1-20 von 68 Publikationen

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      Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa 

      Siegler, Heike; Valerius, Oliver; Ischebeck, Till; Popko, Jennifer; Tourasse, Nicolas J; Vallon, Olivier; Khozin-Goldberg, Inna; Braus, Gerhard H; Feussner, Ivo
      BMC Plant Biology 2017; 17(1): Art. 98
      Abstract Background Lobosphaera incisa (L. incisa) is an oleaginous microalga that stores triacylglycerol (TAG) rich in arachidonic acid in lipid bodies (LBs). This organelle is gaining attention in algal research, since evidence is accumulating that proteins attached to its surface fulfill important functions in TAG storage and metabolism. Results Here, the composition of the LB proteome in L incisa was investigated by comparing different cell fractions in a semiquantitative proteomics approach. After applying stringent filters to the proteomics data in order to remove contaminating proteins from the list of possible LB proteins (LBPs), heterologous expression of candidate proteins in tobacco pollen tubes, allowed us to confirm 3 true LBPs: A member of the algal Major Lipid Droplet Protein family, a small protein of unknown function and a putative lipase. In addition, a TAG lipase that belongs to the SUGAR DEPENDENT 1 family of TAG lipases known from oilseed plants was identified. Its activity was verified by functional complementation of an Arabidopsis thaliana mutant lacking the major seed TAG lipases. Conclusions Here we describe 3 LBPs as well as a TAG lipase from the oleaginous microalga L. incisa and discuss their possible involvement in LB metabolism. This study highlights the importance of filtering LB proteome datasets and verifying the subcellular localization one by one, so that contaminating proteins can be recognized as such. Our dataset can serve as a valuable resource in the identification of additional LBPs, shedding more light on the intriguing roles of LBs in microalgae.
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      The effect of hypoxia on the lipidome of recombinant Pichia pastoris 

      Adelantado, Núria; Tarazona, Pablo; Grillitsch, Karlheinz; García-Ortega, Xavier; Monforte, Sergi; Valero, Francisco; Feussner, Ivo; Daum, Günther; Ferrer, Pau
      Microbial Cell Factories 2017; 16(1): Art. 86
      Abstract Background Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. Results To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Conclusions Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.
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      Structural insights into the mechanism of the DEAH-box RNA helicase Prp43 

      Tauchert, Marcel J.; Fourmann, Jean-Baptiste; Lührmann, Reinhard; Ficner, Ralf
      eLife 2017; 6: Art. e21510
      The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43.ATP-analog.RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a b-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.
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      Bulk isolation of basidiospores from wild mushrooms by electrostatic attraction with low risk of microbial contaminations 

      Lakkireddy, Kiran; Kües, Ursula
      AMB Express 2017; 7(1): Art. 28
      The basidiospores of most Agaricomycetes are ballistospores. They are propelled off from their basidia at maturity when Buller’s drop develops at high humidity at the hilar spore appendix and fuses with a liquid film formed on the adaxial side of the spore. Spores are catapulted into the free air space between hymenia and fall then out of the mushroom’s cap by gravity. Here we show for 66 different species that ballistospores from mushrooms can be attracted against gravity to electrostatic charged plastic surfaces. Charges on basidiospores can influence this effect. We used this feature to selectively collect basidiospores in sterile plastic Petri-dish lids from mushrooms which were positioned upside-down onto wet paper tissues for spore release into the air. Bulks of 104 to >107 spores were obtained overnight in the plastic lids above the reversed fruiting bodies, between 104 and 106 spores already after 2–4 h incubation. In plating tests on agar medium, we rarely observed in the harvested spore solutions contaminations by other fungi (mostly none to up to in 10% of samples in different test series) and infrequently by bacteria (in between 0 and 22% of samples of test series) which could mostly be suppressed by bactericides. We thus show that it is possible to obtain clean basidiospore samples from wild mushrooms. The technique of spore collection through electrostatic attraction in plastic lids is applicable to fresh lamellate and poroid fruiting bodies from the wild, to shortlived deliquescent mushrooms, to older and dehydrating fleshy fruiting bodies, even to animal-infested mushrooms and also to dry specimens of long-lasting tough species such as Schizophyllum commune.
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      Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data 

      Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Ever, Maurits; Kohler, Christian; Shirneshan, Katayoon; et al.
      Vockerodt, MartinaKlapper, WolframSzczepanowski, MonikaMurray, Paul G.Bastians, HolgerTrümper, LorenzSpang, RainerKube, Dieter
      Oncotarget 2016; 7(30)
      To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery.
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      Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts 

      Zienkiewicz, Krzysztof; Zienkiewicz, Agnieszka; Poliner, Eric; Du, Zhi-Yan; Vollheyde, Katharina; Herrfurth, Cornelia; Marmon, Sofia; Farré, Eva M.; Feussner, Ivo; Benning, Christoph
      Biotechnology for Biofuels 2017; 10(1): Art. 8
      Abstract Background Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. Results Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Conclusions Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.
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      Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates 

      Lorenzi, Isotta; Oeljeklaus, Silke; Ronsör, Christin; Bareth, Bettina; Warscheid, Bettina; Rehling, Peter; Dennerlein, Sven
      Molecular and Cellular Biology 2016; 36(22) p.2782-2793
      The three conserved core subunits of the cytochrome c oxidase are encoded by mitochondria in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing the N and C termini to the intermembrane space. For this, the N terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae. Little is known about the translocation of the C terminus, but Cox18 has been identified to be a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner. The Mba1-Cox20 complex accumulates when export of the C terminus of Cox2 is blocked by the loss of the Cox18 protein. While Cox20 engages with Cox18, Mba1 is no longer present at this stage. Our analyses indicate that Cox20 associates with nascent Cox2 and Mba1 to promote Cox2 maturation cotranslationally. We suggest that Mba1 stabilizes the Cox20-ribosome complex and supports the handover of Cox2 to the Cox18 tail export machinery.
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      Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum 

      Peter, Martina; Kohler, Annegret; Ohm, Robin A.; Kuo, Alan; Krützmann, Jennifer; Morin, Emmanuelle; Arend, Matthias; Barry, Kerrie W.; Binder, Manfred; Choi, Cindy; et al.
      Clum, AliciaCopeland, AlexGrisel, NadineHaridas, SajeetKipfer, TabeaLaButti, KurtLindquist, ErikaLipzen, AnnaMaire, RenaudMeier, BarbaraMihaltcheva, SirmaMolinier, VirginieMurat, ClaudePöggeler, StefanieQuandt, C. AlishaSperisen, ChristophTritt, AndrewTisserant, EmilieCrous, Pedro W.Henrissat, BernardNehls, UweEgli, SimonSpatafora, Joseph W.Grigoriev, Igor V.Martin, Francis M.
      Nature Communications 2016; 7
      The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.
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      Morphological and Transcriptomic Analysis of a Beetle Chemosensory System Reveals a Gnathal Olfactory Center 

      Dippel, Stefan; Kollmann, Martin; Oberhofer, Georg; Montino, Alice; Knoll, Carolin; Krala, Milosz; Rexer, Karl-Heinz; Frank, Sergius; Kumpf, Robert; Schachtner, Joachim; et al.
      Wimmer, Ernst A.
      BMC Biology 2016; 14(1): Art. 90
      Abstract Background The red flour beetle Tribolium castaneum is an emerging insect model organism representing the largest insect order, Coleoptera, which encompasses several serious agricultural and forest pests. Despite the ecological and economic importance of beetles, most insect olfaction studies have so far focused on dipteran, lepidopteran, or hymenopteran systems. Results Here, we present the first detailed morphological description of a coleopteran olfactory pathway in combination with genome-wide expression analysis of the relevant gene families involved in chemoreception. Our study revealed that besides the antennae, also the mouthparts are highly involved in olfaction and that their respective contribution is processed separately. In this beetle, olfactory sensory neurons from the mouthparts project to the lobus glomerulatus, a structure so far only characterized in hemimetabolous insects, as well as to a so far non-described unpaired glomerularly organized olfactory neuropil in the gnathal ganglion, which we term the gnathal olfactory center. The high number of functional odorant receptor genes expressed in the mouthparts also supports the importance of the maxillary and labial palps in olfaction of this beetle. Moreover, gustatory perception seems equally distributed between antenna and mouthparts, since the number of expressed gustatory receptors is similar for both organs. Conclusions Our analysis of the T. castaneum chemosensory system confirms that olfactory and gustatory perception are not organotopically separated to the antennae and mouthparts, respectively. The identification of additional olfactory processing centers, the lobus glomerulatus and the gnathal olfactory center, is in contrast to the current picture that in holometabolous insects all olfactory inputs allegedly converge in the antennal lobe. These findings indicate that Holometabola have evolved a wider variety of solutions to chemoreception than previously assumed.
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      Metabolome Analysis Reveals Betaine Lipids as Major Source for Triglyceride Formation, and the Accumulation of Sedoheptulose during Nitrogen-Starvation of Phaeodactylum tricornutum. 

