Applying proteomics to signaling networks

The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.     

Duodenal expression of the epithelial calcium transporter gene TRPV6: is there evidence for Vitamin D-dependence in humans?

Intestinal absorption of dietary calcium is regulated by 1,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) in humans and in experimental animals but interspecies differences in responsiveness to 1,25(OH)(2)D(3) are found, possibly due to differences in the promoters of genes for intestinal calcium transport proteins or of the Vitamin D receptor (VDR). The epithelial calcium transporter, known as ECAC2 or CAT1, the product of the TRPV6 gene expressed in proximal intestinal enterocytes, is the first step in calcium absorption and studies in mice have shown that its expression is Vitamin D-dependent. In contrast in man, we showed that duodenal TRPV6 mRNA expression was independent of blood 1,25(OH)(2)D(3), although in Caco-2 cells, 1,25(OH)(2)D(3)-dependent changes have been demonstrated. We sought to explain these findings. A consensus Vitamin D response element in the mouse gene is absent in the human gene. We re-analysed our duodenal expression data according to a CDX2-site polymorphism in the VDR promoter. Mean TRPV6 expression was the same, but there was evidence of different responsiveness to 1,25(OH)(2)D(3). In the GG genotype group, but not the AG, duodenal TRPV6 expression increased with 1,25(OH)(2)D(3). We postulate that lower levels of expression of VDR in the GG group produce greater sensitivity to 1,25(OH)(2)D(3).     

Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system

The aim of this study was to establish an in vitro system that supports development and differentiation of bovine blastocysts. Agar gel tunnels were covered with modified synthetic oviduct fluid medium supplemented with 10% fetal calf serum and 3g/l D-glucose. Of the total 67 blastocysts loaded individually into the tunnels, 46 continued expansion to 1mm and reached the walls of the gel on Day 10. On Day 12, 35 blastocysts elongated to minimum 1.6 mm while filling completely the space between the walls of the tunnel, and 16 still continued growth and reached an average of 4.3 mm length on Day 14. The largest blastocyst on Day 16 was 12 mm long. On Day 12, in 31 of the 35 elongated blastocysts a second cell layer occurred beneath the trophoblast and formed a complete cover in surviving Day 14 embryos. In most proliferating embryos the inner cell mass was prominent, however, the detection of signs of embryonic disc formation will require further studies. The established system was suitable to induce in vitro elongation, rapid growth and further differentiation, and may have considerable theoretical and practical value for studies of development and differentiation of bovine embryos.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.     

Extracts and sesquiterpene derivatives from the red alga Laurencia chondrioides with antibacterial activity against fish and human pathogenic bacteria

During screening of seaweeds from different places in Europe for antimicrobial activities against human and fish pathogenic bacteria, Laurencia chondrioides was identified as a promising species. By bioassay-guided isolation, followed by structure elucidation by mass spectrometry and 1H- and 13C-NMR spectrometry, two sesquiterpenoides of the chamigrene-type from the selected red seaweed Laurencia chondrioides were identified. Both compounds inhibit the growth of some fish and human pathogenic bacteria.     

Biochemical mechanisms of cyclosporine neurotoxicity

Proper management of chemotoxicity in transplant patients requires detailed knowledge of the biochemical mechanisms underlying immunosuppressant toxicity. Neurotoxicity is one of the most significant clinical side effects of the immunosuppressive undecapeptide cyclosporine, occurring at some degree in up to 60% of transplant patients. The clinical symptoms of cyclosporine-mediated neurotoxicity consist of decreased responsiveness, hallucinations, delusions, seizures, cortical blindness, and stroke-like episodes that mimic those clinical symptoms of mitochondrial encephalopathy. Clinical computed tomography (CT) and magnetic resonance imaging (MRI) studies have revealed a correlation between clinical symptoms of cyclosporine-mediated neurotoxicity and morphological changes in the brain, such as hypodensity of white matter, cerebral edema, metabolic encephalopathy, and hypoxic damages. Paradoxically, in animal models cyclosporine protects the brain from ischemia-reperfusion (I/R) injury. Interestingly, cyclosporine appears to mediate both neurotoxicity (under normoxic conditions) and I/R protection across the same range of drug concentration. Both toxicity and protection might arise from the intersection of cyclosporine with mitochondrial energy metabolism. This review addresses basic biochemical mechanisms of: 1) cyclosporine toxicity in normoxic brain, and 2) its protective effects in the same organ during I/R. The marked and unparallel potential of magnetic resonance spectroscopy (MRS) as a novel quantitative approach to evaluate metabolic drug toxicity is described.     

Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility

BACKGROUND: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.RESULTS: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.CONCLUSIONS: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.