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Guo W, Apte SS, Dickinson MS, Kim SY, Kutsch M, Coers J. Human giant GTPase GVIN1 forms an antimicrobial coatomer around the intracellular bacterial pathogen Burkholderia thailandensis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.24.645074. [PMID: 40196472 PMCID: PMC11974893 DOI: 10.1101/2025.03.24.645074] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
Several human pathogens exploit the kinetic forces generated by polymerizing actin to power their intracellular motility. Human cell-autonomous immune responses activated by the cytokine interferon-gamma (IFNγ) interfere with such microbial actin-based motility, yet the underlying molecular mechanisms are poorly defined. Here, we identify the IFNγ-inducible human giant GTPases GVIN1 as a novel host defense protein that blocks the bacterial pathogen Burkholderia thailandensis from high-jacking the host's actin polymerization machinery. We found that GVIN1 proteins form a coatomer around cytosolic bacteria and prevent Burkholderia from establishing force-generating actin comet tails. Coatomers formed by a second IFNγ-inducible GTPase, human guanylate binding protein 1 (GBP1), constitute a GVIN1-independent but mechanistically related anti-motility pathway. We show that coating with either GVIN1 or GBP1 displaces the Burkholderia outer membrane protein BimA, an actin nucleator that is essential for actin tail formation. Both GVIN1 and GBP1 coatomers require additional IFNγ-inducible co-factors to disrupt the membrane localization of BimA, demonstrating the existence of two parallel-acting IFNγ-inducible defense modules that evolved to target a virulence trait critical for the pathogenesis of numerous bacterial infectious agents.
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Affiliation(s)
- Weilun Guo
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
| | - Shruti S Apte
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
| | - Mary S Dickinson
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
| | - So Young Kim
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
| | - Miriam Kutsch
- Institute of Molecular Pathogenicity, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany
| | - Jörn Coers
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
- Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA
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2
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Tessema MB, Feng S, Enosi Tuipulotu D, Farrukee R, Ngo C, Gago da Graça C, Yamomoto M, Utzschneider DT, Brooks AG, Londrigan SL, Man SM, Reading PC. Mouse guanylate-binding proteins of the chromosome 3 cluster do not mediate antiviral activity in vitro or in mouse models of infection. Commun Biol 2024; 7:1050. [PMID: 39183326 PMCID: PMC11345437 DOI: 10.1038/s42003-024-06748-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Accepted: 08/16/2024] [Indexed: 08/27/2024] Open
Abstract
Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies.
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Affiliation(s)
- Melkamu B Tessema
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
- WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Shouya Feng
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
- The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
| | - Daniel Enosi Tuipulotu
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
- School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, Australia
| | - Rubaiyea Farrukee
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Chinh Ngo
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Catarina Gago da Graça
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Masahiro Yamomoto
- Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daniel T Utzschneider
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Andrew G Brooks
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Sarah L Londrigan
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia
| | - Si Ming Man
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Patrick C Reading
- Department of Microbiology and Immunology, University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia.
- WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Victoria, 3000, Australia.
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3
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Gu T, Qu S, Zhang J, Ying Q, Zhang X, Lv Y, Liu R, Feng Y, Wang F, Wu X. Guanylate-binding protein 1 inhibits Hantaan virus infection by restricting virus entry. J Med Virol 2024; 96:e29730. [PMID: 38860570 DOI: 10.1002/jmv.29730] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 05/08/2024] [Accepted: 06/01/2024] [Indexed: 06/12/2024]
Abstract
Hantaan virus (HTNV) infection can cause hemorrhagic fever with renal syndrome (HFRS) in humans, and currently, there are no long-standing protective vaccines or specific antivirals available. Guanylate-binding protein 1 (GBP1) is an interferon-stimulated gene that defends against various pathogen infections. However, the function of GBP1 in HTNV infection remains unknown. Here, we describe how GBP1 prevents HTNV infection by obstructing virus entry. We found that HTNV infection induced GBP1 expression and that overexpression of GBP1 inhibited HTNV infection, while knockout of GBP1 had the opposite effect. Interestingly, GBP1 did not affect interferon (IFN) signaling during HTNV infection. Instead, GBP1 prevented HTNV from entering cells through clathrin-mediated endocytosis (CME). We also discovered that GBP1 specifically interacted with actin but not dynamin 2 (DNM2) and made it difficult for DNM2 to be recruited by actin, which may account for the suppression of CME during HTNV infection. These findings establish an antiviral role for GBP1 in inhibiting HTNV infection and help us better understand how GBP1 regulates HTNV entry and could potentially aid in developing treatments for this virus.
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Affiliation(s)
- Tianle Gu
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
- Department of Pathogen Biology, College of Basic Medical Science, Chongqing Medical University, Chongqing, China
| | - Sirui Qu
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
- College of Life Sciences, Yan'an University, Yan'an, China
| | - Junmei Zhang
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
- College of Life Sciences, Yan'an University, Yan'an, China
| | - Qikang Ying
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
| | - Xiaoxiao Zhang
- Department of Pulmonary and Critical Care Medicine, Xijing Hospital, Xi'an, China
| | - Yunhua Lv
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
| | - Rongrong Liu
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
| | - Yunan Feng
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
| | - Fang Wang
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
| | - Xingan Wu
- Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, China
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4
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Weismehl M, Chu X, Kutsch M, Lauterjung P, Herrmann C, Kudryashev M, Daumke O. Structural insights into the activation mechanism of antimicrobial GBP1. EMBO J 2024; 43:615-636. [PMID: 38267655 PMCID: PMC10897159 DOI: 10.1038/s44318-023-00023-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 12/11/2023] [Accepted: 12/14/2023] [Indexed: 01/26/2024] Open
Abstract
The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.
