Recent Submissions

  • The zlog value as a basis for the standardization of laboratory results

    Hoffmann, Georg; Klawonn, Frank; Lichtinghagen, Ralf; Orth, Matthias; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-26)
    Abstract Background: With regard to the German E-Health Law of 2016, the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) has been invited to develop a standard procedure for the storage and transmission of laboratory results. We suggest the commonly used z-transformation. Methods: This method evaluates by how many standard deviations (SDs) a given result deviates from the mean of the respective reference population. We confirm with real data that laboratory results of healthy individuals can be adjusted to a normal distribution by logarithmic transformation. Results: Thus, knowing the lower and upper reference limits LL and UL, one can transform any result x into a zlog value using the following equation: $\eqalign{ {\rm{zlog}} = & {\rm{(log(x)}}-{\rm{(log(LL)}} + {\rm{log(UL))/2)\cdot3}}{\rm{.92/(log(UL)}} \cr -{\bf{ }}{\rm{log(LL))}} \cr} $ The result can easily be interpreted, as its reference interval (RI) is –1.96 to +1.96 by default, and very low or high results yield zlog values around –5 and +5, respectively. For intuitive data presentation, the zlog values may be transformed into a continuous color scale, e.g. from blue via white to orange. Using the inverse function, any zlog value can then be translated into the theoretical result of an analytical method with another RI: (1) $${\rm{x}} = {\rm{L}}{{\rm{L}}^{0.5 - {\rm{zlog}}/3.92}} \cdot {\rm{U}}{{\rm{L}}^{0.5 + {\rm{zlog}}/3.92}}$$ Conclusions: Our standardization proposal can easily be put into practice and may effectively contribute to data quality and patient safety in the frame of the German E-health law. We suggest for the future that laboratories should provide the zlog value in addition to the original result, and that the data transmission protocols (e.g. HL7, LDT) should contain a special field for this additional value.
  • 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.
  • 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; Brune, Wolfram; Jänsch, Lothar; Whitehouse, Adrian; Brinkmann, Melanie M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (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.
  • Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.

    Füller, Janis J; Röpke, René; Krausze, Joern; Rennhack, Kim E; Daniel, Nils P; Blankenfeldt, Wulf; Schulz, Stefan; Jahn, Dieter; Moser, Jürgen; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.
  • Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins.

    Romero Pastrana, Francisco; Neef, Jolanda; Koedijk, Dennis G A M; de Graaf, Douwe; Duipmans, José; Jonkman, Marcel F; Engelmann, Susanne; van Dijl, Jan Maarten; Buist, Girbe; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02-19)
    Human antibody responses to pathogens, like Staphylococcus aureus, are important indicators for in vivo expression and immunogenicity of particular bacterial components. Accordingly, comparing the antibody responses to S. aureus components may serve to predict their potential applicability as antigens for vaccination. The present study was aimed at assessing immunoglobulin G (IgG) responses elicited by non-covalently cell surface-bound proteins of S. aureus, which thus far received relatively little attention. To this end, we applied plasma samples from patients with the genetic blistering disease epidermolysis bullosa (EB) and healthy S. aureus carriers. Of note, wounds of EB patients are highly colonized with S. aureus and accordingly these patients are more seriously exposed to staphylococcal antigens than healthy individuals. Ten non-covalently cell surface-bound proteins of S. aureus, namely Atl, Eap, Efb, EMP, IsaA, LukG, LukH, SA0710, Sle1 and SsaA2, were selected by bioinformatics and biochemical approaches. These antigens were recombinantly expressed, purified and tested for specific IgG responses using human plasma. We show that high exposure of EB patients to S. aureus is mirrored by elevated IgG levels against all tested non-covalently cell wall-bound staphylococcal antigens. This implies that these S. aureus cell surface proteins are prime targets for the human immune system.
  • Sweep Dynamics (SD) plots: Computational identification of selective sweeps to monitor the adaptation of influenza A viruses.

