NF-κB RelB Forms an Intertwined Homodimer

SummaryThe X-ray structure of the RelB dimerization domain (DD) reveals that the RelBDD assumes an unexpected intertwined fold topology atypical of other NF-κB dimers. All typical NF-κB dimers are formed by the association of two independently folded immunoglobulin (Ig) domains. In RelBDD, two polypeptides reconstruct both Ig domains in the dimer with an extra β sheet connecting the two domains. Residues most critical to NF-κB dimer formation are invariant in RelB, and Y300 plays a positive role in RelBDD dimer formation. The presence of RelB-specific nonpolar residues at the surface removes several intradomain surface hydrogen bonds that may render the domain fold unstable. Intertwining may stabilize the RelBDD homodimer by forming the extra β sheet. We show that, as in the crystal, RelB forms an intertwined homodimer in solution. We suggest that the transiently stable RelB homodimer might prevent its rapid degradation, allowing for heterodimer formation with p50 and p52

NF-κB Potentiates Caspase Independent Hydrogen Peroxide Induced Cell Death

The pro-survival activity of NF-κB in response to a variety of stimuli has been extensively characterized. Although there have been a few reports addressing the pro-cell death role of NF-κB, the precise mechanism of NF-κB's pro-cell death function still remains elusive.In the present study, we investigated the role of NF-κB in cell death induced by chronic insult with hydrogen peroxide (H(2)O(2)). Here, we show that NF-κB promotes H(2)O(2) induced caspase independent but PARP dependent fibroblast cell death. The pro-death activity of NF-κB is due to the DNA binding activity of RelA, which is induced through IKK- mediated IκBα degradation. NF-κB dependent pro-survival genes, Bcl-2 and XIAP, were significantly repressed, while NF-κB dependent pro-death genes, TNFα and Fas Ligand, were induced in response to H(2)O(2).We discovered an unexpected function of NF-κB, in that it potentiates chronic H(2)O(2) exposure induced cell death, and suggest that NF-κB mediates cell death through the repression of pro-survival genes and induction of pro-death genes. Since unremitting exposure of tissues to H(2)O(2) and other reactive oxygen species can lead to several degenerative disorders and diseases, our results have important implications for the use of NF-κB inhibitors in therapeutic drug design

A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.

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

Dissecting the regulatory strategies of NF-kB RelA target genes in the inflammatory response reveals differential transactivation logics

Nuclear factor κB (NF-κB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each gene’s regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-κB RelA core response

An NFkB activity calculator to delineate signaling crosstalk: type I and II interferons enhance NFkB via distinct mechanisms

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

Identifying Critical Non-Catalytic Residues that Modulate Protein Kinase A Activity

Distal interactions between discrete elements of an enzyme are critical for communication and ultimately for regulation. However, identifying the components of such interactions has remained elusive due to the delicate nature of these contacts. Protein kinases are a prime example of an enzyme with multiple regulatory sites that are spatially separate, yet communicate extensively for tight regulation of activity. Kinase misregulation has been directly linked to a variety of cancers, underscoring the necessity for understanding intramolecular kinase regulation.A genetic screen was developed to identify suppressor mutations that restored catalytic activity in vivo from two kinase-dead Protein Kinase A mutants in S. cerevisiae. The residues defined by the suppressors provide new insights into kinase regulation. Many of the acquired mutations were distal to the nucleotide binding pocket, highlighting the relationship of spatially dispersed residues in regulation.The suppressor residues provide new insights into kinase regulation, including allosteric effects on catalytic elements and altered protein-protein interactions. The suppressor mutations identified in this study also share overlap with mutations identified from an identical screen in the yeast PKA homolog Tpk2, demonstrating functional conservation for some residues. Some mutations were independently isolated several times at the same sites. These sites are in agreement with sites previously identified from multiple cancer data sets as areas where acquired somatic mutations led to cancer progression and drug resistance. This method provides a valuable tool for identifying residues involved in kinase activity and for studying kinase misregulation in disease states

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

Mutations that impair activity of the ER stress response kinase Ire1 inhibit resolution of the unfolded protein response (see also a related paper by Rubio et al. in this issue)