Recent Submissions

  • 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.
  • Common pre-diagnostic features in individuals with different rare diseases represent a key for diagnostic support with computerized pattern recognition?

    Grigull, Lorenz; Mehmecke, Sandra; Rother, Ann-Katrin; Blöß, Susanne; Klemann, Christian; Schumacher, Ulrike; Mücke, Urs; Kortum, Xiaowei; Lechner, Werner; Klawonn, Frank; et al. (Public Library of Science (PLoS), 2019-10-10)
    BACKGROUND: Rare diseases (RD) result in a wide variety of clinical presentations, and this creates a significant diagnostic challenge for health care professionals. We hypothesized that there exist a set of consistent and shared phenomena among all individuals affected by (different) RD during the time before diagnosis is established. OBJECTIVE: We aimed to identify commonalities between different RD and developed a machine learning diagnostic support tool for RD. METHODS: 20 interviews with affected individuals with different RD, focusing on the time period before their diagnosis, were performed and qualitatively analyzed. Out of these pre-diagnostic experiences, we distilled key phenomena and created a questionnaire which was then distributed among individuals with the established diagnosis of i.) RD, ii.) other common non-rare diseases (NRO) iii.) common chronic diseases (CD), iv.), or psychosomatic/somatoform disorders (PSY). Finally, four combined single machine learning methods and a fusion algorithm were used to distinguish the different answer patterns of the questionnaires. RESULTS: The questionnaire contained 53 questions. A total sum of 1763 questionnaires (758 RD, 149 CD, 48 PSY, 200 NRO, 34 healthy individuals and 574 not evaluable questionnaires) were collected. Based on 3 independent data sets the 10-fold stratified cross-validation method for the answer-pattern recognition resulted in sensitivity values of 88.9% to detect the answer pattern of a RD, 86.6% for NRO, 87.7% for CD and 84.2% for PSY. CONCLUSION: Despite the great diversity in presentation and pathogenesis of each RD, patients with RD share surprisingly similar pre-diagnosis experiences. Our questionnaire and data-mining based approach successfully detected unique patterns in groups of individuals affected by a broad range of different rare diseases. Therefore, these results indicate distinct patterns that may be used for diagnostic support in RD.
  • Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.

    Meyerholz, M M; Rohmeier, L; Eickhoff, T; Hülsebusch, A; Jander, S; Linden, M; Macias, L; Koy, M; Heimes, A; Gorríz-Martín, L; et al. (Elsevier, 2019-11-01)
    The susceptibility of animals to periparturient diseases has a great effect on the economic efficiency of dairy industries, on the frequency of antibiotic treatment, and on animal welfare. The use of selection for breeding cows with reduced susceptibility to diseases offers a sustainable tool to improve dairy cattle farming. Several studies have focused on the association of distinct bovine chromosome 18 genotypes or haplotypes with performance traits. The aim of this study was to test whether selection of Holstein Friesian heifers via SNP genotyping for alternative paternal chromosome 18 haplotypes associated with favorable (Q) or unfavorable (q) somatic cell scores influences postpartum reproductive and metabolic diseases. Thirty-six heifers (18 Q and 18 q) were monitored from 3 wk before calving until necropsy on d 39 (± 4 d) after calving. Health status and rectal temperature were measured daily, and body condition score and body weight were assessed once per week. Blood samples were drawn twice weekly, and levels of insulin, nonesterified fatty acids, insulin-like growth factor-I, growth hormone, and β-hydroxybutyrate were measured. Comparisons between the groups were performed using Fisher's exact test, chi-squared test, and the GLIMMIX procedure in SAS. Results showed that Q-heifers had reduced incidence of metritis compared with q-heifers and were less likely to develop fever. Serum concentrations of β-hydroxybutyrate were lower and insulin-like growth factor-I plasma concentrations were higher in Q- compared with q-heifers. However, the body condition score and withers height were comparable between haplotypes, but weight loss tended to be lower in Q-heifers compared with q-heifers. No differences between the groups were detected concerning retained fetal membranes, uterine involution, or onset of cyclicity. In conclusion, selection of chromosome 18 haplotypes associated with a reduced somatic cell score resulted in a decreased incidence of postpartum reproductive and metabolic diseases in this study. The presented data add to the existing knowledge aimed at avoiding negative consequences of genetic selection strategies in dairy cattle farming. The underlying causal mechanisms modulated by haplotypes in the targeted genomic region and immune competence necessitate further investigation.
  • Crystal structure of -aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis.