    Klingen, Thorsten R; Reimering, Susanne; Loers, Jens; Mooren, Kyra; Klawonn, Frank; Krey, Thomas; Gabriel, Gülsah; McHardy, Alice Carolyn; BRICS, Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106 Braunschweig, Germany. (2018-01-10)
    Monitoring changes in influenza A virus genomes is crucial to understand its rapid evolution and adaptation to changing conditions e.g. establishment within novel host species. Selective sweeps represent a rapid mode of adaptation and are typically observed in human influenza A viruses. We describe Sweep Dynamics (SD) plots, a computational method combining phylogenetic algorithms with statistical techniques to characterize the molecular adaptation of rapidly evolving viruses from longitudinal sequence data. SD plots facilitate the identification of selective sweeps, the time periods in which these occurred and associated changes providing a selective advantage to the virus. We studied the past genome-wide adaptation of the 2009 pandemic H1N1 influenza A (pH1N1) and seasonal H3N2 influenza A (sH3N2) viruses. The pH1N1 influenza virus showed simultaneous amino acid changes in various proteins, particularly in seasons of high pH1N1 activity. Partially, these changes resulted in functional alterations facilitating sustained human-to-human transmission. In the evolution of sH3N2 influenza viruses, we detected changes characterizing vaccine strains, which were occasionally revealed in selective sweeps one season prior to the WHO recommendation. Taken together, SD plots allow monitoring and characterizing the adaptive evolution of influenza A viruses by identifying selective sweeps and their associated signatures.
  • Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

    Ramos-Espiritu, Lavoisier; Kleinboelting, Silke; Navarrete, Felipe A; Alvau, Antonio; Visconti, Pablo E; Valsecchi, Federica; Starkov, Anatoly; Manfredi, Giovanni; Buck, Hannes; Adura, Carolina; Zippin, Jonathan H; van den Heuvel, Joop; Glickman, J Fraser; Steegborn, Clemens; Levin, Lonny R; Buck, Jochen; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.
  • Staphylococcal serine protease-like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus.

    Stentzel, Sebastian; Teufelberger, Andrea; Nordengrün, Maria; Kolata, Julia; Schmidt, Frank; van Crombruggen, Koen; Michalik, Stephan; Kumpfmüller, Jana; Tischer, Sebastian; Schweder, Thomas; Hecker, Michael; Engelmann, Susanne; Völker, Uwe; Krysko, Olga; Bachert, Claus; Bröker, Barbara M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02)
    A substantial subgroup of asthmatic patients have "nonallergic" or idiopathic asthma, which often takes a severe course and is difficult to treat. The cause might be allergic reactions to the gram-positive pathogen Staphylococcus aureus, a frequent colonizer of the upper airways. However, the driving allergens of S aureus have remained elusive.
  • Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-05)
    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.
  • Optimization of protein samples for NMR using thermal shift assays.

    Kozak, Sandra; Lercher, Lukas; Karanth, Megha N; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.
  • Effects of pathogen dependency in a multi-pathogen infectious disease system including population level heterogeneity - a simulation study.

    Bakuli, Abhishek; Klawonn, Frank; Karch, André; Mikolajczyk, Rafael T; Helmholtz-Zentrum für Infektionsforschung, GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-12-13)
    Increased computational resources have made individual based models popular for modelling epidemics. They have the advantage of incorporating heterogeneous features, including realistic population structures (like e.g. households). Existing stochastic simulation studies of epidemics, however, have been developed mainly for incorporating single pathogen scenarios although the effect of different pathogens might directly or indirectly (e.g. via contact reductions) effect the spread of each pathogen. The goal of this work was to simulate a stochastic agent based system incorporating the effect of multiple pathogens, accounting for the household based transmission process and the dependency among pathogens.
  • Structural characterization of the Asf1-Rtt109 interaction and its role in histone acetylation.

    Lercher, Lukas; Danilenko, Nataliya; Kirkpatrick, John; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-12-29)
    Acetylation of histone H3 at lysine-56 by the histone acetyltransferase Rtt109 in lower eukaryotes is important for maintaining genomic integrity and is required for C. albicans pathogenicity. Rtt109 is activated by association with two different histone chaperones, Vps75 and Asf1, through an unknown mechanism. Here, we reveal that the Rtt109 C-terminus interacts directly with Asf1 and elucidate the structural basis of this interaction. In addition, we find that the H3 N-terminus can interact via the same interface on Asf1, leading to a competition between the two interaction partners. This, together with the recruitment and position of the substrate, provides an explanation of the role of the Rtt109 C-terminus in Asf1-dependent Rtt109 activation.
  • Effects of drift and noise on the optimal sliding window size for data stream regression models

    Tschumitschew, Katharina; Klawonn, Frank; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124Braunschweig, Germany. (2016-05-27)
  • Determining the bacterial cell biology of Planctomycetes.

