Parallel changes in metabolite and expression profiles in crooked-tail mutant and folate-reduced wild-type mice.

Anomalies in homocysteine (HCY) and folate metabolism are associated with common birth defects and adult diseases, several of which can be suppressed with dietary folate supplementation. Although supplementation reduces the occurrence and severity of neural tube defects (NTDs), many cases are resistant to these beneficial effects. The basis for variable response and biomarkers that predict responsiveness are unknown. Crooked-tail (Cd) mutant mice are an important model of folate-responsive NTDs. To identify features that are diagnostic for responsiveness versus resistance to dietary folate supplementation, we surveyed metabolite and expression levels in liver samples from folate-supplemented, folate-reduced and control diets in Cd mutant and wild-type adult females. Cd homozygotes had normal total homocysteine (tHcy) levels suggesting that folate suppresses NTDs through a mechanism that does not involve modulating serum tHcy levels. Instead, parallel changes in metabolite and expression profiles in folate-supplemented Cd/Cd homozygotes and folate-reduced+/+and Cd/+mice suggest that Crooked-tail homozygotes have a defect in the utilization of intracellular folate. Then, by combining these expression and metabolite profile results with published results for other models and their controls, two clusters were found, one of which included several folate-responsive NTD models and the other previously untested and presumably folate-resistant models. The predictive value of these profiles was verified by demonstrating that NTDs of Ski-/-mutant mice, whose profile suggested resistance to folate supplementation, were not suppressed with dietary folate supplementation. These results raise the possibility of using metabolite and expression profiles to distinguish folate-responsive and resistance adult females who are at risk for bearing fetuses with an NTD.

The AKT/I kappa B kinase pathway promotes angiogenic/metastatic gene expression in colorectal cancer by activating nuclear factor-kappa B and beta-catenin.

Our laboratory has delineated that the phosphatidylinositol 3' kinase (PI3K)/AKT/I kappa B kinase (IKK) pathway positively regulates NF kappa B and beta-catenin, both important transcriptional regulators in colorectal cancer (CRC). Therefore, we investigated the effect of inhibiting the PI3K/AKT/IKK alpha pathway in regulating the inappropriate constitutive activation of NF kappa B and beta-catenin in CRC cell lines. SW480 and RKO CRC cell lines demonstrate constitutive activation of AKT as well as both NF kappa B- and beta-catenin-dependent transcription. The constitutive activation of NF kappa B- and beta-catenin-dependent transcription is inhibited by transiently transfecting either kinase dead (KD) IKK alpha, which blocks IKK alpha kinase activity, KD AKT, which blocks AKT activity, or wildtype (WT) PTEN, which inhibits PI3K and AKT activity. The ability of KD IKK alpha, KD AKT or WT PTEN to decrease beta-catenin-dependent transcription is independent of their effects on NF kappa B. Inducible expression of either KD IKK alpha or WT PTEN strongly inhibits both the constitutive NF kappa B- and beta-catenin-dependent promoter and endogenous gene activation. Targeted array-based gene expression analysis of this inducible system reveals that many of the genes downregulated upon inhibition of this pathway are involved in tumor angiogenesis and metastasis. The activation of this pathway and the expression of the three most repressed genes was further analysed in samples of CRC. These results indicate a role of this pathway in controlling gene expression important in tumor progression and metastasis.

Inhibitor of kappaB kinase is required to activate a subset of interferon gamma-stimulated genes.

IkappaB kinase (IKK), discovered as the major activator of NF-kappaB, plays additional roles in signaling. By using mouse embryo fibroblasts (MEFs) lacking both the alpha and beta subunits of IKK, we find that these proteins are required for induction of a major subset of IFNgamma-stimulated genes and that this requirement is independent of NF-kappaB activation. Furthermore, there is no defect in IFNgamma-stimulated signal transducer and activator of transcription 1 (Stat1) activation or function in the IKKalpha/beta-null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFNgamma, they are not sufficient. These results reveal an important additional pathway for IFNgamma-stimulated gene expression in which an NF-kappaB-independent function of IKK is required.

The crystal structure of a novel unmethylated form of C-phycocyanin, a possible connector between cores and rods in pycobilisomes.

A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized. The crystals belong to the P63 space group with cell dimensions of 153 A x 153 A x 59 A with a single (alphabeta) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 A. The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (Rfree = 27.8%). The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC612 structure shows that the alpha subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnbeta72 residue is not methylated, explaining the blue-shift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core.

Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure.

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 A resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.

Distinct roles of the Ikappa B kinase alpha and beta subunits in liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and in phosphorylating the p65 subunit of NF-kappa B.

Phosphatidylinositol 3'-kinase (PI3K) and the serine/threonine kinase AKT have critical roles in phosphorylating and transactivating the p65 subunit of nuclear factor kappaB (NF-kappaB) in response to the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Mouse embryo fibroblasts (MEFs) lacking either the alpha or beta subunit of IkappaB kinase (IKK) were deficient in NF-kappaB-dependent transcription following treatment with IL-1 or TNF. However, in contrast to IKKbeta-null MEFs, IKKalpha-null MEFs were not substantially defective in the cytokine-stimulated degradation of Ikappabetaalpha or in the nuclear translocation of NF-kappaB. The IKK complexes from IKKalpha- or IKKbeta-null MEFs were both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NF-kappaB in response to IL-1 and TNF, and constitutively activated forms of PI3K or AKT did not potentiate cytokine-stimulated activation of NF-kappaB in either IKKalpha- or IKKbeta-null MEFs. Collectively, these data indicate that, in contrast to IKKbeta, which is required for both NF-kappaB liberation and p65 phosphorylation, IKKalpha is required solely for the cytokine-induced phosphorylation and activation of the p65 subunit of NF-kappaB that are mediated by the PI3K/AKT pathway.