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Christian Herrmann - One of the best experts on this subject based on the ideXlab platform.
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The Molecular Mechanism of Polymer Formation of Farnesylated Human Guanylate-Binding Protein 1
Journal of molecular biology, 2020Co-Authors: Linda Sistemich, Michael Stürzl, Nathalie Britzen-laurent, Sergii Shydlovskyi, Miriam Kutsch, Benjamin Hämisch, Ping Zhang, Klaus Huber, Christian HerrmannAbstract: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 Forster 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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Farnesylation of human Guanylate-Binding Protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization.
The FEBS journal, 2019Co-Authors: Charlotte Lorenz, Christian Herrmann, Semra Ince, Tao Zhang, Anneliese Cousin, Renu Batra-safferling, Luitgard Nagel-steger, Andreas M. StadlerAbstract:Human Guanylate-Binding Protein 1 (hGBP1) belongs to the family of dynamin-like Proteins and is activated by addition of nucleotides, leading to Protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified Protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.
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Human Guanylate-Binding Protein 1 Tethers Giant Unilamellar Vesicles in a Nucleotide-Dependent Manner
Biophysical Journal, 2014Co-Authors: Sergii Shydlovskyi, Annika Hohendahl, Gerrit J. K. Praefcke, Aurélien Roux, Christian HerrmannAbstract:Human Guanylate-Binding Protein 1 (hGBP1) is the most studied Protein within the family of Guanylate-Binding Proteins (GBPs), which has seven isoforms in humans. GBPs belong to the dynamin superfamily of large GTPases and are thought to act as mechanoenzymes. All members of the GPBs family are expressed to high level after treatment of the cells with interferons, and hGBP1 in particular is mostly expressed by interferon γ, and, similar to the family of Mx Proteins, is involved in antiviral response. However, the molecular mechanism of antiviral activity of hGBP1 is poorly understood. In the course of posttranslational modification the Protein is coupled to a lipid anchor (isoprenoid), which might be crucial for performing its function within the cell. We address the question of the molecular function of hGBP1 by studies of its farnesylated form in vitro in the presence and absence of lipid systems. We can show nucleotide-dependent polymerization of the farnesylated form of hGBP1 and, moreover, we can show that the non-farnesylated form of hGBP1 disturbs the latter processes giving a hypothesis of possible regulation of the biological function of the Protein by other isoforms from GBPs family, which cannot undergo lipid modification, through the heterointeraction. Previous studies of Protein interaction with lipids show the binding of the Protein to the liposomes only in the active state of the Protein. In contrast, by using the lipid model of giant unilamellar vesicles (GUV), we can show that the Protein, which carries the farnesyl anchor, binds to the vesicles directly after nucleotide binding and does not require GTP hydrolysis. Also we can show that it tethers vesicles in a nucleotide-dependent manner and we assume this to be related to the biological function of the Protein.
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From Single-Molecule Fluorescence to Quantiative Structural Models of Dynamic Proteins: Human Guanylate Binding Protein 1
Biophysical Journal, 2013Co-Authors: Thomas-otavio Peulen, Carola S. Hengstenberg, Markus Richert, Alessandro Valeri, Alexander Schug, Abhinav Verma, Claus A.m. Seidel, Christian HerrmannAbstract:MFD (multiparameter fluorescence detection) is based on simultaneous recording of fluorescence lifetime, polarization, and color. hpFRET (high precision Forster resonance energy transfer) used in single-molecule experiment can show an existence of diverse conformations adopted by macromolecules.fFCS (filtered Fluorescence Correlation Spectroscopy) is extended analysis method of time, polarization and color resolved FCS. The method uses MFD data to obtain information on the kinetics of transitions between fluorescence species on the multiple timescales (ns to ms).The above techniques were applied to hGBP1 (the human guanylate binding Protein 1) which is a GTP binding interferon-g induced Protein important immuno defense. hGBP1 consists of three domains: a GTP-binding domain (gray), a middle domain (blue) and a helical domain (green). In solution it undergoes conformational transitions between a minor and a major state. The timescale of motion and structural models of the states were resolved by fFCS, MFD and hpFRET.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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The guanine cap of human Guanylate-Binding Protein 1 is responsible for dimerization and self-activation of GTP hydrolysis.
