• Crystal Structure of the HMG-CoA Synthase MvaS from the Gram-Negative Bacterium Myxococcus xanthus.

      Bock, Tobias; Kasten, Janin; Müller, Rolf; Blankenfeldt, Wulf; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-07-01)
      A critical step in bacterial isoprenoid production is the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) catalyzed by HMG-CoA synthase (HMGCS). In myxobacteria, this enzyme is also involved in a recently discovered alternative and acetyl-CoA-dependent isovaleryl CoA biosynthesis pathway. Here we present crystal structures of MvaS, the HMGCS from Myxococcus xanthus, in complex with CoA and acetylated active site Cys115, with the second substrate acetoacetyl CoA and with the product of the condensation reaction, 3-hydroxy-3-methylglutaryl CoA. With these structures, we show that MvaS uses the common HMGCS enzymatic mechanism and provide evidence that dimerization plays a role in the formation and stability of the active site. Overall, MvaS shows features typical of the eukaryotic HMGCS and exhibits differences from homologues from Gram-positive bacteria. This study provides insights into myxobacterial alternative isovaleryl CoA biosynthesis and thereby extends the toolbox for the biotechnological production of renewable fuel and chemicals.
    • Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability.

      Mayer, Janine; Pippel, Jan; Günther, Gabriele; Müller, Carolin; Lauermann, Anna; Knuuti, Tobias; Blankenfeldt, Wulf; Jahn, Dieter; Biedendieck, Rebekka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2019-06-21)
      Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes.
    • Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From in the Pre-assembled State.

      Schwemmlein, Nina; Pippel, Jan; Gazdag, Emerich-Mihai; Blankenfeldt, Wulf (2018-01-01)
      iffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.
    • Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

      Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter; Dept of structure and functions of proteins, Hemholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-05)
      Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.
    • Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA.

      Hellert, Jan; Krausze, Joern; Schulz, Thomas F; Lührs, Thorsten; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2014-11)
      The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposi's sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.
    • CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering.

      Bracco, Paula; Wijma, Hein J; Nicolai, Bastian; Rodriguez Buitrago, Jhon Alexander; Klünemann, Thomas; Vila, Agustina; Schrepfer, Patrick; Blankenfeldt, Wulf; Janssen, Dick B; Schallmey, Anett; et al. (Wiley, 2020-11-04)
      CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16a-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5a-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
    • Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

      Schaks, Matthias; Singh, Shashi P; Kage, Frieda; Thomason, Peter; Klünemann, Thomas; Steffen, Anika; Blankenfeldt, Wulf; Stradal, Theresia E; Insall, Robert H; Rottner, Klemens; et al. (2018-10-25)
      Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
    • An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity

      Haase-Aschoff, Paul; Linke, Diana; Nimtz, Manfred; Popper, Lutz; Berger, Ralf G.; Dept. of structure and function of proteins, Helmhotz Centre for infection research, D-38124 Braunschweig, Germany (2014-01-14)
    • ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

      Büssow, Konrad; Themann, Philipp; Luu, Sabine; Pentrowski, Paul; Harting, Claudia; Majewski, Mira; Vollmer, Veith; Köster, Mario; Grashoff, Martina; Zawatzky, Rainer; et al. (Plos, 2019-01-01)
      Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
    • ER-targeted intrabodies mediating specific in vivo knockdown of transitory proteins in comparison to RNAi

      Backhaus, Oliver; Böldicke, Thomas; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-10-25)
      In animals and mammalian cells, protein function can be analyzed by nucleotide sequence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases, or the RNAi technique. Alternatively, protein knockdown approaches are available based on direct interference of the target protein with the inhibitor.Among protein knockdown techniques, the endoplasmic reticulum (ER) intrabodies arepotent molecules for protein knockdown in vitro and in vivo. These molecules are increasingly used for protein knockdown in living cells and transgenic mice. ER intrabody knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In contrast to nucleotide sequence-based methods, the intrabody-mediated knockdown actsonly on the posttranslational level. In this review, the ER intrabody technology has been compared with the RNAi technique on the molecular level. The generation of intrabodies and RNAi has also been discussed. Specificity and off-target effects (OTE) of these molecules as well as the therapeutic potential of ER intrabodies and RNAi have been compared.
    • Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.