      Popko, Jennifer; Herrfurth, Cornelia; Feussner, Kirstin; Ischebeck, Till; Iven, Tim; Haslam, Richard; Hamilton, Mary; Sayanova, Olga; Napier, Jonathan; Khozin-Goldberg, Inna; et al.
      Feussner, Ivo
      PloS one 2016; 11(10): Art. e0164673
      Oleaginous microalgae are considered as a promising resource for the production of biofuels. Especially diatoms arouse interest as biofuel producers since they are most productive in carbon fixation and very flexible to environmental changes in the nature. Naturally, triacylglycerol (TAG) accumulation in algae only occurs under stress conditions like nitrogen-limitation. We focused on Phaeodactylum strain Pt4 (UTEX 646), because of its ability to grow in medium with low salinity and therefore being suited when saline water is less available or for wastewater cultivation strategies. Our data show an increase in neutral lipids during nitrogen-depletion and predominantly 16:0 and 16:1(n-7) accumulated in the TAG fraction. The molecular species composition of TAG suggests a remodeling primarily from the betaine lipid diacylglyceroltrimethylhomoserine (DGTS), but a contribution of the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) cannot be excluded. Interestingly, the acyl-CoA pool is rich in 20:5(n-3) and 22:6(n-3) in all analyzed conditions, but these fatty acids are almost excluded from TAG. Other metabolites most obviously depleted under nitrogen-starvation were amino acids, lyso-phospholipids and tricarboxylic acid (TCA) cycle intermediates, whereas sulfur-containing metabolites as dimethylsulfoniopropionate, dimethylsulfoniobutyrate and methylsulfate as well as short acyl chain carnitines, propanoyl-carnitine and butanoyl-carnitine increased upon nitrogen-starvation. Moreover, the Calvin cycle may be de-regulated since sedoheptulose accumulated after nitrogen-depletion. Together the data provide now the basis for new strategies to improve lipid production and storage in Phaeodactylum strain Pt4.
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      SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus. 

      Jöhnk, Bastian; Bayram, Özgür; Abelmann, Anja; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.
      PLoS pathogens 2016-09-01; 12(9): Art. e1005899
      F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.
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      Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress. 

      Saini, Priyanka; Li, Yizhu; Dobbelstein, Matthias
      Oncotarget 2015-05-30; 6(15) p.13072-13087
      The therapeutic efficacy of nucleoside analogues, e.g. gemcitabine, against cancer cells can be augmented by inhibitors of checkpoint kinases, including Wee1, ATR, and Chk1. We have compared the chemosensitizing effect of these inhibitors in cells derived from pancreatic cancer, a tumor entity where gemcitabine is part of the first-line therapeutic regimens, and in osteosarcoma-derived cells. As expected, all three inhibitors rendered cancer cells more sensitive to gemcitabine, but Wee1 inhibition proved to be particularly efficient in this context. Investigating the reasons for this potent sensitizing effect, we found that Wee1 inhibition or knockdown not only blocked Wee1 activity, but also reduced the activation of ATR/Chk1 in gemcitabine-treated cells. Combination of several inhibitors revealed that Wee1 inhibition requires Cyclin-dependent kinases 1 and 2 (Cdk1/2) and Polo-like kinase 1 (Plk1) to reduce ATR/Chk1 activity. Through activation of Cdks and Plk1, Wee1 inhibition reduces Claspin and CtIP levels, explaining the impairment in ATR/Chk1 activity. Taken together, these results confer a consistent signaling pathway reaching from Wee1 inhibition to impaired Chk1 activity, mechanistically dissecting how Wee1 inhibitors not only dysregulate cell cycle progression, but also enhance replicative stress and chemosensitivity towards nucleoside analogues.
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      The complex portal--an encyclopaedia of macromolecular complexes. 

