NEMO-binding domains of both IKKalpha and IKKbeta regulate IkappaB kinase complex assembly and classical NF-kappaB activation.
ABSTRACT Proinflammatory NF-kappaB activation requires the IkappaB (inhibitor of NF-kappaB) kinase (IKK) complex that contains two catalytic subunits named IKKalpha and IKKbeta and a regulatory subunit named NF-kappaB essential modulator (NEMO). NEMO and IKKbeta are essential for tumor necrosis factor (TNF)-induced NF-kappaB activation, and we recently demonstrated that NEMO and IKKalpha are sufficient for interleukin (IL)-1-induced signaling. IKKalpha and IKKbeta both contain a functional NEMO-binding domain (NBD); however, the role of NEMO association with each kinase in NF-kappaB signaling and IKK complex formation remains unclear. To address this question, we stably reconstituted IKKalpha(-/-) and IKKbeta(-/-) murine embryonic fibroblasts (MEFs) with wild-type (WT) or NBD-deficient (DeltaNBD) versions of IKKalpha and IKKbeta, respectively. TNF-induced classical NF-kappaB activation in IKKbeta(-/-) MEFs was rescued by IKKbeta(WT) but not IKKbeta(DeltaNBD), whereas neither IKKbeta(WT) nor IKKbeta(DeltaNBD) affected IL-1-induced NF-kappaB signaling. As previously described, classical NF-kappaB transcriptional activity was absent in IKKalpha(-/-) cells. Reconstitution with either IKKalpha(WT) or IKKalpha(DeltaNBD) rescued both IL-1 and TNF-induced transcription, demonstrating that NEMO association is not required for IKKalpha-dependent regulation of NF-kappaB-dependent transcription. Stably expressed IKKalpha(WT) or IKKbeta(WT) associated with endogenous IKKs and NEMO in IKKalpha(-/-) or IKKbeta(-/-) MEFs, respectively, resulting in formation of the heterotrimeric IKKalpha-IKKbeta-NEMO complex. In contrast, although the IKKalpha(DeltaNBD) and IKKbeta(DeltaNBD) mutants associated with endogenous IKKs containing an NBD, these dimeric endogenous IKK-IKK(DeltaNBD) complexes did not associate with NEMO. These findings therefore demonstrate that formation of the heterotrimeric IKKalpha-IKKbeta-NEMO holocomplex absolutely requires two intact NEMO-binding domains.
SourceAvailable from: Qin Zhang[Show abstract] [Hide abstract]
ABSTRACT: Detecting genes associated with milk fat composition could provide valuable insights into the complex genetic networks of genes underling variation in fatty acids synthesis and point towards opportunities for changing milk fat composition via selective breeding. In this study, we conducted a genome-wide association study (GWAS) for 22 milk fatty acids in 784 Chinese Holstein cows with the PLINK software. Genotypes were obtained with the Illumina BovineSNP50 Bead chip and a total of 40,604 informative, high-quality single nucleotide polymorphisms (SNPs) were used. Totally, 83 genome-wide significant SNPs and 314 suggestive significant SNPs associated with 18 milk fatty acid traits were detected. Chromosome regions that affect milk fatty acid traits were mainly observed on BTA1, 2, 5, 6, 7, 9, 13, 14, 18, 19, 20, 21, 23, 26 and 27. Of these, 146 SNPs were associated with more than one milk fatty acid trait; most of studied fatty acid traits were significant associated with multiple SNPs, especially C18:0 (105 SNPs), C18 index (93 SNPs), and C14 index (84 SNPs); Several SNPs are close to or within the DGAT1, SCD1 and FASN genes which are well-known to affect milk composition traits of dairy cattle. Combined with the previously reported QTL regions and the biological functions of the genes, 20 novel promising candidates for C10:0, C12:0, C14:0, C14:1, C14 index, C18:0, C18:1n9c, C18 index, SFA, UFA and SFA/UFA were found, which composed of HTR1B, CPM, PRKG1, MINPP1, LIPJ, LIPK, EHHADH, MOGAT1, ECHS1, STAT1, SORBS1, NFKB2, AGPAT3, CHUK, OSBPL8, PRLR, IGF1R, ACSL3, GHR and OXCT1. Our findings provide a groundwork for unraveling the key genes and causal mutations affecting milk fatty acid traits in dairy cattle.PLoS ONE 05/2014; 9(5):e96186. DOI:10.1371/journal.pone.0096186 · 3.53 Impact Factor
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ABSTRACT: Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.PLoS ONE 12/2014; 9(12):e114129. DOI:10.1371/journal.pone.0114129 · 3.53 Impact Factor
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ABSTRACT: NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-ĸB pathway. Rational drug design targeting the IKK binding site on NEMO would benefit from structural insight, but to date the structure determination of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled-coil at the N-terminus, C-terminus or both ends of NEMO(44-111) present high thermal stability and cooperative melting, and most importantly restore IKKß binding affinity. We examined the consequences on structural content and stability by circular dichoism and nuclear magnetic resonance and measured binding affinity of each construct for IKKβ(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method to engineer short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile the rescuing of the binding affinity implies that a pre-ordered IKK-binding region of NEMO is compatible with IKK binding and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.Biochemistry 10/2014; 53(43). DOI:10.1021/bi500861x · 3.19 Impact Factor