A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold

Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.     

Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation

Activating mutations in the genes for fibroblast growth factor receptors 1-3 (FGFR1-3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a "crossover" disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth.     

Protein kinase C (PKC) betaII induces cell invasion through a Ras/Mek-, PKC iota/Rac 1-dependent signaling pathway

Protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. Expression of PKCbetaII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCbetaII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCbetaII. Here we show that RIE/PKCbetaII cells acquire an invasive phenotype that is blocked by the PKCbeta inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCbetaII, indicating that PKCbetaII activity is required for the invasive phenotype. PKCbetaII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCbetaII cells. PKCbetaII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCiota. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCbetaII-mediated invasion. Taken together, our results define a novel PKCbetaII --> Ras --> PKCiota /Rac1 --> Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCbetaII promotes colon carcinogenesis.     

Protein kinase CbetaII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo

Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.     

Extragonadal luteinizing hormone receptors in the reproductive tract of domestic animals

Binding sites for LH/hCG and/or its mRNA are found in the uterus of several species, including human, primate, pig, cow, and turkey. Activation of LH receptors around Day 15 of the estrous cycle is associated with increased prostaglandin F(2alpha) production in the bovine, porcine, and ovine uterus. Activation of uterine LH receptors is also associated with increased levels of prostaglandins in human and primate. The presence of gonadotropin receptors with a dynamic pattern in the oviduct, endometrium, myometrium, and cervix of different species provides evidence that gonadotropins play a substantial role in molecular autocrine-paracrine regulation of the estrous cycle and implantation.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.     

A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase

CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.     

Advances in understanding neuron-glia interactions

Recent advances in the field of neuron-glia interactions were presented at the 27th International Symposium of the University of Montreal Center de Recherche en Sciences Neruologiques. Topics included synaptogenesis, regulation of synaptic strength by glia at the neuromuscular junction and hippocampus; myelin formation, structure, and maintenance; involvement of glia in nervous-system response to injury, hypoxia, and ischemia; neurogenesis and apoptosis, and microglial involvement in chronic pain.     

A pooling-deconvolution strategy for biological network elucidation

The generation of large-scale data sets is a fundamental requirement of systems biology. But despite recent advances, generation of such high-coverage data remains a major challenge. We developed a pooling-deconvolution strategy that can dramatically decrease the effort required. This strategy, pooling with imaginary tags followed by deconvolution (PI-deconvolution), allows the screening of 2(n) probe proteins (baits) in 2 x n pools, with n replicates for each bait. Deconvolution of baits with their binding partners (preys) can be achieved by reading the prey's profile from the 2 x n experiments. We validated this strategy for protein-protein interaction mapping using both proteome microarrays and a yeast two-hybrid array, demonstrating that PI-deconvolution can be used to identify interactions accurately with fewer experiments and better coverage. We also show that PI-deconvolution can be used to identify protein-small molecule interactions inferred from profiling the yeast deletion collection. PI-deconvolution should be applicable to a wide range of library-against-library approaches and can also be used to optimize array designs.