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Affiliation(s)
- Marius Weismehl
- Structural Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, 14195, Berlin, Germany
| | - Xiaofeng Chu
- In Situ Structural Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
| | - Miriam Kutsch
- Institute of Molecular Pathogenicity, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany
- Institute of Medical Microbiology and Hospital Hygiene, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany
- Institute of Biochemistry, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany
- Department of Molecular Genetics and Microbiology, Duke University, 27710, Durham, NC, USA
| | - Paul Lauterjung
- Faculty of Chemistry and Biochemistry, Physical Chemistry I, Ruhr-University Bochum, 44801, Bochum, Germany
- Institute of Molecular Physical Chemistry, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany
| | - Christian Herrmann
- Faculty of Chemistry and Biochemistry, Physical Chemistry I, Ruhr-University Bochum, 44801, Bochum, Germany
| | - Misha Kudryashev
- In Situ Structural Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
- Institute of Medical Physics and Biophysics, Charité-Universitätsmedizin Berlin, 10117, Berlin, Germany
| | - Oliver Daumke
- Structural Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, 14195, Berlin, Germany.
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5
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Schumann W, Loschwitz J, Reiners J, Degrandi D, Legewie L, Stühler K, Pfeffer K, Poschmann G, Smits SHJ, Strodel B. Integrative modeling of guanylate binding protein dimers. Protein Sci 2023; 32:e4818. [PMID: 37916607 PMCID: PMC10683561 DOI: 10.1002/pro.4818] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Revised: 09/27/2023] [Accepted: 09/30/2023] [Indexed: 11/03/2023]
Abstract
Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1's GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations. Our investigation begins by comparing the protein dynamics of hGBP1, mGBP2, and mGBP7. We observe that the M/E domain in all three proteins exhibits significant mobility and hinge motion, with mGBP7 displaying a slightly less pronounced motion but greater flexibility in its GTPase domain. These dynamic distinctions can be attributed to variations in the sequences of mGBP7 and hGBP1/mGBP2, resulting in different dimerization modes. Unlike hGBP1 and its close ortholog mGBP2, which exclusively dimerize through their GTPase domains, we find that mGBP7 exhibits three equally probable alternative dimer structures. The GTPase domain of mGBP7 is only partially involved in its dimerization, primarily due to an accumulation of negative charge, allowing mGBP7 to dimerize independently of GTP. Instead, mGBP7 exhibits a strong tendency to dimerize in an antiparallel arrangement across its stalks. The results of this work go beyond the sequence-structure-function relationship, as the sequence differences in mGBP7 and mGBP2/hGBP1 do not lead to different structures, but to different protein dynamics and dimerization. The distinct GBP dimer structures are expected to encode specific functions crucial for disrupting pathogen membranes.
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Affiliation(s)
- Wibke Schumann
- Institute of Theoretical and Computational ChemistryHeinrich Heine University DüsseldorfDüsseldorfGermany
- Institute of Biological Information Processing: Structural BiochemistryForschungszentrum JülichJülichGermany
| | - Jennifer Loschwitz
- Institute of Theoretical and Computational ChemistryHeinrich Heine University DüsseldorfDüsseldorfGermany
- Institute of Biological Information Processing: Structural BiochemistryForschungszentrum JülichJülichGermany
| | - Jens Reiners
- Center for Structural StudiesHeinrich Heine University DüsseldorfDüsseldorfGermany
| | - Daniel Degrandi
- Institute of Medical Microbiology and Hospital HygieneHeinrich Heine UniversityDüsseldorfGermany
| | - Larissa Legewie
- Institute of Medical Microbiology and Hospital HygieneHeinrich Heine UniversityDüsseldorfGermany
| | - Kai Stühler
- Institute of Molecular Medicine, Proteome ResearchMedical Faculty and University Hospital Düsseldorf, Heinrich Heine University DüsseldorfDüsseldorfGermany
- Molecular Proteomics Laboratory, Biomedical Research Centre (BMFZ)Heinrich Heine University DüsseldorfDüsseldorfGermany
| | - Klaus Pfeffer
- Institute of Medical Microbiology and Hospital HygieneHeinrich Heine UniversityDüsseldorfGermany
| | - Gereon Poschmann
- Institute of Molecular Medicine, Proteome ResearchMedical Faculty and University Hospital Düsseldorf, Heinrich Heine University DüsseldorfDüsseldorfGermany
| | - Sander H. J. Smits
- Center for Structural StudiesHeinrich Heine University DüsseldorfDüsseldorfGermany
- Institute for BiochemistryHeinrich Heine University DüsseldorfDüsseldorfGermany
| | - Birgit Strodel
- Institute of Theoretical and Computational ChemistryHeinrich Heine University DüsseldorfDüsseldorfGermany
- Institute of Biological Information Processing: Structural BiochemistryForschungszentrum JülichJülichGermany
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6
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Tian Z, Zhang H, Yu R, Du J, Gao S, Wang Q, Guan G, Yin H. The GTPase activity and isoprenylation of Swine GBP1 are critical for inhibiting the production of Japanese Encephalitis Virus. Vet Microbiol 2023; 284:109843. [PMID: 37540998 DOI: 10.1016/j.vetmic.2023.109843] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2022] [Revised: 07/28/2023] [Accepted: 07/30/2023] [Indexed: 08/06/2023]
Abstract
Japanese encephalitis virus (JEV) is a flavivirus that cause severe neurological deficits. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on many RNA and DNA viruses via diverse mechanisms, however, the roles and the action modes of GBP1 in the antiviral effect on the production of JEV RNA and infectious virions remain to be clarified. In this study, we found that the RNA levels of swine GBP1 (sGBP1) in PK15 cells were up-regulated at the late stage of JEV infection. The overexpression of sGBP1 significantly inhibited the production of JEV while the knockdown of sGBP1 promoted the production of JEV. The GTPase activity and isoprenylation of sGBP1 both are critical for anti-JEV activity. The GTPase activity of sGBP1 is responsible for inhibiting the production of JEV genomic RNA. The isoprenylation of sGBP1 inhibited the expression and cleavage of JEV prM to decrease the yields of infectious virions, which may be associated with the interaction between sGBP1 and cellular proprotein convertase furin. Taken together, the study dissected the action modes of sGBP1with potent anti-JEV activity in more details.
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Affiliation(s)
- Zhancheng Tian
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China.
| | - Hongge Zhang
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Ruiming Yu
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Junzheng Du
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Shandian Gao
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Qiongjie Wang
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Guiquan Guan
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China
| | - Hong Yin
- State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, PR China
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7
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Valeva SV, Degabriel M, Michal F, Gay G, Rohde JR, Randow F, Lagrange B, Henry T. Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements. Pathog Dis 2023; 81:ftad005. [PMID: 37012222 DOI: 10.1093/femspd/ftad005] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 03/31/2023] [Indexed: 04/05/2023] Open
Abstract
Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.