The FEBS journal, 2011Co-Authors: Mark Wehner, Simone Kunzelmann, Christian HerrmannAbstract:Human Guanylate-Binding Protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62–Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation. Structured digital abstract • hGBP1 and hGBP1 bind by molecularsieving (Viewinteraction)
Michael Stürzl - One of the best experts on this subject based on the ideXlab platform.
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The Molecular Mechanism of Polymer Formation of Farnesylated Human Guanylate-Binding Protein 1
Journal of molecular biology, 2020Co-Authors: Linda Sistemich, Michael Stürzl, Nathalie Britzen-laurent, Sergii Shydlovskyi, Miriam Kutsch, Benjamin Hämisch, Ping Zhang, Klaus Huber, Christian HerrmannAbstract: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 Forster 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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nucleotide dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate binding Protein 1
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Sergii Shydlovskyi, Christine Dovengerds, Anke Y Zienert, Semra Ince, Annika Hohendahl, Julia M Dargazanli, Ailisa Blum, Saskia D Gunther, Nikolay Kladt, Michael StürzlAbstract:Dynamin-like Proteins (DLPs) mediate various membrane fusion and fission processes within the cell, which often require the polymerization of DLPs. An IFN-inducible family of DLPs, the Guanylate-Binding Proteins (GBPs), is involved in antimicrobial and antiviral responses within the cell. Human Guanylate-Binding Protein 1 (hGBP1), the founding member of GBPs, is also engaged in the regulation of cell adhesion and migration. Here, we show how the GTPase cycle of farnesylated hGBP1 (hGBP1 F ) regulates its self-assembly and membrane interaction. Using vesicles of various sizes as a lipid bilayer model, we show GTP-dependent membrane binding of hGBP1 F . In addition, we demonstrate nucleotide-dependent tethering ability of hGBP1 F . Furthermore, we report nucleotide-dependent polymerization of hGBP1 F , which competes with membrane binding of the Protein. Our results show that hGBP1 F acts as a nucleotide-controlled molecular switch by modulating the accessibility of its farnesyl moiety, which does not require any supportive Proteins.
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Processing and secretion of guanylate binding Protein-1 depend on inflammatory caspase activity.
Journal of cellular and molecular medicine, 2017Co-Authors: Elisabeth Naschberger, Michael Stürzl, Philipp Tripal, Walter Geißdörfer, Christian Bogdan, Elisabeth Kremmer, Nathalie Britzen-laurentAbstract:Human guanylate binding Protein-1 (GBP-1) belongs to the family of large GTPases. The expression of GBP-1 is inducible by inflammatory cytokines, and the Protein is involved in inflammatory processes and host defence against cellular pathogens. GBP-1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP-1 with GMP-agarose from cell culture supernatants co-purified a 47-kD fragment of GBP-1 (p47-GBP-1) in addition to the 67-kD full-length form. MALDI-TOF sequencing revealed that p47-GBP-1 corresponds to the C-terminal helical part of GBP-1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67-GBP-1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47-GBP-1. Furthermore, the secretion of p47-GBP-1 was found to occur via a non-classical secretion pathway and to be dependent on caspase-1 activity but independent of inflammasome activation. Finally, we showed that p47-GBP-1 represents the predominant form of secreted GBP-1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP-1. These findings confirm the involvement of caspase-1 in non-classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP-1.
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guanylate binding Protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue engineered construct
BMC Biotechnology, 2012Co-Authors: Oliver Bleiziffer, Matthias Hammon, Christian D Taeger, Elisabeth Naschberger, Andreas Arkudas, Michael Stürzl, Justus P. Beier, Raymund E. Horch, Kerstin Amann, Ulrich KneserAbstract:Background Guanylate binding Protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo.
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increased expression of guanylate binding Protein 1 in lesional skin of patients with cutaneous lupus erythematosus
Experimental Dermatology, 2011Co-Authors: Elisabeth Naschberger, Michael Stürzl, Martin Herrmann, Jorg Wenzel, Cosima C Kretz, Annegret KuhnAbstract:: The large GTPase human guanylate binding Protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation and is induced by interferon (IFN)-α and IFN-γ in endothelial cells (ECs). The aim of this study was to investigate whether GBP-1 is a marker of skin lesions in patients with cutaneous lupus erythematosus (CLE). Western blotting revealed that GBP-1 was in vitro induced by IFN-α and -γ in primary keratinocytes obtained from healthy controls. Moreover, we found that this Protein was expressed by keratinocytes and ECs in primary and ultraviolet (UV)-induced skin lesions from patients with various subtypes of CLE, when compared to non-lesional skin. No GBP-1 expression was noted in skin biopsy specimens 24 or 72 h after UV irradiation prior to lesion formation in patients with CLE or in healthy control specimens with or without UV irradiation. Initial findings suggest that GBP-1 is not expressed in other skin diseases with different inflammatory aetiology, such as atopic dermatitis. We conclude that GBP-1 expression is closely associated with skin lesions in patients with CLE, suggesting a contribution of GBP-1 in the pathogenesis of this disease.
