Recent Submissions

  • Journal Article

    Evolution of intron splicing towards optimized gene expression is based on various Cis- and Trans-molecular mechanisms 

    Frumkin, Idan; Yofe, Ido; Bar-Ziv, Raz; Gurvich, Yonat; Lu, Yen-Yun; Voichek, Yoav; Towers, Ruth; Schirman, Dvir; Krebber, Heike; Pilpel, Yitzhak
    PLOS Biology 2019; 17(8): Art. e3000423
    Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.
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  • Journal Article

    Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis 

    Reuß, Daniel R.; Faßhauer, Patrick; Mroch, Philipp Joel; Ul-Haq, Inam; Koo, Byoung-Mo; Pöhlein, Anja; Gross, Carol A.; Daniel, Rolf; Brantl, Sabine; Stülke, Jörg
    Nucleic Acids Research 2019; 47(10) p.5231-5242
    DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.
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  • Journal Article

    Structural basis for RNA translocation by DEAH-box ATPases 

    Hamann, Florian; Enders, Marieke; Ficner, Ralf
    Nucleic Acids Research 2019; 47(8) p.4349-4362
    DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3'-5' direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.
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  • Journal Article

    Membrane Lipids, Waxes and Oxylipins in the Moss Model Organism Physcomitrella patens 

    Resemann, Hanno C.; Lewandowska, Milena; Gömann, Jasmin; Feussner, Ivo
    Plant and Cell Physiology 2019; 60(6) p.1166-1175
    The moss Physcomitrella patens receives increased scientific interest since its genome was sequenced a decade ago. As a bryophyte, it represents the first group of plants that evolved in a terrestrial habitat still without a vascular system that developed later in tracheophytes. It is easily transformable via homologous recombination, which enables the formation of targeted loss-of-function mutants. Even though genetics, development and life cycle in Physcomitrella are well studied nowadays, research on lipids in Physcomitrella is still underdeveloped. This review aims on presenting an overview on the state of the art of lipid research with a focus on membrane lipids, surface lipids and oxylipins. We discuss in this review that Physcomitrella possesses very interesting features regarding its membrane lipids. Here, the presence of very-long-chain polyunsaturated fatty acids (VLC-PUFA) still shows a closer similarity to marine microalgae than to vascular plants. Unlike algae, Physcomitrella has a cuticle comparable to vascular plants composed of cutin and waxes. The presence of VLC-PUFA in Physcomitrella also leads to a greater variability of signaling lipids even though the phytohormone jasmonic acid is not present in this organism, which is different to vascular plants. In summary, the research on lipids in Physcomitrella is still in its infancy, especially considering membrane lipids. We hope that this review will help to promote the further advancement of lipid research in this important model organism in the future, so we can better understand how lipids are involved in the evolution of land plants.
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  • Journal Article

    COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development 

    Meister, Cindy; Thieme; Karl G.; Thieme, Sabine; Köhler, Anna M.; Schmitt, Kerstin; Valerius, Oliver; Braus, Gerhard H.
    Biomolecules 2019; 9(6): Art. 238
    COP9 signalosome (CSN) and Den1/A deneddylases physically interact and promote multicellular development in fungi. CSN recognizes Skp1/cullin-1/Fbx E3 cullin-RING ligases (CRLs) without substrate and removes their posttranslational Nedd8 modification from the cullin scaffold. This results in CRL complex disassembly and allows Skp1 adaptor/Fbx receptor exchange for altered substrate specificity. We characterized the novel ubiquitin-specific protease UspA of the mold Aspergillus nidulans, which corresponds to CSN-associated human Usp15 and interacts with six CSN subunits. UspA reduces amounts of ubiquitinated proteins during fungal development, and the uspA gene expression is repressed by an intact CSN. UspA is localized in proximity to nuclei and recruits proteins related to nuclear transport and transcriptional processing, suggesting functions in nuclear entry control. UspA accelerates the formation of asexual conidiospores, sexual development, and supports the repression of secondary metabolite clusters as the derivative of benzaldehyde (dba) genes. UspA reduces protein levels of the fungal NF-kappa B-like velvet domain protein VeA, which coordinates differentiation and secondary metabolism. VeA stability depends on the Fbx23 receptor, which is required for light controlled development. Our data suggest that the interplay between CSN deneddylase, UspA deubiquitinase, and SCF-Fbx23 ensures accurate levels of VeA to support fungal development and an appropriate secondary metabolism.
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  • Journal Article