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Affiliation(s)
- Stanimira V Valeva
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
| | - Manon Degabriel
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
| | - Fanny Michal
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
| | - Gabrielle Gay
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
| | - John R Rohde
- Department of Microbiology and Immunology, Dalhousie University, Halifax, B3H 4R2, NS, Canada
| | - Felix Randow
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, CB2 0QH, Cambridge, United Kingdom
- Department of Medicine, University of Cambridge, Addenbrooke's Hospital, CB2 0QH, Cambridge, United Kingdom
| | - Brice Lagrange
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
| | - Thomas Henry
- CIRI, Centre International de Recherche en Infectiologie, Univ Lyon, Inserm U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, Univ Lyon, F-69007, Lyon, France
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8
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Loschwitz J, Steffens N, Wang X, Schäffler M, Pfeffer K, Degrandi D, Strodel B. Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2. Sci Rep 2023; 13:679. [PMID: 36639389 PMCID: PMC9839784 DOI: 10.1038/s41598-023-27520-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Accepted: 01/03/2023] [Indexed: 01/15/2023] Open
Abstract
Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-[Formula: see text] and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 μs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface.
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Affiliation(s)
- Jennifer Loschwitz
- grid.411327.20000 0001 2176 9917Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany ,grid.8385.60000 0001 2297 375XInstitute of Biological Information Processing: Structural Biochemistry (IBI-7), Forschungszentrum Jülich, 52425 Jülich, Germany
| | - Nora Steffens
- grid.411327.20000 0001 2176 9917Institute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | - Xue Wang
- grid.411327.20000 0001 2176 9917Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany ,grid.8385.60000 0001 2297 375XInstitute of Biological Information Processing: Structural Biochemistry (IBI-7), Forschungszentrum Jülich, 52425 Jülich, Germany
| | - Moritz Schäffler
- grid.8385.60000 0001 2297 375XInstitute of Biological Information Processing: Structural Biochemistry (IBI-7), Forschungszentrum Jülich, 52425 Jülich, Germany
| | - Klaus Pfeffer
- grid.411327.20000 0001 2176 9917Institute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | - Daniel Degrandi
- Institute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
| | - Birgit Strodel
- Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany. .,Institute of Biological Information Processing: Structural Biochemistry (IBI-7), Forschungszentrum Jülich, 52425, Jülich, Germany.
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9
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Kutsch M, Coers J. Human guanylate binding proteins: nanomachines orchestrating host defense. FEBS J 2021; 288:5826-5849. [PMID: 33314740 PMCID: PMC8196077 DOI: 10.1111/febs.15662] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 11/27/2020] [Accepted: 12/07/2020] [Indexed: 02/06/2023]
Abstract
Disease-causing microorganisms not only breach anatomical barriers and invade tissues but also frequently enter host cells, nutrient-enriched environments amenable to support parasitic microbial growth. Protection from many infectious diseases is therefore reliant on the ability of individual host cells to combat intracellular infections through the execution of cell-autonomous defense programs. Central players in human cell-autonomous immunity are members of the family of dynamin-related guanylate binding proteins (GBPs). The importance of these interferon-inducible GTPases in host defense to viral, bacterial, and protozoan pathogens has been established for some time; only recently, cell biological and biochemical studies that largely focused on the prenylated paralogs GBP1, GBP2, and GBP5 have provided us with robust molecular frameworks for GBP-mediated immunity. Specifically, the recent characterization of GBP1 as a bona fide pattern recognition receptor for bacterial lipopolysaccharide (LPS) disrupting the integrity of bacterial outer membranes through LPS aggregation, the discovery of a link between hydrolysis-induced GMP production by GBP1 and inflammasome activation, and the classification of GBP2 and GBP5 as inhibitors of viral envelope glycoprotein processing via suppression of the host endoprotease furin have paved the way for a vastly improved conceptual understanding of the molecular mechanisms by which GBP nanomachines execute cell-autonomous immunity. The herein discussed models incorporate our current knowledge of the antimicrobial, proinflammatory, and biochemical properties of human GBPs and thereby provide testable hypotheses that will guide future studies into the intricacies of GBP-controlled host defense and their role in human disease.
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Affiliation(s)
- Miriam Kutsch
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 22710, USA
| | - Jörn Coers
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 22710, USA
- Department of Immunology, Duke University Medical Center, Durham, North Carolina 22710, USA
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10
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Sistemich L, Dimitrov Stanchev L, Kutsch M, Roux A, Günther Pomorski T, Herrmann C. Structural requirements for membrane binding of human guanylate-binding protein 1. FEBS J 2021; 288:4098-4114. [PMID: 33405388 DOI: 10.1111/febs.15703] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 11/25/2020] [Accepted: 12/29/2020] [Indexed: 12/14/2022]
Abstract
Human guanylate-binding protein 1 (hGBP1) is a key player in innate immunity and fights diverse intracellular microbial pathogens. Its antimicrobial functions depend on hGBP1's GTP binding- and hydrolysis-induced abilities to form large, structured polymers and to attach to lipid membranes. Crucial for both of these biochemical features is the nucleotide-controlled release of the C terminally located farnesyl moiety. Here, we address molecular details of the hGBP1 membrane binding mechanism by employing recombinant, fluorescently labeled hGBP1, and artificial membranes. We demonstrate the importance of the GTPase activity and the resulting structural rearrangement of the hGBP1 molecule, which we term the open state. This open state is supported and stabilized by homodimer contacts involving the middle domain of the protein and is further stabilized by binding to the lipid bilayer surface. We show that on the surface of the lipid bilayer a hGBP1 monolayer is built in a pins in a pincushion-like arrangement with the farnesyl tail integrated in the membrane and the N-terminal GTPase domain facing outwards. We suggest that similar intramolecular contacts between neighboring hGBP1 molecules are responsible for both polymer formation and monolayer formation on lipid membranes. Finally, we show that tethering of large unilamellar vesicles occurs after the vesicle surface is fully covered by the monolayer. Both hGBP1 polymer formation and hGBP1-induced vesicle tethering have implications for understanding the molecular mechanism of combating bacterial pathogens. DATABASES: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6K1Z, 2D4H.
