• Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1.

      Schenke, Maren; Prause, Hélène-Christine; Bergforth, Wiebke; Przykopanski, Adina; Rummel, Andreas; Klawonn, Frank; Seeger, Bettina; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-08-22)
      The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.
    • Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis.

      Lehrke, Markus; Rump, Steffen; Heidenreich, Torsten; Wissing, Josef; Mendel, Ralf R; Bittner, Florian; Department of Plant Biology, Braunschweig University of Technology, Humboldtstrasse 1, 38023 Braunschweig, Germany. (2012-02-01)
      The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys⁴³⁰, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys⁴³⁰. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys²⁰⁶, was identified. Furthermore, the active-site Cys⁴³⁰ was found to be located on top of a loop structure, formed by the two flanking residues Cys⁴²⁸ and Cys⁴³⁵, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys⁴²⁸ and Cys⁴³⁵ are within disulfide bond distance and that a persulfide transfer from Cys⁴³⁰ to Cys²⁰⁶ is indeed possible.
    • Improving the Decision Support in Diagnostic Systems using Classifier Probability Calibration

      Kortum, Xiaowei; Grigull, Lorenz; Lechner, Werner; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2018-11-09)
      In modern medical diagnoses, classifying a patient’s disease is often realized with the help of a system-aided symptoms interpreter. Most of these systems rely on supervised learning algorithms, which can statistically extend the doctor’s logic capabilities for interpreting and examining symptoms, thus supporting the doctor to find the correct diagnosis. Besides, these algorithms compute classifier scores and class labels that are used to statistically characterize the system’s confidence level on a patient’s type of disease. Unfortunately, most classifier scores are based on an arbitrary scale but not uniformed, thus the interpretations often lack of clinical significance and evaluation criterion. Especially combining multiple classifier scores within a diagnostic system, it is essential to apply a calibration process to make the different scores comparable. As a frequently used calibration technique, we adapted isotonic regression for our medical diagnostic support system, to provide a flexible and effective scaling process that consequently calibrates the arbitrary scales of classifiers’ scores. In a comparative evaluation, we show that our disease diagnostic system with isotonic regression can actively improve the diagnostic result based on an ensemble of classifiers, also effectively remove outliers from data, thus optimize the decision support system to obtain better diagnostic results.
    • In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

      Kumar, Suresh; Koenig, Johannes; Schneider, Andreas; Wermeling, Fredrik; Boddul, Sanjaykumar; Theobald, Sebastian J; Vollmer, Miriam; Kloos, Doreen; Lachmann, Nico; Klawonn, Frank; et al. (MDPI, 2021-08-05)
      Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.
    • Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

      Baumgärtner, Maja; Kärst, Uwe; Gerstel, Birgit; Loessner, Martin; Wehland, Jürgen; Jänsch, Lothar; Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), D-38124 Braunschweig, Germany. (2007-01)
      Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.
    • Induced B Cell Development in Adult Mice.

      Brennecke, Anne-Margarete; Düber, Sandra; Roy, Bishnudeo; Thomsen, Irene; Garbe, Annette I; Klawonn, Frank; Pabst, Oliver; Kretschmer, Karsten; Weiss, Siegfried; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.
    • Infection- and procedure-dependent effects on pulmonary gene expression in the early phase of influenza A virus infection in mice