      Meldal, Birgit H. M.; Forner-Martinez, Oscar; Costanzo, Maria C.; Dana, Jose; Demeter, Janos; Dumousseau, Marine; Dwight, Selina S.; Gaulton, Anna; Licata, Luana; Melidoni, Anna N.; et al.
      Ricard-Blum, SylvieRoechert, BerndSkyzypek, Marek S.Tiwari, ManuVelankar, SameerWong, Edith D.Hermjakob, HenningOrchard, Sandra
      Nucleic acids research 2015; 43(Database issue) p.D479-D484
      The IntAct molecular interaction database has created a new, free, open-source, manually curated resource, the Complex Portal (www.ebi.ac.uk/intact/complex), through which protein complexes from major model organisms are being collated and made available for search, viewing and download. It has been built in close collaboration with other bioinformatics services and populated with data from ChEMBL, MatrixDB, PDBe, Reactome and UniProtKB. Each entry contains information about the participating molecules (including small molecules and nucleic acids), their stoichiometry, topology and structural assembly. Complexes are annotated with details about their function, properties and complex-specific Gene Ontology (GO) terms. Consistent nomenclature is used throughout the resource with systematic names, recommended names and a list of synonyms all provided. The use of the Evidence Code Ontology allows us to indicate for which entries direct experimental evidence is available or if the complex has been inferred based on homology or orthology. The data are searchable using standard identifiers, such as UniProt, ChEBI and GO IDs, protein, gene and complex names or synonyms. This reference resource will be maintained and grow to encompass an increasing number of organisms. Input from groups and individuals with specific areas of expertise is welcome.
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      Comparative Genomics Uncovers Unique Gene Turnover and Evolutionary Rates in a Gene Family Involved in the Detection of Insect Cuticular Pheromones 

      Torres-Oliva, Montserrat; Almeida, Francisca C.; Sa´nchez-Gracia, Alejandro; Rozas, Julio
      Genome Biology and Evolution 2016; 8(6) p.1734-1747
      Chemoreception is an essential process for the survival and reproduction of animals.Many of the proteins responsible for recognizing and transmitting chemical stimuli in insects are encoded by genes that are members of moderately sized multigene families. The members of the CheB family are specialized in gustatory-mediated detection of long-chain hydrocarbon pheromones in Drosophila melanogaster and play a central role in triggering andmodulating mating behavior in this species. Here,we present a comprehensive comparative genomic analysis of the CheB family across 12 species of the Drosophila genus.We have identified a total of 102 new CheB genes in the genomes of these species, including a functionally divergent member previously uncharacterized in D. melanogaster. We found that, despite its relatively small repertory size, the CheB family has undergone multiple gain and loss events and various episodes of diversifying selection during the divergence of the surveyed species. Present estimates of gene turnover and coding sequence substitution rates showthat this family is evolving faster than any knownDrosophila chemosensory family. To date, only other insect gustatory-related genes among these families had shown evolutionary dynamics close to those observed in CheBs. Our findings reveal the high adaptive potential of molecular componentsof the gustatory system ininsects and anticipate a key role of genes involved in this sensory modality in species adaptation and diversification.
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      A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity. 

      Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M.; Tauchert, Marcel J.; Agafonov, Dmitry E.; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L.; Lührmann, Reinhard
      Nature communications 2016; 7: Art. 11997
      The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.
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      Functional Characterization of CYP94-Genes and Identification of a Novel Jasmonate Catabolite in Flowers. 