    Formation and development of the male copulatory organ in the spider Parasteatoda tepidariorum involves a metamorphosis-like process 

    Quade, Felix Simon Christian; Holtzheimer, Jana; Frohn, Jasper; Töpperwien, Mareike; Salditt, Tim; Prpic, Nikola-Michael
    Scientific Reports 2019; 9(1): Art. 6945
    Spiders have evolved a unique male copulatory organ, the pedipalp bulb. The morphology of the bulb is species specific and plays an important role in species recognition and prezygotic reproductive isolation. Despite its importance for spider biodiversity, the mechanisms that control bulb development are virtually unknown. We have used confocal laser scanning microscopy (CLSM) and diffusible iodine-based contrast-enhanced micro computed tomography (dice-µCT) to study bulb development in the spider Parasteatoda tepidariorum. These imaging technologies enabled us to study bulb development in situ, without the use of destructive procedures for the first time. We show here that the inflated pedipalp tip in the subadult stage is filled with haemolymph that rapidly coagulates. Coagulation indicates histolytic processes that disintegrate tibia and tarsus, similar to histolytic processes during metamorphosis in holometabolous insects. The coagulated material contains cell inclusions that likely represent the cell source for the re-establishment of tarsus and tibia after histolysis, comparable to the histoblasts in insect metamorphosis. The shape of the coagulated mass prefigures the shape of the adult tarsus (cymbium) like a blueprint for the histoblasts. This suggests a unique role for controlled coagulation after histolysis in the metamorphosis-like morphogenesis of the male pedipalp.
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  • Journal Article

    Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis 

    Pinter, Niko; Hach, Christina Andrea; Hampel, Martin; Rekhter, Dmitrij; Zienkiewicz, Krzysztof; Feussner, Ivo; Poehlein, Anja; Daniel, Rolf; Finkernagel, Florian; Heimel, Kai
    PLOS Pathogens 2019; 15(4): Art. e1007734
    The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.
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  • Journal Article

    Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence 

    Bonnin, Edith; Cabochette, Pauline; Filosa, Alessandro; Jühlen, Ramona; Komatsuzaki, Shoko; Hezwani, Mohammed; Dickmanns, Achim; Martinelli, Valérie; Vermeersch, Marjorie; Supply, Lynn; et al.
    Martins, NunoPirenne, LaurenceRavenscroft, GianinaLombard, MarcusPort, SarahSpillner, ChristianeJanssens, SandraRoets, EllenVan Dorpe, JoLammens, MartinKehlenbach, Ralph HFicner, RalfLaing, Nigel GHoffmann, KatrinVanhollebeke, BenoitFahrenkrog, Birthe
    PLOS Genetics 2018; 14(12): Art. e1007845
    Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.
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  • Journal Article

    Targeting of LRRC59 to the Endoplasmic Reticulum and the Inner Nuclear Membrane. 

    Blenski, Marina; Kehlenbach, Ralph H.
    International Journal of Molecular Sciences 2019; 20(2): Art. 334
    LRRC59 (leucine-rich repeat-containing protein 59) is a tail-anchored protein with a single transmembrane domain close to its C-terminal end that localizes to the endoplasmic reticulum (ER) and the nuclear envelope. Here, we investigate the mechanisms of membrane integration of LRRC59 and its targeting to the inner nuclear membrane (INM). Using purified microsomes, we show that LRRC59 can be post-translationally inserted into ER-derived membranes. The TRC-pathway, a major route for post-translational membrane insertion, is not required for LRRC59. Like emerin, another tail-anchored protein, LRRC59 reaches the INM, as demonstrated by rapamycin-dependent dimerization assays. Using different approaches to inhibit importin α/β-dependent nuclear import of soluble proteins, we show that the classic nuclear transport machinery does not play a major role in INM-targeting of LRRC59. Instead, the size of the cytoplasmic domain of LRRC59 is an important feature, suggesting that targeting is governed by passive diffusion.
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  • Journal Article

    The green microalga Lobosphaera incisa harbours an arachidonate 15 S‐lipoxygenase 

    Djian, B.; Hornung, E.; Ischebeck, T.; Feussner, I.
    Plant Biology 2018; 21(S1) p.131-142
    The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules. • Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity. • The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n-6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration. • LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms.
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  • Journal Article

    A perforated anodised aluminium slide for improved specimen clearing and imaging for confocal laser scanning microscopy 