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Affiliation(s)
- Linda Sistemich
- Faculty of Chemistry and Biochemistry, Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany
| | - Lyubomir Dimitrov Stanchev
- Faculty of Chemistry and Biochemistry, Molecular Biochemistry, Ruhr University Bochum, Bochum, Germany.,Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Miriam Kutsch
- Faculty of Chemistry and Biochemistry, Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany.,Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA
| | - Aurélien Roux
- Biochemistry Department, University of Geneva, Geneva, Switzerland
| | - Thomas Günther Pomorski
- Faculty of Chemistry and Biochemistry, Molecular Biochemistry, Ruhr University Bochum, Bochum, Germany.,Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Christian Herrmann
- Faculty of Chemistry and Biochemistry, Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany
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11
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Ye Y, Xiong Y, Huang H. Substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of bacterial ubiquitin ligase IpaH9.8. Commun Biol 2020; 3:752. [PMID: 33303953 PMCID: PMC7728815 DOI: 10.1038/s42003-020-01492-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Accepted: 11/17/2020] [Indexed: 12/21/2022] Open
Abstract
IpaH enzymes are bacterial E3 ligases targeting host proteins for ubiquitylation. Two autoinhibition modes of IpaH enzymes have been proposed based on the relative positioning of the Leucine-rich repeat domain (LRR) with respect to the NEL domain. In mode 1, substrate-binding competitively displaces the interactions between theLRR and NEL to relieve autoinhibition. However, the molecular basis for mode 2 is unclear. Here, we present the crystal structures of Shigella IpaH9.8 and the LRR of IpaH9.8 in complex with the substrate of human guanylate-binding protein 1 (hGBP1). A hydrophobic cluster in the C-terminus of IpaH9.8LRR forms a hydrophobic pocket involved in binding the NEL domain, and the binding is important for IpaH9.8 autoinhibition. Substrate-binding destabilizes the hydrophobic cluster by inducing conformational changes of IpaH9.8LRR. Arg166 and Phe187 in IpaH9.8LRR function as sensors for substrate-binding. Collectively, our findings provide insights into the molecular mechanisms for the actication of IpaH9.8 in autoinhibition mode 2. Ye, Xiong et al. present crystal structures of bacterial E3 ubiquitin ligase IpaH9.8 and IpaH9.8LRR–hGBP1. They find that substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of IpaH9.8. This study provides insights into the mechanisms underlying substrate-induced activation of IpaH9.8.
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Affiliation(s)
- Yuxin Ye
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 518055, Shenzhen, China. .,Shenzhen Bay Laboratory Pingshan Translational Medicine Center, Shenzhen, China. .,Laboratory of Structural Biology and Drug Discovery, Peking University Shenzhen Graduate School, 518055, Shenzhen, China.
| | - Yuxian Xiong
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 518055, Shenzhen, China.,Laboratory of Structural Biology and Drug Discovery, Peking University Shenzhen Graduate School, 518055, Shenzhen, China
| | - Hao Huang
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 518055, Shenzhen, China. .,Laboratory of Structural Biology and Drug Discovery, Peking University Shenzhen Graduate School, 518055, Shenzhen, China.
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12
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Raninga N, Nayeem SM, Gupta S, Mullick R, Pandita E, Das S, Deep S, Sau AK. Stimulation of GMP formation in hGBP1 is mediated by W79 and its effect on the antiviral activity. FEBS J 2020; 288:2970-2988. [PMID: 33113220 DOI: 10.1111/febs.15611] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 10/08/2020] [Accepted: 10/21/2020] [Indexed: 11/30/2022]
Abstract
Interferon-inducible large GTPases are critical for innate immunity. The distinctive feature of a large GTPase, human guanylate binding protein-1 (hGBP1), is the sequential hydrolysis of GTP into GMP via GDP. Despite several structural and biochemical studies, the underlying mechanism of assembly-stimulated GMP formation by hGBP1 and its role in immunity are not fully clarified. Using a series of biochemical, biophysical, and in silico experiments, we studied four tryptophan residues, located near switch I-II (in and around the active site) to understand the conformational changes near these regions and also to investigate their effect on enhanced GMP formation. The W79A mutation showed significantly reduced GMP formation, whereas the W81A and W180A substitutions exhibited only a marginal defect. The W114A mutation showed a long-range effect of further enhanced GMP formation, which was mediated through W79. We also observed that after first phosphate cleavage, the W79-containing region undergoes a conformational change, which is essential for stimulated GMP formation. We suggest that this conformational change helps to reposition the active site for the next cleavage step, which occurs through a stable contact between the indole moiety of W79 and the main chain carbonyl of K76. We also showed that stimulated GMP formation is crucial for antiviral activity against hepatitis C. Thus, the present study not only provides new insight for the stimulation of GMP formation in hGBP1, but also highlights the importance of the enhanced second phosphate cleavage product in the antiviral activity.
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Affiliation(s)
| | - Shahid M Nayeem
- Department of Chemistry, Aligarh Muslim University, Aligarh, India
| | | | - Ranajoy Mullick
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
| | - Esha Pandita
- National Institute of Immunology, New Delhi, India
| | - Saumitra Das
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
| | - Shashank Deep
- Department of Chemistry, Indian Institute of Technology, New Delhi, India
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13
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Biochemical and structural characterization of murine GBP7, a guanylate binding protein with an elongated C-terminal tail. Biochem J 2020; 476:3161-3182. [PMID: 31689351 DOI: 10.1042/bcj20190364] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Revised: 10/08/2019] [Accepted: 10/09/2019] [Indexed: 12/23/2022]
Abstract
Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20 min-1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22 µM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii.
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14
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Ince S, Zhang P, Kutsch M, Krenczyk O, Shydlovskyi S, Herrmann C. Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain. FEBS J 2020; 288:582-599. [PMID: 32352209 DOI: 10.1111/febs.15348] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 04/10/2020] [Accepted: 04/27/2020] [Indexed: 12/21/2022]
Abstract
Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the β-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.