      Preusse, Matthias; Tantawy, Mohamed A; Klawonn, Frank; Schughart, Klaus; Pessler, Frank (2013-12-17)
      Abstract Background Investigating the host response in the early stage of influenza A virus (IAV) infection is of considerable interest. However, it is conceivable that effects due to the anesthesia and/or intranasal infection procedure might introduce artifacts. We therefore aimed to evaluate the effects of anesthesia and/or intranasal infection on transcription of selected pulmonary mRNAs in two inbred mouse strains with differential susceptibility to IAV infection. Results DBA/2J and C57BL/6J mice were evaluated in a time course experiment in which lung tissue was sampled after 6, 12, 18, 24, 48 and 120 h. After anesthesia with ketamine and xylazine, a suspension of mouse-adapted IAV strain PR8_Mun in 20 μl sterile buffer, or 20 μl sterile buffer only, was instilled intranasally. The mice receiving anesthesia and PBS only were designated the “mock treatment” group. Pulmonary expression of 10 host mRNAs (Fos, Retnla, Irg1, Il6, Il1b, Cxcl10, Stat1, Ifng, Ifnl2, and Mx1) and viral hemagglutinin (HA) mRNA were determined at the designated time points. As expected, weight loss and viral replication were greater in the DBA/2J strain (which is more susceptible to IAV infection). Four mRNAs (Retnla, Irg1, Il6, and Cxcl10) were procedure-dependently regulated in DBA/2J mice between 6 and 24 h, and two (Retnla and Il6) in C57BL/6J mice, although to a lesser extent. All 10 mRNAs rose after infection, but one (Fos) only in DBA/2J mice. These infection-dependent effects could be separated from procedure-dependent effects beginning around 12 h in DBA/2J and 18 h in C57BL/6J mice. The interferon-related mRNAs Stat1, Ifng, Infl2, and Mx1 were unaffected by mock treatment in either mouse strain. Mx1 and Infl2 correlated best with HA mRNA expression (r = 0.97 and 0.93, respectively, in DBA/2J). Conclusions These results demonstrate effects of the anesthesia and/or intranasal infection procedure on pulmonary gene expression, which are detectable between approximately 6 and 24 h post procedure and vary in intensity and temporal evolution depending on the mouse strain used. Mock infection controls should be included in all studies on pulmonary gene expression in the early phase of infection with IAV and, likely, other respiratory pathogens.
    • The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein.

      Bussey, Kendra A; Lau, Ulrike; Schumann, Sophie; Gallo, Antonio; Osbelt, Lisa; Stempel, Markus; Arnold, Christine; Wissing, Josef; Gad, Hans Henrik; Hartmann, Rune; et al. (2018-03)
      Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.
    • The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding.

      Solbak, Sara M; Reksten, Tove R; Wray, Victor; Bruns, Karsten; Horvli, Ole; Raae, Arnt J; Henklein, Petra; Henklein, Peter; Röder, Rene; Mitzner, David; et al. (2010)
      Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.
    • Iron affinity gel and gallium immobilized metal affinity chromatographic technique for phosphopeptide enrichment: a comparative study

      Biswas, Sagarika; Sarkar, Ashish; Misra, Richa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany.; Department of Genomics and Molecular Medicine, CSIR – Institute of Genomics and Integrative Biology, Delhi, India; Department of Genomics and Molecular Medicine, CSIR – Institute of Genomics and Integrative Biology, Delhi, India; Department of Genomics and Molecular Medicine, CSIR – Institute of Genomics and Integrative Biology, Delhi, India (2017-02-28)
    • Künstliche Intelligenz zur diagnostischen Unterstützung ausgewählter seltener lysosomaler Speichererkrankungen: Ergebnisse einer Pilotstudie.

      Sieg, Anna-Lena; Anibh, Martin; Muschol, Nicole Maria; Köhn, Anja; Lampe, Christina; Kortum, Xiauwei; Mehmecke, Sandra; Blöß, Susanne; Lechner, Werner; Klawonn, Frank; et al. (Thieme, 2019-02-10)
      Hintergrund: Die Diagnosestellung einer seltenen Stoffwechselerkrankung stellt eine Herausforderung für Familien und betreuende Ärzte dar. Um den Weg zur Diagnose zu unterstützen, wurde ein diagnostisches Werkzeug entwickelt, welches die Erfahrungen Betroffener nutzt.
    • LC/MS Based Monitoring of Endogenous Decay Markers for Quality Assessment of Serum Specimens