    Quade, Felix S. C.; Preitz, Beate; Prpic, Nikola-Michael
    BMC Research Notes. 2018 Oct 10;11(1):716
    Objective The bleaching, clearing and handling of tiny specimens with soft tissue and cuticular components for confocal laser scanning microscopy is difficult, because after cuticle bleaching and tissue clearing the specimens are virtually invisible. We have adjusted the design of the specimen container described by Smolla et al. (Arthropod Struct Dev 43:175–81, 2014) to handle tiny specimens. Results We describe a perforated and anodised aluminium slide that was designed to hold the distal tips of the pedipalp appendages of the spider Parasteatoda tepidariorum during clearing, and that can then be used directly for confocal laser scanning microscopy. We believe that this slide design will be helpful for others who want to visualise specimens between 500 and 800 µm with confocal laser scanning microscopy.
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  • Journal Article

    The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis. 

    Lorenzi, Isotta; Oeljeklaus, Silke; Aich, Abhishek; Ronsör, Christin; Callegari, Sylvie; Dudek, Jan; Warscheid, Bettina; Dennerlein, Sven; Rehling, Peter
    Biochimica et Biophysica Acta 2018; 1865(2) p.323-333
    The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the CuA-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of CuA-site formation.
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  • Journal Article

    Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function 

    Roitman, Sheila; Hornung, Ellen; Flores-Uribe, José; Sharon, Itai; Feussner, Ivo; Béjà, Oded
    The ISME Journal 2018; 12(2) p.343-355
    Cyanobacteria are among the most abundant photosynthetic organisms in the oceans; viruses infecting cyanobacteria (cyanophages) can alter cyanobacterial populations, and therefore affect the local food web and global biochemical cycles. These phages carry auxiliary metabolic genes (AMGs), which rewire various metabolic pathways in the infected host cell, resulting in increased phage fitness. Coping with stress resulting from photodamage appears to be a central necessity of cyanophages, yet the overall mechanism is poorly understood. Here we report a novel, widespread cyanophage AMG, encoding a fatty acid desaturase (FAD), found in two genotypes with distinct geographical distribution. FADs are capable of modulating the fluidity of the host's membrane, a fundamental stress response in living cells. We show that both viral FAD (vFAD) families are Δ9 lipid desaturases, catalyzing the desaturation at carbon 9 in C16 fatty acid chains. In addition, we present a comprehensive fatty acid profiling for marine cyanobacteria, which suggests a unique desaturation pathway of medium- to long-chain fatty acids no longer than C16, in accordance with the vFAD activity. Our findings suggest that cyanophages are capable of fiddling with the infected host's membranes, possibly leading to increased photoprotection and potentially enhancing viral-encoded photosynthetic proteins, resulting in a new viral metabolic network.
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  • Journal Article

    Sumoylation Protects Against β-Synuclein Toxicity in Yeast. 

    Popova, Blagovesta; Kleinknecht, Alexandra; Arendarski, Patricia; Mischke, Jasmin; Wang, Dan; Braus, Gerhard H.
    Frontiers in molecular neuroscience 2018; 11 p.1-17: Art. 94
    Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, β and γ isoforms. β-synuclein (βSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of βSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and βSyn exacerbates the cytotoxicity. This suggests an important role of βSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and βSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing βSyn. This corroborates a protective role of the cellular sumoylation machinery against βSyn-induced toxicity. Upregulation of sumoylation significantly reduced βSyn aggregate formation. This is an indirect molecular process, which is not directly linked to βSyn sumoylation because amino acid substitutions in the lysine residues required for βSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for βSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of βSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect βSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular βSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between βSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to βSyn on eukaryotic cells.
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  • Journal Article

    High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes. 

    Yu, Dan; Hornung, Ellen; Iven, Tim; Feussner, Ivo
    Biotechnology for Biofuels 2018; 11 p.1-14: Art. 53
    Background: Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient. Results: For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed-1 of wax esters, containing 27-34 mol% oleyl oleate. Conclusions: The overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.
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  • Journal Article

    Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant. 

    Kuhn, Hannah; Lorek, Justine; Kwaaitaal, Mark; Consonni, Chiara; Becker, Katia; Micali, Cristina; Ver Loren van Themaat, Emiel; Bednarek, Paweł; Raaymakers, Tom M.; Appiano, Michela; et al.
    Bai, YulingMeldau, DorotheaBaum, StephaniConrath, UweFeussner, IvoPanstruga, Ralph
    Frontiers in plant science 2017; 8: Art. 1006
    Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.
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  • Journal Article

    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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  • Journal Article

    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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  • Journal Article

    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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  • Journal Article

    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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