1-20 von 74 Publikationen

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      Bayesian refinement of protein structures and ensembles against SAXS data using molecular dynamics. 

      Shevchuk, Roman; Hub, Jochen S.
      PLoS computational biology 2017-10; 13(10): Art. e1005800
      Small-angle X-ray scattering is an increasingly popular technique used to detect protein structures and ensembles in solution. However, the refinement of structures and ensembles against SAXS data is often ambiguous due to the low information content of SAXS data, unknown systematic errors, and unknown scattering contributions from the solvent. We offer a solution to such problems by combining Bayesian inference with all-atom molecular dynamics simulations and explicit-solvent SAXS calculations. The Bayesian formulation correctly weights the SAXS data versus prior physical knowledge, it quantifies the precision or ambiguity of fitted structures and ensembles, and it accounts for unknown systematic errors due to poor buffer matching. The method further provides a probabilistic criterion for identifying the number of states required to explain the SAXS data. The method is validated by refining ensembles of a periplasmic binding protein against calculated SAXS curves. Subsequently, we derive the solution ensembles of the eukaryotic chaperone heat shock protein 90 (Hsp90) against experimental SAXS data. We find that the SAXS data of the apo state of Hsp90 is compatible with a single wide-open conformation, whereas the SAXS data of Hsp90 bound to ATP or to an ATP-analogue strongly suggest heterogenous ensembles of a closed and a wide-open state.
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      INA complex liaises the F1Fo-ATP synthase membrane motor modules. 

      Naumenko, Nataliia; Morgenstern, Marcel; Rucktäschel, Robert; Warscheid, Bettina; Rehling, Peter
      Nature communications 2017-11-01; 8(1): Art. 1237
      The F1F0-ATP synthase translates a proton flux across the inner mitochondrial membrane into a mechanical rotation, driving anhydride bond formation in the catalytic portion. The complex's membrane-embedded motor forms a proteinaceous channel at the interface between Atp9 ring and Atp6. To prevent unrestricted proton flow dissipating the H(+)-gradient, channel formation is a critical and tightly controlled step during ATP synthase assembly. Here we show that the INA complex (INAC) acts at this decisive step promoting Atp9-ring association with Atp6. INAC binds to newly synthesized mitochondrial-encoded Atp6 and Atp8 in complex with maturation factors. INAC association is retained until the F1-portion is built on Atp6/8 and loss of INAC causes accumulation of the free F1. An independent complex is formed between INAC and the Atp9 ring. We conclude that INAC maintains assembly intermediates of the F1 F0-ATP synthase in a primed state for the terminal assembly step-motor module formation.
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      Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2 

      Fourmann, Jean-Baptiste; Tauchert, Marcel J.; Ficner, Ralf; Fabrizio, Patrizia; Lührmann, Reinhard
      Nucleic Acids Research 2016; 45(7) p.4068-4080
      The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly.
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      BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells 

      Nagarajan, Sankari; Bedi, Upasana; Budida, Anusha; Hamdan, Feda H.; Mishra, Vivek Kumar; Najafova, Zeynab; Xie, Wanhua; Alawi, Malik; Indenbirken, Daniela; Knapp, Stefan; et al.
      Chiang, Cheng-MingGrundhoff, AdamKari, VijayalakshmiScheel, Christina H.Wegwitz, FlorianJohnsen, Steven A.
      Nucleic Acids Research 2016; 45(6) p.3130-3145
      Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.
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      The house spider genome reveals an ancient whole-genome duplication during arachnid evolution 

      Schwager, Evelyn E.; Sharma, Prashant P.; Clarke, Thomas; Leite, Daniel J.; Wierschin, Torsten; Pechmann, Matthias; Akiyama-Oda, Yasuko; Esposito, Lauren; Bechsgaard, Jesper; Bilde, Trine; et al.
      Buffry, Alexandra D.Chao, HsuDinh, HuyenDoddapaneni, HarshaVardhanDugan, ShannonEibner, CorneliusExtavour, Cassandra G.Funch, PeterGarb, JessicaGonzalez, Luis B.Gonzalez, Vanessa L.Griffiths-Jones, SamHan, YiHayashi, CherylHilbrant, MaartenHughes, Daniel S. T.Janssen, RalfLee, Sandra L.Maeso, IgnacioMurali, Shwetha C.Muzny, Donna M.Nunes da Fonseca, RodrigoPaese, Christian L. B.Qu, JiaxinRonshaugen, MatthewSchomburg, ChristophSchönauer, AnnaStollewerk, AngelikaTorres-Oliva, MontserratTuretzek, NataschaVanthournout, BramWerren, John H.Wolff, CarstenWorley, Kim C.Bucher, GregorGibbs, Richard A.Coddington, JonathanOda, HirokiStanke, MarioAyoub, Nadia A.Prpic, Nikola-MichaelFlot, Jean-FrançoisPosnien, NicoRichards, StephenMcGregor, Alistair P.
      BMC Biology 2017; 15(62) p.1-27
      The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.
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      Rapid diversification of homothorax expression patterns after gene duplication in spiders 

      Turetzek, Natascha; Khadjeh, Sara; Schomburg, Christoph; Prpic, Nikola-Michael
      BMC Evolutionary Biology 2017; 17(1)
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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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    • Zeitschriftenartikel

      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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