    Chen, Fangfang; Lukat, Peer; Iqbal, Azeem Ahmed; Saile, Kyrill; Kaever, Volkhard; van den Heuvel, Joop; Blankenfeldt, Wulf; Büssow, Konrad; Pessler, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (National Academy of Sciences, 2019-09-23)
    cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.
  • The N‐terminal peptide of the transglutaminase‐activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross‐linking sites

    Juettner, Norbert E.; Schmelz, Stefan; Anderl, Anita; Colin, Felix; Classen, Moritz; Pfeifer, Felicitas; Scrima, Andrea; Fuchsbauer, Hans‐Lothar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2019-08-29)
    Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (
  • Histone chaperone exploits intrinsic disorder to switch acetylation specificity

    Danilenko, Nataliya; Lercher, Lukas; Kirkpatrick, John; Gabel, Frank; Codutti, Luca; Carlomagno, Teresa; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany (Springer Science and Business Media LLC, 2019-08-06)
    Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.
  • Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    Böldicke, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2017-01-01)
    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant‐ and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell‐penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell‐specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb‐DNA or –protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. Keywords: intrabodies, single domain antibodies, scFv fragment, cytosolic/nuclear intrabodies, camelid VHHs, shark vNARs, human VH, human VL
  • Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

    Heimes, A; Brodhagen, J; Weikard, R; Hammon, H M; Meyerholz, M M; Petzl, W; Zerbe, H; Engelmann, S; Schmicke, M; Hoedemaker, M; et al. (BioMedCentral, 2019-07-11)
    BACKGROUND: A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. RESULTS: In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (< 10,000 cells / ml) compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. This might result in impaired mammary gland sentinel function towards invading pathogens. Furthermore, across the course of the first lactation, there was indication that q half-sib heifers showed higher somatic cell counts, a surrogate trait for udder health, in whole milkings compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. Moreover, heifers with the haplotype Q had a higher feed intake and higher milk yield compared to those with the q haplotype. Results of this study indicate that differences in milk production and calculated energy balance per se are not the main drivers of the genetically determined differences between the BTA18 Q and q groups of heifers. CONCLUSIONS: The paternally inherited haplotype from a targeted BTA18 genomic region affect somatic cell count in udder quarters during the early postpartum period and might also contribute to further aspects of animal's health and performance traits due to indirect effects on feed intake and metabolism.
  • Two FtsH Proteases Contribute to Fitness and Adaptation of Clone C Strains.

    Kamal, Shady Mansour; Rybtke, Morten Levin; NIMTZ, MANFRED; Sperlein, Stefanie; Giske, Christian; Trček, Janja; Deschamps, Julien; Briandet, Romain; Dini, Luciana; Jänsch, Lothar; et al. (Frontiers, 2019-01-01)
    Pseudomonas aeruginosa is an environmental bacterium and a nosocomial pathogen with clone C one of the most prevalent clonal groups. The P. aeruginosa clone C specific genomic island PACGI-1 harbors a xenolog of ftsH encoding a functionally diverse membrane-spanning ATP-dependent metalloprotease on the core genome. In the aquatic isolate P. aeruginosa SG17M, the core genome copy ftsH1 significantly affects growth and dominantly mediates a broad range of phenotypes, such as secretion of secondary metabolites, swimming and twitching motility and resistance to aminoglycosides, while the PACGI-1 xenolog ftsH2 backs up the phenotypes in the ftsH1 mutant background. The two proteins, with conserved motifs for disaggregase and protease activity present in FtsH1 and FtsH2, have the ability to form homo- and hetero-oligomers with ftsH2 distinctively expressed in the late stationary phase of growth. However, mainly FtsH1 degrades a major substrate, the heat shock transcription factor RpoH. Pull-down experiments with substrate trap-variants inactive in proteolytic activity indicate both FtsH1 and FtsH2 to interact with the inhibitory protein HflC, while the phenazine biosynthesis protein PhzC was identified as a substrate of FtsH1. In summary, as an exception in P. aeruginosa, clone C harbors two copies of the ftsH metallo-protease, which cumulatively are required for the expression of a diversity of phenotypes.
  • 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 Structure of Dihydro-Heme d Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis.

    Klünemann, Thomas; Preuß, Arne; Adamczack, Julia; Rosa, Luis F M; Harnisch, Falk; Layer, Gunhild; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-06-04)
    Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d1-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.
  • Identifying parameters to improve the reproducibility of transient gene expression in High Five cells.

