 on DNA binding by p52:p52 also relieved by a C-terminal deletion of Bcl3. Overall, this study revealed mechanistic bases of how Bcl3 phosphorylation regulates transcriptional potential of NF-κB and intricate cell physiology, a dysregulation of which can lead to cancers.

Chromogranin A (CgA) Deficiency Attenuates Tauopathy by Altering Epinephrine-Alpha-Adrenergic Receptor Signaling.

Our previous studies have indicated that insulin resistance, hyperglycemia, and hypertension in aged wild-type (WT) mice can be reversed in mice lacking chromogranin-A (CgA-KO mice). These health conditions are associated with a higher risk of Alzheimer's disease (AD). CgA, a neuroendocrine secretory protein has been detected in protein aggregates in the brains of AD patients. Here, we determined the role of CgA in tauopathies, including AD (secondary tauopathy) and corticobasal degeneration (CBD, primary tauopathy). We found elevated levels of CgA in both AD and CBD brains, which were positively correlated with increased phosphorylated tau in the frontal cortex. Furthermore, CgA ablation in a human P301S tau (hTau) transgenic mice (CgA-KO/hTau) exhibited reduced tau aggregation, resistance to tau spreading, and an extended lifespan, coupled with improved cognitive function. Transcriptomic analysis of mice cortices highlighted altered levels of alpha-adrenergic receptors (Adra) in hTau mice compared to WT mice, akin to AD patients. Since CgA regulates the release of the Adra ligands epinephrine (EPI) and norepinephrine (NE), we determined their levels and found elevated EPI levels in the cortices of hTau mice, AD and CBD patients. CgA-KO/hTau mice exhibited reversal of EPI levels in the cortex and the expression of several affected genes, including Adra1 and 2, nearly returning them to WT levels. Treatment of hippocampal slice cultures with EPI or an Adra1 agonist intensified, while an Adra1 antagonist inhibited, tau hyperphosphorylation and aggregation. These findings reveal a critical role of CgA in regulation of tau pathogenesis via the EPI-Adra signaling axis.

Transient interactions modulate the affinity of NF-κB transcription factors for DNA.

The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 µs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.

Gut microbial DNA and immune checkpoint gene Vsig4/CRIg are key antagonistic players in healthy aging and age-associated development of hypertension and diabetes.

Aims: Aging is associated with the development of insulin resistance and hypertension which may stem from inflammation induced by accumulation of toxic bacterial DNA crossing the gut barrier. The aim of this study was to identify factors counter-regulating these processes. Taking advantage of the Chromogranin A (CgA) knockout (CgA-KO) mouse as a model for healthy aging, we have identified Vsig4 (V-set and immunoglobulin domain containing 4) as the critical checkpoint gene in offsetting age-associated hypertension and diabetes. Methods and Results: The CgA-KO mice display two opposite aging phenotypes: hypertension but heightened insulin sensitivity at young age, whereas the blood pressure normalizes at older age and insulin sensitivity further improves. In comparison, aging WT mice gradually lost glucose tolerance and insulin sensitivity and developed hypertension. The gut barrier, compromised in aging WT mice, was preserved in CgA KO mice leading to major 35-fold protection against bacterial DNA-induced inflammation. Similarly, RNA sequencing showed increased expression of the Vsig4 gene (which removes bacterial DNA) in the liver of 2-yr-old CgA-KO mice, which may account for the very low accumulation of microbial DNA in the heart. The reversal of hypertension in aging CgA-KO mice likely stems from (i) low accumulation of microbial DNA, (ii) decreased spillover of norepinephrine in the heart and kidneys, and (iii) reduced inflammation. Conclusion: We conclude that healthy aging relies on protection from bacterial DNA and the consequent low inflammation afforded by CgA-KO. Vsig4 also plays a crucial role in "healthy aging" by counteracting age-associated insulin resistance and hypertension.

PABP1 Drives the Selective Translation of Influenza A Virus mRNA.

Influenza A virus (IAV) is a human-infecting pathogen with a history of causing seasonal epidemics and on several occasions worldwide pandemics. Infection by IAV causes a dramatic decrease in host mRNA translation, whereas viral mRNAs are efficiently translated. The IAV mRNAs have a highly conserved 5'-untranslated region (5'UTR) that is rich in adenosine residues. We show that the human polyadenylate binding protein 1 (PABP1) binds to the 5'UTR of the viral mRNAs. The interaction of PABP1 with the viral 5'UTR makes the translation of viral mRNAs more resistant to canonical cap-dependent translation inhibition than model mRNAs. Additionally, PABP1 bound to the viral 5'UTR can recruit eIF4G in an eIF4E-independent manner. These results indicate that PABP1 bound to the viral 5'UTR may promote eIF4E-independent translation initiation.

Intrapulmonary administration of purified NEIL2 abrogates NF-κB-mediated inflammation.

Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB-mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.

LINC00261 Is an Epigenetically Regulated Tumor Suppressor Essential for Activation of the DNA Damage Response.

Lung cancer is the leading cause of cancer-related death in the United States. Long noncoding RNAs (lncRNA) are a class of regulatory molecules whose role in lung carcinogenesis is poorly understood. In this study, we profiled lncRNA expression in lung adenocarcinoma (LUAD) cell lines, compared their expression with that of purified alveolar epithelial type II cells (the purported cell of origin for LUAD), cross-referenced these with lncRNAs altered in the primary human tumors, and interrogated for lncRNAs whose expression correlated with patient survival. We identified LINC00261, a lncRNA with unknown function in LUAD, adjacent to the pioneering transcription factor FOXA2. Loss of LINC00261 was observed in multiple tumor types, including liver, breast, and gastric cancer. Reintroduction of LINC00261 into human LUAD cell lines inhibited cell migration and slowed proliferation by inducing G2-M cell-cycle arrest, while upregulating DNA damage pathway genes and inducing phosphorylation-mediated activation of components of the DNA damage pathway. FOXA2 was able to induce LINC00261 expression, and the entire locus underwent hypermethylation in LUAD, leading to loss of expression. We have thus identified an epigenetically deregulated lncRNA, whose loss of expression in LUAD promotes the malignant phenotype and blocks activation of the DNA damage machinery, predisposing lung cells to cancer development. SIGNIFICANCE: These findings identify LINC00261 as a tumor suppressor that blocks cellular proliferation by activating the DNA damage response and suggest that epigenetic therapy to inhibit DNA methylation may enhance treatment of LUAD. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/12/3050/F1.large.jpg.See related commentary by Davalos and Esteller, p. 3028.

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

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.