Molecular and regulatory properties of glutamine synthetase from the phototrophic bacterium Rhodopseudomonas capsulata E1F1.

The glutamine synthetase of the phototrophic bacterium Rhodopseudomonas capsulata E1F1 was purified to homogeneity by a procedure which used a single affinity chromatography step. Like enzymes from other photosynthetic procaryotes, native glutamine synthetase from R. capsulata E1F1 was found to be a dodecameric protein of approximately 660 kilodaltons with identical subunits of about 55 kilodaltons each. The Stokes radius and S20,w of the native enzyme were 8.35 nm and 19.20, respectively. The enzyme exhibited different aggregation states with detectable oligomers of 1, 2, 3, 4, 6, 8, 10, and 12 subunits. Disaggregation of the glutamine synthetase occurred after the native protein was subjected to electrophoresis in polyacrylamide gels, as well as occurring spontaneously at low ionic strength. Glutamine synthetase from R. capsulata E1F1 was regulated by an adenylylation-deadenylylation mechanism, and the adenylylation state of the protein depended on the nitrogen source, growth phase, and light intensity. Ammonia repressed glutamine synthetase, whereas glycine, serine, alanine, valine, and aspartate were noncompetitive inhibitors of the glutamine synthetase biosynthetic activity.     

The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus,plays a critical role in capsid assembly

Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells.     

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.     

Proteomic and metabolomic profiling of Valencia orange fruit after natural frost exposure

The aim of this study was to evaluate the response of orange fruit (Citrus sinensis var. Valencia Late) to freezing stress in planta, both immediately after the natural event and after a week, in order to understand the biochemical and molecular basis of the changes that later derive in internal and external damage symptoms. Using two‐dimensional differential gel electrophoresis to analyze exposed and non‐exposed fruit, 27 differential protein spots were detected in juice sacs and flavedo, among all comparisons made. Also, primary and secondary metabolites relative contents were analyzed in both tissues by gas chromatography‐mass spectrometry and liquid chromatography‐mass spectrometry, respectively. Proteins and compounds involved in regulatory functions, iron metabolism, oxidative damage and carbohydrate metabolism were the most affected. Interestingly, three glycolytic enzymes were induced by cold, and there was an increase in fermentation products (volatiles); all of that suggests that more energy generation might be required from glycolysis to counter the cold stress. Moreover, a notable increase in sugar levels was observed after frost, but it was not at the expense of organic acids utilization. Consequently, these results suggest a probable redistribution of photoassimilates in the frost‐exposed plants, tending to restore the homeostasis altered by that severe type of stress. Isosinensetin was the most cold‐sensitive secondary metabolite because it could not be detected at all after the frost, constituting a possible tool to early diagnose freezing damage.     

Towards the prediction of protein interaction partners using physical docking

Deciphering the whole network of protein interactions for a given proteome (‘interactome’) is the goal of many experimental and computational efforts in Systems Biology. Separately the prediction of the structure of protein complexes by docking methods is a well‐established scientific area. To date, docking programs have not been used to predict interaction partners. We provide a proof of principle for such an approach. Using a set of protein complexes representing known interactors in their unbound form, we show that a standard docking program can distinguish the true interactors from a background of 922 non‐redundant potential interactors. We additionally show that true interactions can be distinguished from non‐likely interacting proteins within the same structural family. Our approach may be put in the context of the proposed ‘funnel‐energy model’; the docking algorithm may not find the native complex, but it distinguishes binding partners because of the higher probability of favourable models compared with a collection of non‐binders. The potential exists to develop this proof of principle into new approaches for predicting interaction partners and reconstructing biological networks.     

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina^® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ^c?2j /J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.     

Rainfall stochasticity controls the distribution of invasive crayfish and its impact on amphibian guilds in Mediterranean temporary waters

Invasive crayfish have severely impacted invaded aquatic ecosystems worldwide. We studied temporal and spatial variation in the range expansion of the red swamp crayfish at one of the first European localities to which it was introduced: Doñana National Park (SW Spain). In contrast to the rapid range expansion witnessed in other areas, this invasive crayfish has not spread across the entire park. Instead, its distribution has expanded during wet periods, but contracted during drought periods. The red swamp crayfish has caused steep amphibian declines in other invaded areas. However, after approximately 35 years of crayfish presence in Doñana National Park, we have yet to detect a reduction in the number or occurrence of amphibian species. Amphibians may thus be protected by the large abundance of temporary ponds in the area, which provides them with an effective refuge network. We show that natural fluctuations in annual rainfall and in the number of ponds filled can temporarily eliminate invasive crayfish from particular areas. This fact should be taken into account when attempting to reduce the impact of crayfish on aquatic communities, intensifying crayfish removal during particularly dry years, when it is most effective.     

Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis

We have investigated the role of glycans on Trichinella spiralisantigens in recognition by rat monoclonal antibodies (mAbs)which protect rat pups against challenge with the parasite.In pups born to infected dams or pups passively immunized withmAbs, antibodies eliminate a challenge dose from the intestinewithin hours (‘rapid expulsion’). Because such dramaticprotection can be afforded by mAbs, we have sought to characterizethe parasite antigens they target In this report we show thatprotective antibodies were unable to bind excretory/secretory(ES) antigens de-glycosylated with trifluoromethanesulphonicacid (TFMS). In addition, oligosaccharides isolated from glycoproteinsby alkaline hydrolysis or peptlde: Nglycosidase F (PNGase F)digestion were bound by protective, but not non-protective,mAbs. Glycans affinity purified with protective mAb 9D boundto all but one protective mAb. These antibodies have been shownpreviously to bind to the surfaces of intact larvae, indicatingthat the glycan is exposed on the parasite surface. Candidateglycans that may be involved in binding protective mAbs haveunusual tri- and tetra-antennary structures with terminal tyvelosemoieties (Reason et al., Glycobiology, 4, 000-000, 1994). Coatingof the larval surface with such glycans may serve to protectthe parasite and its secreted products from enzymatic attackas the parasite travels to and resides in its epithelial niche. glycans protective antibodies Trichinella spiralis