      Thumfart, Jörg Oliver; Abidi, Nada; Mindt, Sonani; Costani, Victor; Hofheinz, Ralf; Klawonn, Frank; Findeisen, Peter; 1Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany (2015-05-04)
      Preanalytical variations have major impact on most biological assays. Specifically MS-based multiparametric proteomics analyses of blood specimens are seriously affected by limited protein stability due to high intrinsic proteolytic activity of serum and plasma. However, the direct analysis of sample quality (DASQ) for serum specimens is not readily available. Here we propose the mass spectrometry based monitoring of peptide patterns that are ex vivo changing in a time dependent manner to alleviate these constrains.
    • Letter to the editor by Winter et al.: Reply

      Hoffmann, Georg E.; Klawonn, Frank; Orth, Matthias; Lichtinghagen, Ralf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (De Gruyter, 2018-04-03)
    • Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection.

      Worthmann, Hans; Tryc, Anita B; Dirks, Meike; Schuppner, Ramona; Brand, Korbinian; Klawonn, Frank; Lichtinghagen, Ralf; Weissenborn, Karin; Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623, Hannover, Germany. (2015)
      Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection.
    • Mass-spectrometric profiling of cerebrospinal fluid reveals metabolite biomarkers for CNS involvement in varicella zoster virus reactivation.

      Kuhn, Maike; Sühs, Kurt-Wolfram; Akmatov, Manas K; Klawonn, Frank; Wang, Junxi; Skripuletz, Thomas; Kaever, Volkhard; Stangel, Martin; Pessler, Frank; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-01-17)
      Varicella zoster virus (VZV) reactivation spans the spectrum from uncomplicated segmental herpes zoster to life-threatening disseminated CNS infection. Moreover, in the absence of a small animal model for this human pathogen, studies of pathogenesis at the organismal level depend on analysis of human biosamples. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuroinfection and neuroinflammation. We therefore applied a targeted metabolomics screen of CSF to three clinically distinct forms of VZV reactivation and infectious and non-infectious disease controls in order to identify biomarkers for CNS involvement in VZV reactivation.
    • Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.

      Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; et al. (2013)
      The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.
    • Methicillin-resistant Staphylococcus pseudintermedius synthesizes deoxyadenosine to cause persistent infection.

      Bünsow, Dorothea; Tantawy, Eshraq; Ostermeier, Tjorven; Bähre, Heike; Garbe, Annette; Larsen, Jesper; Winstel, Volker; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2021-03-29)
      Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging zoonotic pathogen of canine origin that causes an array of fatal diseases, including bacteremia and endocarditis. Despite large-scale genome sequencing projects have gained substantial insights into the genomic landscape of MRSP, current knowledge on virulence determinants that contribute to S. pseudintermedius pathogenesis during human or canine infection is very limited. Using a panel of genetically engineered MRSP variants and a mouse abscess model, we here identified the major secreted nuclease of S. pseudintermedius designated NucB and adenosine synthase A (AdsA) as two synergistically acting enzymes required for MRSP pathogenesis. Similar to Staphylococcus aureus, S. pseudintermedius requires nuclease secretion along with the activity of AdsA to degrade mammalian DNA for subsequent biosynthesis of cytotoxic deoxyadenosine. In this manner, S. pseudintermedius selectively kills macrophages during abscess formation thereby antagonizing crucial host immune cell responses. Ultimately, bioinformatics analyses revealed that NucB and AdsA are widespread in the global S. pseudintermedius population. Together, these data suggest that S. pseudintermedius deploys the canonical Nuc/AdsA pathway to persist during invasive disease and may aid in the development of new therapeutic strategies to combat infections caused by MRSP.
    • Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

      Wiesselmann, Milan; Hebecker, Stefanie; Borrero-de Acuña, José M; NIMTZ, MANFRED; Bollivar, David; Jänsch, Lothar; Moser, Jürgen; Jahn, Dieter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press, 2020-11-19)
      During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.
    • 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.
    • The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

      Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04-26)
      Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.