Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor kappaB

The DNA binding protein nuclear factor kappaB (NF-kappaB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-kappaB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (ii) nuclear extracts, and (iii) whole cell lysates--we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.     

A requirement of nuclear factor-kappaB activation in fear-potentiated startle

Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-kappaB (NF-kappaB) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IkappaB-alpha were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-kappaB in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IkappaB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of kappaB decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-kappaB inhibitors blocked IkappaB-alpha degradation and startle response. These results provide the first evidence of a requirement of NF-kappaB activation in the amygdala for consolidation of fear memory.     

X-ray structure of a NF-kappaB p50/RelB/DNA complex reveals assembly of multiple dimers on tandem kappaB sites

We describe here the X-ray crystal structure of NF-kappaB p50/RelB heterodimer bound to a kappaB DNA. Although the global modes of subunit association and kappaB DNA recognition are similar to other NF-kappaB/DNA complexes, this complex reveals distinctive features not observed for non-RelB complexes. For example, Lys274 of RelB is removed from the protein-DNA interface whereas the corresponding residues in all other subunits make base-specific contacts. This mode of binding suggests that RelB may allow the recognition of more diverse kappaB sequences. Complementary surfaces on RelB and p50, as revealed by the crystal contacts, are highly suggestive of assembly of multiple p50/RelB heterodimers on tandem kappaB sites in solution. Consistent with this model our in vitro binding experiments reveal optimal assembly of two wild-type p50/RelB heterodimers on tandem HIV kappaB DNA with 2 bp spacing but not by a mutant heterodimer where one of the RelB packing surface is altered. We suggest that multiple NF-kappaB dimers assemble at diverse kappaB promoters through direct interactions utilizing unique protein-protein interaction surfaces.     

Targeted deletion of MKK4 gene potentiates TNF-induced apoptosis through the down-regulation of NF-kappa B activation and NF-kappa B-regulated antiapoptotic gene products

MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-x(L), and cFLIP) gene products, all regulated by NF-kappaB. Indeed we found that TNF-induced NF-kappaB activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced I kappaB alpha kinase activation, I kappaB alpha phosphorylation, I kappaB alpha degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-kappaB reporter assay revealed that NF-kappaB activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and I kappaB alpha kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-kappaB-regulated gene products.     

Insertion of nuclear factor-kappaB binding sequence into plasmid DNA for increased transgene expression in colon carcinoma cells

To increase plasmid DNA (pDNA)-based transgene expression, 5, 10 or 20 repeats of nuclear factor kappaB (NF-kappaB) binding sequences were inserted upstream of the cytomegalovirus (CMV) promoter region of a conventional pDNA encoding firefly luciferase (pCMV-Luc) to obtain pCMV-kappaB5-Luc, pCMV-kappaB10-Luc and pCMV-kappaB20-Luc. Murine carcinoma colon 26 cells, in which NF-kappaB was constitutively activated, were co-transfected with a firefly luciferase-expressing pDNA and a renilla luciferase-expressing pDNA having no NF-kappaB binding sequences using cationic liposomes. The expression efficiency of pCMV-kappaB(n)-Luc was evaluated using the ratio of the luciferase activities. Increasing numbers of NF-kappaB binding sequences significantly increased transgene expression. The expression was increased by NF-kappaB activators and the effects were marked with pDNA having many NF-kappaB binding sequences. These results indicate that insertion of NF-kappaB binding sequences into pDNA is an effective approach to increase transgene expression in cancer cells in which NF-kappaB is activated.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).