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Item Open Access Patterns of autologous and nonautologous interactions between core nuclear egress complex (NEC) proteins of α-, β- and γ-herpesviruses(2020) Häge, Sigrun; Sonntag, Eric; Borst, Eva Maria; Tannig, Pierre; Seyler, Lisa; Bäuerle, Tobias; Bailer, Susanne M.; Lee, Chung-Pei; Müller, Regina; Wangen, Christina; Milbradt, Jens; Marschall, ManfredNuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.Item Open Access A universal polyphosphate kinase : PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate(2020) Hildenbrand, Jennie C.; Teleki, Attila; Jendrossek, DieterPolyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP.Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates(2020) Frank, Celina; Teleki, Attila; Jendrossek, DieterAgrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data - in combination with in vivo results - suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification.Item Open Access DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes(2020) Benz, David; Olayioye, Monilola (Prof. Dr.)Item Open Access Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate(2020) Schuhmacher, Maren Kirstin; Beldar, Serap; Khella, Mina S.; Bröhm, Alexander; Ludwig, Jan; Tempel, Wolfram; Weirich, Sara; Min, Jinrong; Jeltsch, AlbertSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.Item Open Access Role of the medial orbitofrontal cortex and ventral tegmental area in effort-related responding(2020) Münster, Alexandra; Votteler, Angeline; Sommer, Susanne; Hauber, WolfgangDie Arbeit untersucht die Interaktion von zwei Gehirnarealen, dem medialen Orbitofrontalcortex und dem Ventralen Tegmentalen Areal, bei der Handlungssteuerung.Item Open Access Loops und Tunnel : unterschätzte Elemente in Enzymen(2020) Heinemann, Peter M.; Rapp, Lea R.; Hauer, BernhardIn enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.Item Open Access Structural optimization through biomimetic-inspired material-specific application of plant-based natural fiber-reinforced polymer composites (NFRP) for future sustainable lightweight architecture(2020) Sippach, Timo; Dahy, Hanaa; Uhlig, Kai; Grisin, Benjamin; Carosella, Stefan; Middendorf, PeterUnder normal conditions, the cross-sections of reinforced concrete in classic skeleton construction systems are often only partially loaded. This contributes to non-sustainable construction solutions due to an excess of material use. Novel cross-disciplinary workflows linking architects, engineers, material scientists and manufacturers could offer alternative means for more sustainable architectural applications with extra lightweight solutions. Through material-specific use of plant-based Natural Fiber-Reinforced Polymer Composites (NFRP), also named Biocomposites, a high-performance lightweight structure with topology optimized cross-sections has been here developed. The closed life cycle of NFRPs promotes sustainability in construction through energy recovery of the quickly generative biomass-based materials. The cooperative design resulted in a development that were verified through a 1:10 demonstrator, whose fibrous morphology was defined by biomimetically-inspired orthotropic tectonics, generated with by the fiber path optimization software tools, namely EdoStructure and EdoPath in combination with the appliance of the digital additive manufacturing technique: Tailored Fiber Placement (TFP).Item Open Access Identification and characterization of IgE‐reactive proteins and a new allergen (Cic a 1.01) from chickpea (Cicer arietinum)(2020) Wangorsch, Andrea; Kulkarni, Anuja; Jamin, Annette; Spiric, Jelena; Bräcker, Julia; Brockmeyer, Jens; Mahler, Vera; Blanca‐López, Natalia; Ferrer, Marta; Blanca, Miguel; Torres, Maria; Gomez, Paqui; Bartra, Joan; García‐Moral, Alba; Goikoetxea, María J.; Vieths, Stefan; Toda, Masako; Zoccatelli, Gianni; Scheurer, StephanChickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.