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Affiliation(s)
- Semra Ince
- Physical Chemistry I, Ruhr-University, Bochum, Germany
| | - Ping Zhang
- Physical Chemistry I, Ruhr-University, Bochum, Germany
| | - Miriam Kutsch
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA
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15
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Sistemich L, Kutsch M, Hämisch B, Zhang P, Shydlovskyi S, Britzen-Laurent N, Stürzl M, Huber K, Herrmann C. The Molecular Mechanism of Polymer Formation of Farnesylated Human Guanylate-binding Protein 1. J Mol Biol 2020; 432:2164-2185. [PMID: 32087202 DOI: 10.1016/j.jmb.2020.02.009] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Revised: 01/30/2020] [Accepted: 02/09/2020] [Indexed: 02/07/2023]
Abstract
The human guanylate-binding protein 1 (hGBP1) belongs to the dynamin superfamily proteins and represents a key player in the innate immune response. Farnesylation at the C-terminus is required for hGBP1's activity against microbial pathogens, as well as for its antiproliferative and antitumor activity. The farnesylated hGBP1 (hGBP1fn) retains many characteristics of the extensively studied nonfarnesylated protein and gains additional abilities like binding to lipid membranes and formation of hGBP1fn polymers. These polymers are believed to serve as a protein depot, making the enzyme immediately available to fight the invasion of intracellular pathogens. Here we study the molecular mechanism of hGBP1 polymer formation as it is a crucial state of this enzyme, allowing for a rapid response demanded by the biological function. We employ Förster resonance energy transfer in order to trace intra and intermolecular distance changes of protein domains. Light scattering techniques yield deep insights into the changes in size and shape. The GTP hydrolysis driven cycling between a closed, farnesyl moiety hidden state and an opened, farnesyl moiety exposed state represents the first phase, preparing the molecule for polymerization. Within the second phase of polymer growth, opened hGBP1 molecules can be incorporated in the growing polymer where the opened structure is stabilized, similar to a surfactant molecule in a micelle, pointing the farnesyl moieties into the hydrophobic center and positioning the head groups at the periphery of the polymer. We contribute the molecular mechanism of polymer formation, paving the ground for a detailed understanding of hGBP1 function.
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Affiliation(s)
- Linda Sistemich
- Physical Chemistry I, Ruhr-University Bochum, 44780, Bochum, Germany
| | - Miriam Kutsch
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC27710, USA
| | - Benjamin Hämisch
- Chemistry Department, University of Paderborn, 33098, Paderborn, Germany
| | - Ping Zhang
- Physical Chemistry I, Ruhr-University Bochum, 44780, Bochum, Germany
| | | | - Nathalie Britzen-Laurent
- Division of Molecular and Experimental Surgery, Translational Research Center, Department of Surgery, University Medical Center Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 91054, Erlangen, Germany
| | - Michael Stürzl
- Division of Molecular and Experimental Surgery, Translational Research Center, Department of Surgery, University Medical Center Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 91054, Erlangen, Germany
| | - Klaus Huber
- Chemistry Department, University of Paderborn, 33098, Paderborn, Germany
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16
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Wang J, Min H, Hu B, Xue X, Liu Y. Guanylate-binding protein-2 inhibits colorectal cancer cell growth and increases the sensitivity to paclitaxel of paclitaxel-resistant colorectal cancer cells by interfering Wnt signaling. J Cell Biochem 2019; 121:1250-1259. [PMID: 31489998 DOI: 10.1002/jcb.29358] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Accepted: 08/20/2019] [Indexed: 12/16/2022]
Abstract
Among the GTPase family members, guanylate-binding protein-1 (GBP-1) is the most thoroughly studied member in a plethora of human cancers. GBP-2, on the other hand, remains limitedly studied. We wonder how GBP-2 participates in colorectal carcinoma (CRC) as well as the paclitaxel (PTX)-resistance of CRC. In this study, the authors are determined to dig into the role that GBP-2 plays in the sensitivity of CRC to PTX, therefore, possibly indicating a promising gene therapy target for CRC. Forced expression of GBP-2 gene was done by plasmid transfection. Reverse transcriptase-polymerase chain reaction and immunoblot were conducted to detect the expression of GBP-2 messenger RNA (mRNA) and protein, respectively. Colony foci formation assay, transwell invasion assay, and flow cytofluorometry were done to determine the proliferation, invasion, and apoptosis of PTX-resistant and PTX-sensitive CRC cell lines, respectively. The level of GBP-2 mRNA and protein in PTX-resistant CRC cell lines was significantly lower than in nonresistant cell lines. Forced exogenous expression of GBP-2 in PTX-resistant CRC cell lines resulted in more sensitivity to PTX because of the demonstration of less cell proliferation, invasion, and more apoptosis. Wnt signaling was suppressed when GBP-2 was upregulated by transfection of GBP-2 overexpression plasmids, and Wnt signaling did not affect GBP-2 expression. GBP-2 upregulation could enhance the killing effect of PTX in both PTX-sensitive CRC cells and PTX-resistant CRC cells by suppressing Wnt signaling.