    Bleckmann, Maren; Schürig, Margitta; Endres, Michelle; Samuels, Anke; Gebauer, Daniela; Konisch, Nadine; van den Heuvel, Joop; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-01-01)
    Virus-free, transient gene expression (TGE) in High Five cells was recently presented as an efficient protein production method. However, published TGE protocols have not been standardized to a general protocol. Therefore, reproducibility and implementation of the method in other labs remains difficult. The aim of this study is to analyse the parameters determining the reproducibility of the TGE in insect cells. Here, we identified that using linear 40 kDa PEI instead of 25 kDa PEI was one of the most important aspects to improve TGE. Furthermore, DNA amount, DNA:PEI ratio, growth phase of the cells before transfection, passage number, the origin of the High-Five cell isolates and the type of cultivation medium were considered. Interestingly, a correlation of the passage number to the DNA content of single cells (ploidy) and to the transfection efficacy could be shown. The optimal conditions for critical parameters were used to establish a robust TGE method. Finally, we compared the achieved product yields in High Five cells using our improved TGE method with both the baculoviral expression system and TGE in the mammalian HEK293-6E cell line. In conclusion, the presented robust TGE protocol in High Five cells is easy to establish and produces ample amounts of high-quality recombinant protein, bridging the gap in expression level of this method to the well-established mammalian TGE in HEK293 cells as well as to the baculoviral expression vector system (BEVS).
  • Still Something to Discover: Novel Insights into Phage Diversity and Taxonomy.

    Korf, Imke H E; Meier-Kolthoff, Jan P; Adriaenssens, Evelien M; Kropinski, Andrew M; Nimtz, Manfred; Rohde, Manfred; van Raaij, Mark J; Wittmann, Johannes; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-05-17)
    The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.
  • Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

    Brodhagen, Johanna; Weikard, Rosemarie; Thom, Ulrike; Heimes, Annika; Günther, Juliane; Hadlich, Frieder; Zerbe, Holm; Petzl, Wolfgang; Meyerholz, Marie M; Hoedemaker, Martina; et al. (BioMed Central (BMC), 2019-05-22)
    Background: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. Results: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challengeassociated biological signaling pathway patterns. Conclusions: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.
  • Pleiotropic Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

    Ünal, Can Murat; Karagöz, Mustafa Safa; Berges, Mareike; Priebe, Christina; Borrero de Acuña, José Manuel; Wissing, Josef; Jänsch, Lothar; Jahn, Dieter; Steinert, Michael; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
    The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.
  • Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

    Solarczek, Jennifer; Klünemann, Thomas; Brandt, Felix; Schrepfer, Patrick; Wolter, Mario; Jacob, Christoph R; Blankenfeldt, Wulf; Schallmey, Anett; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-03-25)
    HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.
  • 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.
  • Modulation of TAP-dependent antigen compartmentalization during human monocyte-to-DC differentiation.

    Döring, Marius; Blees, Hanna; Koller, Nicole; Tischer-Zimmermann, Sabine; Müsken, Mathias; Henrich, Frederik; Becker, Jennifer; Grabski, Elena; Wang, Junxi; Janssen, Hans; et al. (American Society of Hematology, 2019-03-26)
    Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.
  • Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

    Gromova, Kira V; Muhia, Mary; Rothammer, Nicola; Gee, Christine E; Thies, Edda; Schaefer, Irina; Kress, Sabrina; Kilimann, Manfred W; Shevchuk, Olga; Oertner, Thomas G; et al. (Elsevier, 2018-05-29)
    Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.
  • Design and characterization of dietary assessment in the German National Cohort.

    Knüppel, Sven; Clemens, Matthias; Conrad, Johanna; Gastell, Sylvia; Michels, Karin B; Leitzmann, Michael; Krist, Lilian; Pischon, Tobias; Krause, Gerard; Ahrens, Wolfgang; et al. (Springer Nature, 2019-01-15)
    BACKGROUND/OBJECTIVES: The aim of the study was to describe a novel dietary assessment strategy based on two instruments complemented by information from an external population applied to estimate usual food intake in the large-scale multicenter German National Cohort (GNC). As proof of concept, we applied the assessment strategy to data from a pretest study (2012-2013) to assess the feasibility of the novel assessment strategy. SUBJECTS/METHODS: First, the consumption probability for each individual was modeled using three 24 h food lists (24h-FLs) and frequencies from one food frequency questionnaire (FFQ). Second, daily consumed food amounts were estimated from the representative German National Nutrition Survey II (NVS II) taking the characteristics of the participants into account. Usual food intake was estimated using the product of consumption probability and amounts. RESULTS: We estimated usual intake of 41 food groups in 318 men and 377 women. The participation proportion was 100, 84.4, and 68.5% for the first, second, and third 24h-FL, respectively. We observed no associations between the probability of participating and lifestyle factors. The estimated distributions of usual food intakes were plausible and total energy was estimated to be 2707 kcal/day for men and 2103 kcal/day for women. The estimated consumption frequencies did not differ substantially between men and women with only few exceptions. The differences in energy intake between men and women were mostly due to differences in estimated daily amounts. CONCLUSIONS: The combination of repeated 24h-FLs, a FFQ, and consumption-day amounts from a reference population represents a user-friendly dietary assessment approach having generated plausible, but not yet validated, food intake values in the pretest study

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