      Lambertz, Ruth L O; Pippel, Jan; Gerhauser, Ingo; Kollmus, Heike; Anhlan, Darisuren; Hrincius, Eike R; Krausze, Joern; Kühn, Nora; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-01)
      The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2/ knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2/ as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2/ knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2/ knockout mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.
    • Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

      Rodriguez Buitrago, Jhon Alexander; Klünemann, Thomas; Blankenfeldt, Wulf; Schallmey, Anett; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley & Sons, 2020-07-28)
      he ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.
    • Flexible Fragment Growing Boosts Potency of Quorum Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

      Zender, Michael; Witzgall, Florian; Kiefer, Alexander Felix; Kirsch, Benjamin; Maurer, Christine K; Kany, Andreas M; Xu, Ningna; Schmelz, Stefan; Börger, Carsten; Blankenfeldt, Wulf; et al. (Wiley-VCH, 2019-11-11)
      Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-amino pyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa . We pursue an innovative treatment strategy by interfering with the Pseudomonas Quinolone Signal (PQS) Quorum Sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.
    • Gastrointestinal stress as innate defence against microbial attack.

      Panwar, H; Rokana, N; Aparna, S V; Kaur, J; Singh, A; Singh, J; Singh, K S; Chaudhary, V; Puniya, A K; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-08-31)
      A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.
    • High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

      Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas (2013-06-26)
      Abstract Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
    • The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis , is involved in complement evasion.

      Seele, Jana; Beineke, Andreas; Hillermann, Lena-Maria; Jaschok-Kentner, Beate; von Pawel-Rammingen, Ulrich; Valentin-Weigand, Peter; Baums, Christoph Georg; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015)
      Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide Ssuis . The objective of this study was to characterize the function of Ide Ssuis in the host-pathogen interaction. Edman-sequencing revealed that Ide Ssuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide Ssuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide Ssuis . Furthermore, expression of Ide Ssuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide Ssuis . Bactericidal assays confirmed a positive effect of Ide Ssuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide Ssuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
    • Immunotherapy with antibodies: Tumor development, immune defense and therapeutic antibodies

      Böldicke, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Association for Cancer Research (AACR), 2021-01-01)
      Tumor development is based on mutations of genes involved in cell growth (e.g. transcription factors, growth receptors or intracellular signal molecules) or in suppressor genes (e.g. p53). During tumor growth cell clones are selected, which contain driver genes, leading to uncontrolled growth of these cell clones. During all phases of tumor development (immunosurveillance, equilibrium phase, escape of the tumor from the immune system) the interaction between the immune system and the tumor cells and the development of a chronic inflammation in the tumor microenvironment play a crucial role. The aim of cancer immunotherapy is to activate the immune system. A promising immunotherapy is based on antibodies that activate immune cells, inhibit tumor growth or lead to destruction of tumor cells. Applied are recombinant IgG antibodies or genetically engineered antibody fragments against tumor-associated antigens (TAA’s). They are applied singularly or in combination with chemo- or radiotherapy. Promising are checkpoint antibodies, which abrogate blocking of cytotoxic CD8+ T cells and CD4+ T cells by tumor cells and/or dendritic cells. Other successfully applied antibodies are bispecific antibodies (recognize T‑cell and tumor cell), chimeric antigen receptors (CARs) for T cell therapy, immunocytokines (cytokines fused to antibodies) and immunotoxins (toxins fused to antibodies). In addition intracellular antibodies successfully tested in xenograft tumor mouse models have promising therapeutic potential.
    • Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.

      Hercher, Thomas W; Krausze, Joern; Hoffmeister, Sven; Zwerschke, Dagmar; Lindel, Thomas; Blankenfeldt, Wulf; Mendel, Ralf R; Kruse, Tobias; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press, 2020-01-31)
      Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.
    • Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.

      Somplatzki, Stefan; Mühlenhoff, Martina; Kröger, Andrea; Gerardy-Schahn, Rita; Böldicke, Thomas; Helmholtz Centr for infection research (2017-05-12)
      Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo.
    • Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

      Muthukumar, Yazh; Münkemer, Johanna; Mathieu, Daniel; Richter, Christian; Schwalbe, Harald; Steinmetz, Heinrich; Kessler, Wolfgang; Reichelt, Joachim; Beutling, Ulrike; Frank, Ronald; et al. (PLOS, 2018-01-01)
      The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.