Integrating signals from the T-cell receptor and the interleukin-2 receptor

T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.     

Additive effects of the feed-back insensitive bacterial aspartate kinase and the Brazil nut 2S albumin on the methionine content of transgenic narbon bean (Vicia narbonensis L.)

So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.     

Identification and characterisation of regions in the cellular protein LaXp180 and the<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> surface protein ActA necessary for the interaction of the two proteins

The Listeria monocytogenes surface protein ActA is an important virulence factor that plays an essential role in intracellular movement of Listeria cells by inducing actin polymerisation. The ActA protein is known to interact with several mammalian proteins including the phosphoprotein VASP, actin and the Arp2/3 complex. In a search for additional ActA-binding proteins we recently employed the yeast two-hybrid system to search for proteins that interact with ActA, and identified, among others, the mammalian protein LaXp180 as a binding partner. In the present study the interaction of the two proteins was investigated in more detail. A number of variants were tested in the yeast two-hybrid system for their ability to interact. On the basis of these assays, the 14 C-terminal amino acids of LaXp180 were identified as being necessary for the interaction with ActA. The proline-rich repeat (PRR) region of ActA was found to be necessary for the interaction with LaXp180, but upstream or downstream sequences are also required to enhance the specificity of the interaction. The second and third repeats in ActA are especially important, and the minimal sequence of ActA capable of interacting with LaXp180 was a proline- and glutamate-rich stretch of PRR3 fused to part of the N-terminal sequence of ActA. Further analysis using site-specific mutations located in either the C-terminal region of LaXp180 or the proline-rich motif of PRR3 of ActA showed that three positively charged amino acids in LaXp180 and two negatively charged amino acids in ActA are critical for the interaction of the two proteins.     

1.3-A resolution structure of human glutathione S-transferase with S-hexyl glutathione bound reveals possible extended ligandin binding site

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.     

Reprint of "Crystal packing analysis of Rhodopsin crystals" [J. Struct. Biol. 158 (2007) 455-462

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin-rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.     

Chemical and Microbial Composition of Sediments in Reservoirs with Different Trophic State

For the assessment of the metabolic potential of a sediment, the upper 5 cm layer should preferentially be regarded, since this is the horizon with the highest biochemical activity in the entire reservoir. Sediment cores from four different reservoirs were examined. These represent a broad range of trophic conditions (Neunzehnhain – oligotrophic; Muldenberg – oligotrophic, acidic, dystrophic; Saidenbach – mesotrophic; Quitzdorf – highly eutrophic). The vertical concentration gradients in the interstitial water of the examined sediments showed the tendencies characteristic of the trophic state of the reservoirs. The gradients increased with the trophic state, which affected the release of dissolved substances. The internal P load was substantial in all cases, in Quitzdorf it even exceeded the external P load. The total cell numbers of the bacteria in the sediment were only slightly influenced by the trophic state, all being in the same order of magnitude. The reservoir Quitzdorf displayed very high P release rates from the sediment and mass growths of Microcystis with a high content of intracellular polyphosphate granules in the summer months.     

Transport of Algal Cells in Hyporheic Sediments of the River Elbe (Germany)

The advective transport of algal cells into the interstices of the hyporheic zone of the River Elbe was spatially and temporally heterogenous. Even deep sediment layers were reached by large phytoplankton species. Therefore, it is suggested that (i) the advective interstitial transport patterns vary between different algal sizes and morphotypes and (ii) sediment characteristics, expressed by the permeability coefficient k_f of porous media, affect retention and retardation of surface water algae during subsurface transport. The transport behaviour of different green algae (Chlorella sp., Scenedesmus acuminatus, Desmodesmus communis, and Pediastrum duplex) and algal sized microspheres was tested in flow‐through column experiments with hyporheic sediments. The algal cell transport was directly related to the permeability of the column sediments. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Litter Cover Variability Affects Seedling Performance and Herbivory1

We assessed the effects of litter cover on herbivory and performance (annual survival and relative growth rate in height and leaf number) for established seedlings (≥9 cm tall, and ≥4 mo old), of four shade-tolerant, large-seeded, co-occurring Sapotaceae species. Seedlings were divided into three litter treatments: (1) litter addition, (2) control, and (3) continuous litter removal. The mean survival for all treatments in descending order were: Chrysophyllum pomiferum (89%), Pouteria pemviemis (84%), P. caimito (75%), and Micropholis venulosa (40%). For all variables, species differed significantly within litter treatments. In M. venulosa, survival and relative growth rate in leaf number significantly decreased with the removal of litter, whereas herbivory increased. Relative growth rate in height of P. peruviensis decreased significantly with litter removal, while the levels of herbivory were higher with the addition of litter. Conversely, litter quantity had no effect on the performance of C. pomiferum, but herbivory was lower in the removal treatment. Finally, in P. caimito, litter treatment did not affect performance or herbivory. Results suggest that differences in litter quantity differentially affect tree species with similar biology (i.e., large-seeds, shade tolerance) in life stages other than germination and early establishment. The variability in species' responses to litter might be an important factor in determining species richness, abundance, and distribution of tropical rain forest tree species at the seedling level.