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Affiliation(s)
- Jing Wang
- Department of Pharmacy, Xi'an Fourth Hospital, Xi'an, Shaanxi, China.,Department of Pharmacology, School of Pharmacy, Air Force Medical University, Xi'an, Shaanxi, China
| | - Hui Min
- Department of Pharmacy, Xi'an Fourth Hospital, Xi'an, Shaanxi, China
| | - Bin Hu
- Department of Pharmacy, Xi'an Fourth Hospital, Xi'an, Shaanxi, China
| | - Xiaorong Xue
- Department of Pharmacy, Xi'an Fourth Hospital, Xi'an, Shaanxi, China
| | - Yufan Liu
- Department of Pharmacy, Xi'an Fourth Hospital, Xi'an, Shaanxi, China
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17
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Rajan S, Pandita E, Mittal M, Sau AK. Understanding the lower GMP formation in large GTPase hGBP-2 and role of its individual domains in regulation of GTP hydrolysis. FEBS J 2019; 286:4103-4121. [PMID: 31199074 DOI: 10.1111/febs.14957] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2018] [Revised: 04/28/2019] [Accepted: 06/11/2019] [Indexed: 12/24/2022]
Abstract
The interferon γ-inducible large GTPases, human guanylate-binding protein (hGBP)-1 and hGBP-2, mediate antipathogenic and antiproliferative effects in human cells. Both proteins hydrolyse GTP to GDP and GMP through successive cleavages of phosphate bonds, a property that functionally distinguishes them from other GTPases. However, it is unclear why hGBP-2 yields lower GMP than hGBP-1 despite sharing a high sequence identity (~ 78%). We previously reported that the hGBP-1 tetramer is crucial for enhanced GMP formation. We show here that the hGBP-2 tetramer has no role in GMP formation. Using truncated hGBP-2 variants, we found that its GTP-binding domain alone hydrolyses GTP only to GDP. However, this domain along with the intermediate region enabled dimerization and hydrolysed GTP further to GMP. We observed that unlike in hGBP-1, the helical domain of hGBP-2 has an insignificant role in the regulation of GTP hydrolysis, suggesting that the differences in GMP formation between hGBP-2 and hGBP-1 arise from differences in their GTP-binding domains. A large sequence variation seen in the guanine cap may be responsible for the lower GMP formation in hGBP-2. Moreover, we identified the sites in the hGBP-2 domains that are critical for both dimerization and tetramerization. We also found the existence of hGBP-2 tetramer in mammalian cells, which might have a role in the suppression of the carcinomas. Our study suggests that sequence variation near the active site in these two close homologues leads to differential second phosphate cleavage and highlights the role of individual hGBP-2 domains in the regulation of GTP hydrolysis.
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Affiliation(s)
| | - Esha Pandita
- National Institute of Immunology, New Delhi, India
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18
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Kutsch M, Ince S, Herrmann C. Homo and hetero dimerisation of the human guanylate-binding proteins hGBP-1 and hGBP-5 characterised by affinities and kinetics. FEBS J 2018; 285:2019-2036. [PMID: 29618166 DOI: 10.1111/febs.14459] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2017] [Revised: 03/16/2018] [Accepted: 03/30/2018] [Indexed: 01/03/2023]
Abstract
The human guanylate-binding proteins (hGBPs) exhibit diverse antipathogenic and tumour-related functions which make them key players in the innate immune response. The isoforms hGBP-1 to hGBP-5 form homomeric complexes and localise to specific cellular compartments. Upon heteromeric interactions, hGBPs are able to guide each other to their specific compartments. Thus, homo- and heteromeric interactions allow the hGBPs to build a network within the cell which might be important for their diverse biological functions. We characterised homomeric complexes of hGBPs in vitro and presented most recently that nonprenylated hGBP-1 and hGBP-5 form dimers as highest oligomeric species while farnesylated hGBP-1 is able to form polymers. We continued to work on the biochemical characterisation of the heteromeric interactions between hGBPs and present here results for nonprenylated hGBP-1 and hGBP-5. Multiangle light scattering identified the GTP-dependent heteromeric complex as dimer. Also hGBP-5's tumour-associated splice variant (hGBP-5ta) was able to form a hetero dimer with hGBP-1. Intriguingly, both hGBP-5 splice variants were able to induce domain rearrangements within hGBP-1. We further characterised the homo and hetero dimers with Förster resonance energy transfer-based experiments. This allowed us to obtain affinities and kinetics of the homo and hetero dimer formation. Furthermore, we identified that the LG domains of hGBP-1 and hGBP-5 build an interaction site within the hetero dimer. Our in vitro study provides mechanistic insights into the homomeric and heteromeric interactions of hGBP-1 and hGBP-5 and present useful strategies to characterise the hGBP network further.
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Affiliation(s)
- Miriam Kutsch
- Physical Chemistry I, Ruhr-University Bochum, Germany
| | - Semra Ince
- Physical Chemistry I, Ruhr-University Bochum, Germany
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19
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Praefcke GJK. Regulation of innate immune functions by guanylate-binding proteins. Int J Med Microbiol 2017; 308:237-245. [PMID: 29174633 DOI: 10.1016/j.ijmm.2017.10.013] [Citation(s) in RCA: 78] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Revised: 10/27/2017] [Accepted: 10/31/2017] [Indexed: 01/02/2023] Open
Abstract
Guanylate-binding proteins (GBP) are a family of dynamin-related large GTPases which are expressed in response to interferons and other pro-inflammatory cytokines. GBPs mediate a broad spectrum of innate immune functions against intracellular pathogens ranging from viruses to bacteria and protozoa. Several binding partners for individual GBPs have been identified and several different mechanisms of action have been proposed depending on the organisms, the cell type and the pathogen used. Many of these anti-pathogenic functions of GBPs involve the recruitment to and the subsequent destruction of pathogen containing vacuolar compartments, the assembly of large oligomeric innate immune complexes such as the inflammasome, or the induction of autophagy. Furthermore, GBPs often cooperate with immunity-related GTPases (IRGs), another family of dynamin-related GTPases, to exert their anti-pathogenic function, but since most IRGs have been lost in the evolution of higher primates, the anti-pathogenic function of human GBPs seems to be IRG-independent. GBPs and IRGs share biochemical and structural properties with the other members of the dynamin superfamily such as low nucleotide affinity and a high intrinsic GTPase activity which can be further enhanced by oligomerisation. Furthermore, GBPs and IRGs can interact with lipid membranes. In the case of three human and murine GBP isoforms this interaction is mediated by C-terminal isoprenylation. Based on cell biological studies, and in analogy to the function of other dynamins in membrane scission events, it has been postulated that both GBPs and IRGs might actively disrupt the outer membrane of pathogen-containing vacuole leading to the detection and destruction of the pathogen by the cytosolic innate immune system of the host. Recent evidence, however, indicates that GBPs might rather function by mediating membrane tethering events similar to the dynamin-related atlastin and mitofusin proteins, which mediate fusion of the ER and mitochondria, respectively. The aim of this review is to highlight the current knowledge on the function of GBPs in innate immunity and to combine it with the recent progress in the biochemical characterisation of this protein family.
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Affiliation(s)
- Gerrit J K Praefcke
- Division of Haematology / Transfusion Medicine, Paul-Ehrlich-Institut, Langen, Germany; Institute for Genetics, University of Cologne, Cologne, Germany.