    Boedeker, Christian; Schüler, Margarete; Reintjes, Greta; Jeske, Olga; van Teeseling, Muriel C F; Jogler, Mareike; Rast, Patrick; Borchert, Daniela; Devos, Damien P; Kucklick, Martin; Schaffer, Miroslava; Kolter, Roberto; van Niftrik, Laura; Engelmann, Susanne; Amann, Rudolf; Rohde, Manfred; Engelhardt, Harald; Jogler, Christian; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-04-10)
    Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.
  • 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)
  • Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.

    Lukat, Peer; Katsuyama, Yohei; Wenzel, Silke; Binz, Tina; König, Claudia; Blankenfeldt, Wulf; Brönstrup, Mark; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-11-01)
    Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
  • Der zlog-Wert als Basis für die Standardisierung von Laborwerten

    Hoffmann, Georg; Klawonn, Frank; Lichtinghagen, Ralf; Orth, Matthias; Helmholtz-Zentrum für Infektionsforshung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-01)
    Zusammenfassung Hintergrund Im Zuge des deutschen E-Health-Gesetzes von 2016 wurde die DGKL aufgefordert, Vorschläge für die standardisierte Speicherung und Übermittlung von Labordaten zu erarbeiten. Wir schlagen dafür die in der Statistik weit verbreitete z-Transformation vor. Methoden Man erhält mit diesem Verfahren einen Relativwert, der angibt, um wie viele Standardabweichungen ein Messwert vom Mittelwert des Referenzkollektivs abweicht. Anhand realer Daten belegen wir die Annahme, dass die Werte gesunder Referenzpersonen durch logarithmische Transformation einer Normalverteilung angenähert werden können. Ergebnisse Kennt man somit die Unter- und Obergrenze UG und OG des Referenzintervalls, so kann man jedes Laborergebnis mit folgender Gleichung transformieren: Der zlog-Wert ist leicht interpretierbar: Sein Referenzintervall liegt methodenunabhängig stets zwischen –1,96 und +1,96; stark erniedrigte oder erhöhte Laborergebnisse führen zu zlog-Werten um –5 bzw. +5. Für eine intuitive Befunddarstellung kann man zlog-Werte auch in eine kontinuierliche Farbskala, z. B. von Blau über Weiß bis Orange umrechnen. Mithilfe der Umkehrfunktion lässt sich aus dem zlog-Wert auch das theoretische Resultat einer Messmethode mit einem anderen Referenzintervall berechnen: Schlussfolgerung Unser Standardisierungsvorschlag ist ein leicht realisierbarer und effektiver Beitrag zur Verbesserung der Datenqualität und Patientensicherheit im Rahmen des E-Health-Gesetzes. Es wird gefordert, dass alle Labore künftig zusätzlich zum Originalwert den zlog-Wert zur Verfügung stellen und dass in die Protokolle für die elektronische Labordatenübertragung (HL7, LDT) ein eigenes Feld für diesen zusätzlichen Wert eingefügt wird.
  • Identification, biochemical characterization and crystallization of the central region of human ATG16L1.

    Archna, Archna; Scrima, Andrea; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-10-01)
    ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein-protein interactions essential for autophagosome formation. The ATG12~ATG5-ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12~ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L111-307 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5-ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5-ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72-307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L172-307 subfragment are reported.
  • RNA Interference

    Backhaus, Oliver; Böldicke, Thomas; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-10-25)
  • Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.

    DiCara, Danielle M; Chirgadze, Dimitri Y; Pope, Anthony R; Karatt-Vellatt, Aneesh; Winter, Anja; Slavny, Peter; van den Heuvel, Joop; Parthiban, Kothai; Holland, Jane; Packman, Len C; Mavria, Georgia; Hoffmann, Jens; Birchmeier, Walter; Gherardi, Ermanno; McCafferty, John; Helmholtz-Zentrum für Infektionsforschung GmbH. Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-21)
    The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.

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