      Bruckhoff, Viktoria; Haroth, Sven; Feussner, Kirstin; König, Stefanie; Brodhun, Florian; Feussner, Ivo
      PloS one 2016; 11(7): Art. e0159875
      Over the past decades much research focused on the biosynthesis of the plant hormone jasmonyl-isoleucine (JA-Ile). While many details about its biosynthetic pathway as well about its physiological function are established nowadays, knowledge about its catabolic fate is still scarce. Only recently, the hormonal inactivation mechanisms became a stronger research focus. Two major pathways have been proposed to inactivate JA-Ile: i) The cleavage of the jasmonyl-residue from the isoleucine moiety, a reaction that is catalyzed by specific amido-hydrolases, or ii), the sequential oxidation of the ω-end of the pentenyl side-chain. This reaction is catalyzed by specific members of the cytochrome P450 (CYP) subfamily CYP94: CYP94B1, CYP94B3 and CYP94C1. In the present study, we further investigated the oxidative fate of JA-Ile by expanding the analysis on Arabidopsis thaliana mutants, lacking only one (cyp94b1, cyp94b2, cyp94b3, cyp94c1), two (cyp94b1xcyp94b2, cyp94b1xcyp94b3, cyp94b2xcyp94b3), three (cyp94b1xcyp94b2xcyp94b3) or even four (cyp94b1xcyp94b2xcyp94b3xcyp94c1) CYP94 functionalities. The results obtained in the present study show that CYP94B1, CYP94B2, CYP94B3 and CYP94C1 are responsible for catalyzing the sequential ω-oxidation of JA-Ile in a semi-redundant manner. While CYP94B-enzymes preferentially hydroxylate JA-Ile to 12-hydroxy-JA-Ile, CYP94C1 catalyzes primarily the subsequent oxidation, yielding 12-carboxy-JA-Ile. In addition, data obtained from investigating the triple and quadruple mutants let us hypothesize that a direct oxidation of unconjugated JA to 12-hydroxy-JA is possible in planta. Using a non-targeted metabolite fingerprinting analysis, we identified unconjugated 12-carboxy-JA as novel jasmonate derivative in floral tissues. Using the same approach, we could show that deletion of CYP94-genes might not only affect JA-homeostasis but also other signaling pathways. Deletion of CYP94B1, for example, led to accumulation of metabolites that may be characteristic for plant stress responses like systemic acquired resistance. Evaluation of the in vivo function of the different CYP94-enzymes on the JA-sensitivity demonstrated that particularly CYP94B-enzymes might play an essential role for JA-response, whereas CYP94C1 might only be of minor importance.
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      Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora. 

      Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie
      PloS one 2016; 11(6): Art. e0157960
      In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.
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      C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease. 

      Kleinknecht, Alexandra; Popova, Blagovesta; Lázaro, Diana F.; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F.; Braus, Gerhard H.
      PLoS genetics 2016-06-01; 12(6): Art. e1006098
      Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.
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      Chromosome condensation and decondensation during mitosis. 

      Antonin, Wolfram; Neumann, Heinz
      Current opinion in cell biology 2016-06-01; 40 p.15-22
      During eukaryotic cell division, nuclear chromatin undergoes marked changes with respect to shape and degree of compaction. Although already significantly compacted during interphase, upon entry into mitosis chromatin further condenses and individualizes to discrete chromosomes that are captured and moved independently by the mitotic spindle apparatus. Once segregated by the spindle, chromatin decondenses to re-establish its interphase structure competent for DNA replication and transcription. Although cytologically described a long time ago, the underlying molecular mechanisms of mitotic chromatin condensation and decondensation are still ill-defined. Here we summarize our current knowledge of mitotic chromatin restructuring and recent progress in the field.
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      The insect central complex as model for heterochronic brain development-background, concepts, and tools. 

      Koniszewski, Nikolaus Dieter Bernhard; Kollmann, Martin; Bigham, Mahdiyeh; Farnworth, Max; He, Bicheng; Büscher, Marita; Hütteroth, Wolf; Binzer, Marlene; Schachtner, Joachim; Bucher, Gregor
      Development genes and evolution p.1-11
      The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.
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