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20
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Guanylate-Binding Protein 1 Inhibits Nuclear Delivery of Kaposi's Sarcoma-Associated Herpesvirus Virions by Disrupting Formation of Actin Filament. J Virol 2017; 91:JVI.00632-17. [PMID: 28592529 DOI: 10.1128/jvi.00632-17] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Accepted: 05/31/2017] [Indexed: 12/21/2022] Open
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV.IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.
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Abstract
Specialized adaptations for killing microbes are synonymous with phagocytic cells including macrophages, monocytes, inflammatory neutrophils, and eosinophils. Recent genome sequencing of extant species, however, reveals that analogous antimicrobial machineries exist in certain non-immune cells and also within species that ostensibly lack a well-defined immune system. Here we probe the evolutionary record for clues about the ancient and diverse phylogenetic origins of macrophage killing mechanisms and how some of their properties are shared with cells outside the traditional bounds of immunity in higher vertebrates such as mammals.
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Ince S, Kutsch M, Shydlovskyi S, Herrmann C. The human guanylate-binding proteins hGBP-1 and hGBP-5 cycle between monomers and dimers only. FEBS J 2017; 284:2284-2301. [PMID: 28580591 DOI: 10.1111/febs.14126] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Revised: 04/18/2017] [Accepted: 06/01/2017] [Indexed: 12/11/2022]
Abstract
Belonging to the dynamin superfamily of large GTPases, human guanylate-binding proteins (hGBPs) comprise a family of seven isoforms (hGBP-1 to hGBP-7) that are strongly upregulated in response to interferon-γ and other cytokines. Accordingly, several hGBPs are found to exhibit various cellular functions encompassing inhibitory effects on cell proliferation, tumor suppression as well as antiviral and antibacterial activity; however, their mechanism of action is only poorly understood. Often, cellular functions of dynamin-related proteins are closely linked to their ability to form nucleotide-dependent oligomers, a feature that also applies to hGBP-1 and hGBP-5. hGBPs are described as monomers, dimers, tetramers, and higher oligomeric species, the function of which is not clearly established. Therefore, this work focused on the oligomerization capability of hGBP-1 and hGBP-5, which are reported to assemble to homodimers and homotetramers. Employing independent methods such as size-exclusion chromatography, which relies on the hydrodynamic radius, and multiangle light scattering, which relies on the mass of the protein, revealed that previous interpretations regarding the size of the proteins and their complexes have to be revised. Additional studies using inter- and intramolecular Förster resonance energy transfer demonstrated that nucleotide-triggered intramolecular structural changes lead to a more extended shape of hGBP-1 being responsible for the appearance of larger oligomeric species. Thus, previously reported tetrameric and dimeric species of hGBP-1 and hGBP-5 were unmasked as dimers and monomers, respectively, with their shapes depending on both the bound nucleotide and the ionic strength of the solution.
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Affiliation(s)
- Semra Ince
- Physical Chemistry I, Ruhr-University Bochum, Germany
| | - Miriam Kutsch
- Physical Chemistry I, Ruhr-University Bochum, Germany
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Daumke O, Praefcke GJK. Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily. Biopolymers 2016; 105:580-93. [PMID: 27062152 PMCID: PMC5084822 DOI: 10.1002/bip.22855] [Citation(s) in RCA: 64] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Revised: 03/31/2016] [Accepted: 04/01/2016] [Indexed: 12/29/2022]
Abstract
Dynamin superfamily proteins are multidomain mechano-chemical GTPases which are implicated in nucleotide-dependent membrane remodeling events. A prominent feature of these proteins is their assembly- stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin-related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head-to-head fashion. A catalytic arginine finger from the phosphate binding (P-) loop is repositioned toward the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization-dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin-like Irga6 which bears a glycine at the corresponding position in the P-loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano-chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 580-593, 2016.
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Affiliation(s)
- Oliver Daumke
- Kristallographie, Max-Delbrück Centrum Für Molekulare Medizin, Robert-Rössle-Straße 10, Berlin, 13125, Germany
- Institut Für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, Berlin, 14195, Germany
| | - Gerrit J K Praefcke
- Abteilung Hämatologie/Transfusionsmedizin, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, Langen, 63225, Germany
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Britzen-Laurent N, Herrmann C, Naschberger E, Croner RS, Stürzl M. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases. World J Gastroenterol 2016; 22:6434-6443. [PMID: 27605879 PMCID: PMC4968125 DOI: 10.3748/wjg.v22.i28.6434] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Revised: 05/25/2016] [Accepted: 06/13/2016] [Indexed: 02/06/2023] Open
Abstract
Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton.
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Persico M, Petrella L, Orteca N, Di Dato A, Mariani M, Andreoli M, De Donato M, Scambia G, Novellino E, Ferlini C, Fattorusso C. GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1. Eur J Med Chem 2014; 91:132-44. [PMID: 25081641 DOI: 10.1016/j.ejmech.2014.07.093] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2014] [Revised: 07/21/2014] [Accepted: 07/25/2014] [Indexed: 12/31/2022]
Abstract
GBP1 and PIM1 are known to interact with a molar ratio 1:1. GBP1:PIM1 binding initiates a signaling pathway that induces resistance to common chemotherapeutics such as paclitaxel. Since GBP1 is a large GTPase which undergoes conformational changes in a nucleotide-dependent manner, we investigated the effect of GTP/GDP binding on GBP1:PIM1 interaction by using computational and biological studies. It resulted that only GTP decreases the formation of the GBP1:PIM1 complex through an allosteric mechanism, putting the bases for the identification of new compounds potentially able to revert resistance to paclitaxel.
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Affiliation(s)
- Marco Persico
- Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy
| | - Lella Petrella
- Laboratory of Molecular Oncology, Jean Paul II Research Foundation, Campobasso 86100, Italy
| | - Nausicaa Orteca
- Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy
| | - Antonio Di Dato
- Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy
| | - Marisa Mariani
- Danbury Hospital Research Institute, Danbury, CT 06810, USA
| | - Mirko Andreoli
- Danbury Hospital Research Institute, Danbury, CT 06810, USA
| | - Marta De Donato
- Catholic University of the Sacred Heart, Department of Obstetrics and Gynaecology, Rome, Italy
| | - Giovanni Scambia
- Catholic University of the Sacred Heart, Department of Obstetrics and Gynaecology, Rome, Italy
| | - Ettore Novellino
- Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy
| | | | - Caterina Fattorusso
- Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy.
