Association of the mammalian proto-oncoprotein Int-6 with the three protein complexes eIF3, COP9 signalosome and 26S proteasome

The mammalian Int-6 protein has been characterized as a subunit of the eIF3 translation initiation factor and also as a transforming protein when its C-terminal part is deleted. It includes a protein domain, which also exists in various subunits of eIF3, of the 26S proteasome and of the COP9 signalosome (CSN). By performing a two-hybrid screen with Int-6 as bait, we have isolated subunits belonging to all three complexes, namely eIF3-p110, Rpt4, CSN3 and CSN6. The results of transient expression experiments in COS7 cells confirmed the interaction of Int-6 with Rpt4, CSN3 and CSN6, but also showed that Int-6 is able to bind another subunit of the CSN: CSN7a. Immunoprecipitation experiments performed with the endogenous proteins showed that Int-6 binds the entire CSN, but in low amount, and also that Int-6 is associated with the 26S proteasome. Taken together these results show that the Int-6 protein can bind the three complexes with various efficiencies, possibly exerting a regulatory activity in both protein translation and degradation.     

Insulin promotes the cell surface recruitment of the SAT2/ATA2 system A amino acid transporter from an endosomal compartment in skeletal muscle cells

SAT1-3 comprise members of the recently cloned family of System A transporters that mediate the sodium-coupled uptake of short chain neutral amino acids, and their activity is regulated extensively by stimuli such as insulin, growth factors, and amino acid availability. In skeletal muscle, insulin stimulates System A activity rapidly by a presently ill-defined mechanism. Here we demonstrate that insulin induces an increase in the plasma membrane abundance of SAT2 in a phosphatidylinositol 3-kinase-dependent manner and that this increase is derived from an endosomal compartment that is required for the hormonal activation of System A. Chloroquine, an acidotropic weak base that impairs endosomal recycling of membrane proteins, induced a complete inhibition in the insulin-mediated stimulation of System A, which was associated with a loss in SAT2 recruitment to the plasma membrane. The failure to stimulate System A and recruit SAT2 to the cell surface could not be attributed to a block in insulin signaling, as chloroquine had no effect on the insulin-mediated phosphorylation of protein kinase B or glycogen synthase kinase 3 or upon insulin-stimulated GLUT4 translocation and glucose transport. Our data indicate strongly that insulin increases System A transport in L6 cells by stimulating the exocytosis of SAT2 carriers from a chloroquine-sensitive endosomal compartment.     

Solution NMR characterization of an unusual distal H-bond network in the active site of the cyanide-inhibited,human heme oxygenase complex of the symmetric substrate, 2,4-dimethyldeuterohemin

The presence of variable static hemin orientational disorder about the alpha-gamma-meso axis in the substrate complexes of mammalian heme oxygenase, together with the incomplete averaging of a second, dynamic disorder, for each hemin orientation, has led to NMR spectra with severe spectral overlap and loss of key two-dimensional correlations that seriously interfere with structural characterization in solution. We demonstrate that the symmetric substrate, 2,4-dimethyldeuterohemin, yields a single solution species for which the dynamic disorder is sufficiently rapid to allow effective and informative (1)H NMR structural characterization. A much more extensive, effective, and definitive NMR characterization of the cyanide-inhibited, symmetric heme complex of human heme oxygenase shows that the active site structure, with some minor differences, is essentially the same as that for the native protohemin in solution and crystal. A unique distal network that involves particularly strong hydrogen bonds, as well as inter-aromatic contacts, is described that is proposed to stabilize the position of the catalytically critical distal helix Asp-140 carboxylate (Liu, Y., Koenigs Lightning, L., Huang, H., Mo?nne-Loccoz, P., Schuller, D. J., Poulos, T. L., Loehr, T. M., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 34501-34507). The potential role of this network in placing a water molecule to stabilize the hydroperoxy species and as a template for the condensation of the distal helix upon substrate binding are discussed.     

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.     

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.     

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.     

PLA2 and secondary metabolites of arachidonic acid control filopodial behavior in neuronal growth cones

The neuronal growth cone provides the sensory and motor structure that guides neuronal processes to their target. The ability of a growth cone to navigate correctly depends on its filopodia, which sample the environment by continually extending and retracting as the growth cone advances. Several second messengers systems that are activated upon contact with extracellular cues have been reported to affect growth cone morphology by changing the length and number of filopodia. Because recent studies have suggested that guidance cues can signal via G-protein coupled receptors to regulate phospholipases, we here investigated whether phospholipase A2 (PLA2) may control filopodial dynamics and could thereby affect neuronal pathfinding. Employing identified Helisoma neurons in vitro, we demonstrate that inhibition of PLA2 with 2 microM BPB caused a 40.3% increase in average filopodial length, as well as a 37.3% reduction in the number of filopodia on a growth cone. The effect of PLA2 inhibition on filopodial length was mimicked by the inhibition of G-proteins with 500 ng/ml pertussis toxin and was partially blocked by the simultaneous activation of PLA2 with 50 nM melittin. We provide evidence that PLA2 acts via production of arachidonic acid (AA), because (1) the effect of inhibition of PLA2 could be counteracted by supplying AA exogenously, and (2) the inhibition of cyclooxygenase, which metabolizes AA into prostaglandins, also increased filopodial length. We conclude that filopodial contact with extracellular signals that alter the activity of PLA2 can control growth cone morphology and may affect neuronal pathfinding by regulating the sensory radius of navigating growth cones.     

Refolding strategies from inclusion bodies in a structural genomics project

The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.