Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation.

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'-GGGGTGACCCC-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.

The complete structure of the human TFIIH core complex.

Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 Å resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.

Model of the Interaction between the NF-κB Inhibitory Protein p100 and the E3 Ubiquitin Ligase ß-TrCP based on NMR and Docking Experiments.

NF-κB is a major transcription factor whose activation is triggered through two main activation pathways: the canonical pathway involving disruption of IκB-α/NF-κB complexes and the alternative pathway whose activation relies on the inducible proteolysis of the inhibitory protein p100. One central step controlling p100 processing consists in the interaction of the E3 ubiquitin ligase ß-TrCP with p100, thereby leading to its ubiquitinylation and subsequent either complete degradation or partial proteolysis by the proteasome. However, the interaction mechanism between p100 and ß-TrCP is still poorly defined. In this work, a diphosphorylated 21-mer p100 peptide model containing the phosphodegron motif was used to characterize the interaction with ß-TrCP by NMR. In parallel, docking simulations were performed in order to obtain a model of the 21P-p100/ß-TrCP complex. Saturation transfer difference (STD) experiments were performed in order to highlight the residues of p100 involved in the interaction with the ß-TrCP protein. These results highlighted the importance of pSer865 and pSer869 residues in the interaction with ß-TrCP and particularly the Tyr867 that fits inside the hydrophobe ß-TrCP cavity with the Arg474 guanidinium group. Four other arginines, Arg285, Arg410, Arg431, and Arg521, were found essential in the stabilization of p100 on the ß-TrCP surface. Importantly, the requirement for these five arginine residues of ß-TrCP for the interaction with p100 was further confirmed in vivo, thereby validating the docking model through a biological approach.

Pyruvate dehydrogenase kinase 4 (PDK4) could be involved in a regulatory role in apoptosis and a link between apoptosis and insulin resistance.

Pyruvate dehydrogenase kinase 4 (PDK4), a mammalian mitochondrial serine kinase has emerged as an interesting candidate for diabetes therapy. Due to the high prevalence of this disease especially type 2 diabetes (T2D) and the health complications associated with it, there is extensive effort to find the appropriate treatment. Understanding the regulation of PDK4 activity would therefore contribute significantly to the development of therapeutic agents. This research outlines the utilization of bioinformatics tools such as Interweaver, ClustalW and Protein Structure Visualizer, in order to predict proteins that potentially interact with PDK4 and possibly regulate its activity. Interweaver database identified 96 proteins that have possible interaction sites for PDK4. Protein p100/p49, containing a death domain that is known to have a role in suppressing apoptosis, was identified as a potential partner for PDK4. The alignment between p100/p49 primary sequence and that of PDK4 using ClustalW demonstrated sequence similarity between the two proteins. Swiss PDB Viewer then located the positions of the amino acids that are in the hypothetical protein binding motif of p100/p49 within the 3D structure of hPDK4. These amino acids were found to be located in the region of PDK4 which is known to bind protein substrates of PDK4 and may be accessible to other proteins as well. These findings were very interesting as PDK4 has not previously been associated with apoptosis and this could be the link between apoptosis and insulin resistance. Cell biology studies were then performed to verify the relationship between PDK4 and apoptosis. In this regard, HeLa and HepG2 cells were treated with apoptosis-inducing agents such as TNFα, C2-ceramide, and linoleic acid. These cells were then monitored for apoptosis and PDK4 mRNA expression using a DNA laddering assay as well as Real Time PCR. The results showed that these factors induced apoptosis in a concentration dependent manner and suppressed PDK4 mRNA levels. These findings suggested a relationship between PDK4 and apoptosis.

p100/IκBδ sequesters and inhibits NF-κB through kappaBsome formation.

Degradation of I kappaB (κB) inhibitors is critical to activation of dimeric transcription factors of the NF-κB family. There are two types of IκB inhibitors: the prototypical IκBs (IκBα, IκBß, and IκBε), which form low-molecular-weight (MW) IκB:NF-κB complexes that are highly stable, and the precursor IκBs (p105/IκBγ and p100/IκBδ), which form high-MW assemblies, thereby suppressing the activity of nearly half the cellular NF-κB [Savinova OV, Hoffmann A, Ghosh G (2009) Mol Cell 34(5):591-602]. The identity of these larger assemblies and their distinct roles in NF-κB inhibition are unknown. Using the X-ray crystal structure of the C-terminal domain of p100/IκBδ and functional analysis of structure-guided mutants, we show that p100/IκBδ forms high-MW (IκBδ)4:(NF-κB)4 complexes, referred to as kappaBsomes. These IκBδ-centric "kappaBsomes" are distinct from the 2:2 complexes formed by IκBγ. The stability of the IκBδ tetramer is enhanced upon association with NF-κB, and hence the high-MW assembly is essential for NF-κB inhibition. Furthermore, weakening of the IκBδ tetramer impairs both its association with NF-κB subunits and stimulus-dependent processing into p52. The unique ability of p100/IκBδ to stably interact with all NF-κB subunits by forming kappaBsomes demonstrates its importance in sequestering NF-κB subunits and releasing them as dictated by specific stimuli for developmental programs.