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Vöpel T, Hengstenberg CS, Peulen TO, Ajaj Y, Seidel CAM, Herrmann C, Klare JP. Triphosphate induced dimerization of human guanylate binding protein 1 involves association of the C-terminal helices: a joint double electron-electron resonance and FRET study. Biochemistry 2014; 53:4590-600. [PMID: 24991938 DOI: 10.1021/bi500524u] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Human guanylate binding protein 1 (hGBP1) is a member of the dynamin superfamily of large GTPases. During GTP hydrolysis, the protein undergoes structural changes leading to self-assembly. Previous studies have suggested dimerization of the protein by means of its large GTPase (LG) domain and significant conformational changes in helical regions near the LG domain and at its C-terminus. We used site-directed labeling and a combination of pulsed electron paramagnetic resonance and time-resolved fluorescence spectroscopy for structural investigations on hGBP1 dimerization and conformational changes of its C-terminal helix α13. Consistent distance measurements by double electron-electron resonance (DEER, also named pulse double electron resonance = PELDOR) spectroscopy and Förster resonance energy transfer (FRET) measurements using model-free analysis approaches revealed a close interaction of the two α13 helices in the hGBP1 dimer formed upon binding of the nonhydrolyzable nucleoside triphosphate derivate GppNHp. In molecular dynamics (MD) simulations, these two helices form a stable dimer in solution. Our data show that dimer formation of hGBP1 involves multiple spatially distant regions of the protein, namely, the N-terminal LG domain and the C-terminal helices α13. The contacts formed between the two α13 helices and the resulting juxtaposition are expected to be a key step for the physiological membrane localization of hGBP1 through the farnesyl groups attached to the end of α13.
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Affiliation(s)
- Tobias Vöpel
- Physical Chemistry I, Faculty of Chemistry and Biochemistry, Ruhr-University Bochum , Universitätsstrasse 150, 44780 Bochum, Germany
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Ostler N, Britzen-Laurent N, Liebl A, Naschberger E, Lochnit G, Ostler M, Forster F, Kunzelmann P, Ince S, Supper V, Praefcke GJK, Schubert DW, Stockinger H, Herrmann C, Stürzl M. Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor. Mol Cell Biol 2014; 34:196-209. [PMID: 24190970 PMCID: PMC3911287 DOI: 10.1128/mcb.00664-13] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Revised: 07/22/2013] [Accepted: 10/28/2013] [Indexed: 02/07/2023] Open
Abstract
Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.
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Affiliation(s)
- Nicole Ostler
- Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Nathalie Britzen-Laurent
- Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Andrea Liebl
- Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Elisabeth Naschberger
- Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Günter Lochnit
- Faculty of Medicine, Institute of Biochemistry, Justus Liebig University, Giessen, Germany
| | - Markus Ostler
- Institute of Physics, Chemnitz University of Technology, Chemnitz, Germany
| | - Florian Forster
- Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | - Peter Kunzelmann
- Institute of Polymer Materials, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Semra Ince
- Physical Chemistry I, Ruhr University Bochum, Bochum, Germany
| | - Verena Supper
- Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | | | - Dirk W. Schubert
- Institute of Polymer Materials, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
| | - Hannes Stockinger
- Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | | | - Michael Stürzl
- Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany
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Britzen-Laurent N, Lipnik K, Ocker M, Naschberger E, Schellerer VS, Croner RS, Vieth M, Waldner M, Steinberg P, Hohenadl C, Stürzl M. GBP-1 acts as a tumor suppressor in colorectal cancer cells. Carcinogenesis 2013; 34:153-162. [PMID: 23042300 DOI: 10.1093/carcin/bgs310] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.
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Affiliation(s)
- Nathalie Britzen-Laurent
- Division of Molecular and Experimental Surgery, Department of Surgery, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Schwabachanlage 10, 91054 Erlangen, Germany
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Kravets E, Degrandi D, Weidtkamp-Peters S, Ries B, Konermann C, Felekyan S, Dargazanli JM, Praefcke GJK, Seidel CAM, Schmitt L, Smits SHJ, Pfeffer K. The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii. J Biol Chem 2012; 287:27452-66. [PMID: 22730319 DOI: 10.1074/jbc.m112.379636] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.
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Affiliation(s)
- Elisabeth Kravets
- Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine University, D-40225 Dusseldorf, Germany
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Syguda A, Kerstan A, Ladnorg T, Stüben F, Wöll C, Herrmann C. Immobilization of biotinylated hGBP1 in a defined orientation on surfaces is crucial for uniform interaction with analyte proteins and catalytic activity. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2012; 28:6411-6418. [PMID: 22458356 DOI: 10.1021/la3008359] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Guanylate binding proteins (GBPs) belong to the dynamin superfamily of large GTP binding proteins. A biochemical feature common to these proteins is guanosine-triphosphate (GTP) binding leading to self-assembly of the proteins, and this in turn results in higher catalytic GTP hydrolysis activity. In the case of human guanylate binding protein 1 (hGBP1) homodimer formation is observed after binding of nonhydrolyzable GTP analogs like GppNHp. hGBP1 is one of seven GBP isoforms identified in human. While cellular studies suggest heterocomplex formation of various isoforms biochemical binding studies in quantitative terms are lacking. In this work we established a method to study hGBP1 interactions by attaching this protein in a defined orientation to a surface allowing for interaction with molecules from the solution. Briefly, specifically biotinylated hGBP1 is attached to a streptavidin layer on a self-assembled monolayer (SAM) surface allowing for characterization of the packing density of the immobilized protein by surface plasmon resonance (SPR) technology and atomic force microscopy (AFM), respectively. In addition, the enzymatic activity of immobilized hGBP1 and the kinetics of interaction with binding partners in solution are quantified. We present a procedure for attaching an enzyme in a defined orientation to a surface which exposes its active end, the GTPase domain to the solution resulting in a homogeneous population of this enzyme in terms of enzymatic activity and of interaction with soluble proteins.
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Affiliation(s)
- Adrian Syguda
- Department of Physical Chemistry I, University of Bochum, 44801 Bochum, Germany
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