A structural basis for selective dimerization by NF-κB RelB.

Transcription factors of the nuclear factor kappaB (NF-κB) family arise through the combinatorial association of five distinct Rel subunits into functional dimers. However, not every dimer combination is observed in cells. The RelB subunit, for example, does not appear as a homodimer and forms heterodimers exclusively in combination with p50 or p52 subunits. We previously reported that the RelB homodimer could be forced to assemble through domain swapping in vitro. In order to understand the mechanism of selective dimerization among Rel subunits, we have determined the x-ray crystal structures of five RelB dimers. We find that RelB forms canonical side-by-side heterodimers with p50 and p52. We observe that, although mutation of four surface hydrophobic residues that are unique to RelB does not affect its propensity to form homodimers via domain swapping, alteration of two interfacial residues converts RelB to a side-by-side homodimer. Surprisingly, these mutant RelB homodimers remain distinct from canonical side-by-side NF-κB dimers in that the two monomers move away from one another along the 2-fold axis to avoid non-complementary interactions at the interface. The presence of distinct residues buried within the hydrophobic core of the RelB dimerization domain appears to influence the conformations of the surface residues that mediate the dimer interface. This conclusion is consistent with prior observations that alterations of domain core residues change dimerization propensity in the NF-κB family transcription factors. We suggest that RelB has evolved into a specialized NF-κB subunit with unique amino acids optimized for selective formation of heterodimers with p50 and p52.

Balance between NF-κB p100 and p52 regulates T cell costimulation dependence.

c-IAP1 and c-IAP2 are ubiquitin protein ligases (E3s) that repress noncanonical NF-κB activation. We have created mice that bear a mutation in c-IAP2 that inactivates its E3 activity and interferes, in a dominant-negative fashion, with c-IAP1 E3 activity (c-IAP2(H570A)). The immune response of these animals was explored by infecting them with the Th1-inducing parasite Toxoplasma gondii. Surprisingly, c-IAP2(H570A) mice succumbed because of T cell production of high levels of proinflammatory cytokines. Unlike naive wild-type (WT) cells, which require signals generated by the TCR and costimulatory receptors to become fully activated, naive c-IAP2(H570A) T cells proliferated and produced high levels of IL-2 and IFN-γ to stimulation via TCR alone. c-IAP2(H570A) T cells had constitutive noncanonical NF-κB activation, and IκB kinase inhibition reduced their proliferation to anti-TCR alone to WT levels but had no effect when costimulation via CD28 was provided. Notably, T cells from nfkb2(-/-) mice, which cannot generate the p52 component of noncanonical NF-κB, were also costimulation independent, consistent with the negative role of this unprocessed protein in canonical NF-κB activation. Whereas T cells from nfkb2(+/-) mice behaved like WT, coexpression of a single copy of c-IAP2(H570A) resulted in cleavage of p100, upregulation of p52, and T cell costimulation independence. Thus, p100 represses and p52 promotes costimulation, and the ratio regulates T cell dependence on costimulatory signals.

The Nfkb1 and Nfkb2 proteins p105 and p100 function as the core of high-molecular-weight heterogeneous complexes.

Nfkb1 and Nfkb2 proteins p105 and p100 serve both as NF-kappaB precursors and inhibitors of NF-kappaB dimers. In a biochemical characterization of endogenous cytoplasmic and purified recombinant proteins, we found that p105 and p100 assemble into high-molecular-weight complexes that contribute to the regulation of all NF-kappaB isoforms. Unlike the classical inhibitors IkappaBalpha, -beta, and -epsilon, high-molecular-weight complexes of p105 and p100 proteins bind NF-kappaB subunits in two modes: through direct dimerization of Rel homology domain-containing NF-kappaB polypeptides and through interactions of the p105 and p100 ankyrin repeats with preformed NF-kappaB dimers, thereby mediating the bona fide IkappaB activities, IkappaBgamma and IkappaBdelta. Our biochemical evidence suggests an assembly pathway in which kinetic mechanisms control NF-kappaB dimer formation via processing and assembly of large complexes that contain IkappaB activities.

Matrix Metalloproteinase-9 gene induction by a truncated oncogenic NF-kappaB2 protein involves the recruitment of MLL1 and MLL2 H3K4 histone methyltransferase complexes.

Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100. Hut-78, a truncated form of p100, constitutively generates p52 and contributes to the development of T-cell lymphomas but the molecular mechanism underlying this oncogenic potential remains unclear. We show here that MMP9 gene expression is induced through the alternative NF-kappaB-activating pathway in fibroblasts and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78 mutant as well as other truncated NF-kappaB2 proteins that are not processed into p52 failed to induce the expression of this metalloproteinase. Conversely, MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly, MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9 but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells. Moreover, MLL1 and MLL2 are associated with Hut-78 in a native chromatin-enriched extract. Thus, we identified a molecular mechanism by which the recruitment of a H3K4 histone methyltransferase complex on the promoter of a NF-kappaB-dependent gene induces its expression and potentially the invasive potential of lymphoma cells harbouring constitutive activity of the alternative NF-kappaB-activating pathway.

NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes.

The X-ray structure of the nuclear factor-kappaB (NF-kappaB) p52:RelB:kappaB DNA complex reveals a new recognition feature not previously seen in other NF-kappaB:kappaB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of kappaB sites. We found that the Arg 125-sensitive kappaB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein-induced kink observed in this complex, which used an AT-rich kappaB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non-AT-rich kappaB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of kappaB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in kappaB sites when compared with other NF-kappaB dimers.

NF-kappaB p52, RelB and c-Rel are transported into the nucleus via a subset of importin alpha molecules.

In resting cells NF-kappaB transcription factors are retained in the cytoplasm as latent inactive complexes, until they are activated and rapidly transported into the nucleus. We show that all NF-kappaB proteins are imported into the nucleus via a subset of importin alpha isoforms. Our data indicate that the NF-kappaB components of the classical and alternative pathways have somewhat different specifities to importin alpha molecules. Based on the results from binding experiments of in vitro-translated and Sendai virus infection-induced or TNF-alpha-stimulated endogenous NF-kappaB proteins, it can be predicted that the specifity of NF-kappaB proteins to importin alpha molecules is different and changes upon the composition of the imported dimer. p52 protein binds directly to importin alpha3, alpha4, alpha5 and alpha6 and c-Rel binds to importin alpha5, alpha6 and alpha7 via a previously described monopartite nuclear localization signals (NLSs). Here we show that RelB, instead, has a bipartite arginine/lysine-rich NLS that mediates the binding of RelB to importin alpha5 and alpha6 and subsequent nuclear translocation of the protein. Moreover, we show that the nuclear import of p52/RelB heterodimers is mediated exclusively by the NLS of RelB. In addition, we found that the NLS of p52 mediates the nuclear import of p52/p65 heterodimers.

Stabilization of RelB requires multidomain interactions with p100/p52.

The NF-kappaB family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IkappaB proteins. We show that the protein level of RelB is significantly reduced in the absence of p100 and reduced even more when both p100 and p105 are absent. RelB stabilizes itself by directly interacting with p100, p105, and their processed products. However, RelB forms complexes with its partners using different interaction modes. Although the C-terminal ankyrin repeat domain of p105 is not involved in the RelB-p105 complex formation, all domains and flexible regions of each protein are engaged in the RelB-p100 complex. In several respects the RelB-p52 and RelB-p100 complexes are unique in the NF-kappaB family. The N-terminal domain of p100/p52 interacts with RelB but not RelA. The transcriptional activation domain of RelB, but not RelA, directly interacts with the processing region of p100. These unique protein-protein contacts explain why RelB prefers p52 as its dimeric partner for transcriptional activity and is retained in the cytoplasm as an inhibited complex by p100. This association-mediated stabilization of RelB implies a possible role for RelB in the processing of p100 into p52.

In vitro selection of optimal RelB/p52 DNA-binding motifs.

Rel/NF-kB dimers of different subunit composition can activate distinct subsets of target genes in vivo, however, the role of DNA recognition in this specificity is not well understood. We set out to study the DNA-binding ability of RelB/p52, the least studied of all NF-kB proteins and the main transcriptionally active product of the alternative NF-kB signaling pathway. We searched for optimal binding sites for RelB/p52 by random site selection method, using full-length proteins expressed in eukaryotic cells. The subset of RelB/p52-binding sequences defines a consensus which is very similar to the classical RelA/p50 consensus. Importantly, each of these binding sites is also recognized by RelA/p50 heterodimer with comparable affinity, questioning the existence of RelB/p52-specific kappa B sites.

Akt regulates basal and induced processing of NF-kappaB2 (p100) to p52.

NF-kappaB is a family of transcription factors important for innate and adaptive immunity. NF-kappaB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-kappaB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IkappaB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKalpha and IKKbeta. The preferred substrate for IKKbeta is IkappaBalpha, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKalpha activates a non-canonical NF-kappaB pathway in which p100 (NF-kappaB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKalpha and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-kappaB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-kappaB activity.

RelB/p52 NF-kappaB complexes rescue an early delay in mammary gland development in transgenic mice with targeted superrepressor IkappaB-alpha expression and promote carcinogenesis of the mammary gland.

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.