Kuo-wang Tsai - One of the best experts on this subject based on the ideXlab platform.
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Guanylate-Binding Protein 6 is a novel biomarker for tumorigenesis and prognosis in tongue squamous cell carcinoma
Clinical Oral Investigations, 2019Co-Authors: Hung-chih Chen, Huei-han Liou, Jiin-tsuey Cheng, Kuo-wang TsaiAbstract:Objectives Guanylate-Binding Protein 6 (GBP6) is a member of the Guanylate-Binding Protein family, and its role in cancer has not yet been reported. We aimed to investigate the clinical significance of GBP6 in oral squamous cell carcinoma (OSCC). Materials and methods Next-generation sequencing was applied for analyzing differential gene expression profiling between corresponding tumor adjacent normal (CTAN) and tumor tissue from two paired OSCC patients. Real-time PCRs (RT-PCRs) were used to investigate the gene expression level of GBP6 of CTAN and tumor tissue samples from 14 TSCC patients. Immunohistochemistry was used to investigate the Protein expression level of GBP6 in tumor tissues and paired CTAN tissues from 488 OSCC patients, including 183 buccal mucosa squamous cell carcinoma (BMSCC), 245 tongue squamous cell carcinoma (TSCC), and 60 lip squamous cell carcinoma (LSCC) patients. Results Compared with CTAN tissues of OSCC patients, GBP6 is identified as a downregulated gene using the NGS platform, which was confirmed in 14 OSCC patients by RT-PCR. Moreover, Protein expression level of GBP6 in tumor tissues was lower than that in CTAN tissues and the low GBP6 expression was correlated with poor cell differentiation/lymph node metastasis in TSCC patients. In addition, TSCC patients with low expression levels of GBP6 had poor disease-specific survival rate. Conclusion The low expression of GBP6 was associated with tumorigenesis and poor prognosis in OSCC patients, especially in TSCC patients. Clinical relevance GBP6 may serve as a novel favorable diagnostic and prognostic biomarker in TSCC patients.
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Guanylate-Binding Protein 6 is a novel biomarker for tumorigenesis and prognosis in tongue squamous cell carcinoma.
Clinical oral investigations, 2019Co-Authors: Pei-feng Liu, Hung-chih Chen, Chih-wen Shu, Huei-cin Sie, Cheng-hsin Lee, Huei-han Liou, Jiin-tsuey Cheng, Kuo-wang Tsai, Luo-ping GerAbstract:Objectives Guanylate-Binding Protein 6 (GBP6) is a member of the Guanylate-Binding Protein family, and its role in cancer has not yet been reported. We aimed to investigate the clinical significance of GBP6 in oral squamous cell carcinoma (OSCC).
Klaus Pfeffer - One of the best experts on this subject based on the ideXlab platform.
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Biochemical and structural characterization of murine GBP7, a guanylate binding Protein with an elongated C-terminal tail
The Biochemical journal, 2019Co-Authors: Larissa Legewie, Jennifer Loschwitz, Birgit Strodel, Daniel Degrandi, Nora Steffens, Martin Prescher, Xue Wang, Sander H. J. Smits, Lutz Schmitt, Klaus PfefferAbstract: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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guanylate binding Proteins promote caspase 11 dependent pyroptosis in response to cytoplasmic lps
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Danielle M Pilla, Daniel Degrandi, Klaus Pfeffer, Masahiro Yamamoto, Arun K. Haldar, Jon A Hagar, Ashley K Mason, Robert K Ernst, Edward A Miao, Jörn CoersAbstract:Abstract IFN receptor signaling induces cell-autonomous immunity to infections with intracellular bacterial pathogens. Here, we demonstrate that IFN-inducible guanylate binding Protein (Gbp) Proteins stimulate caspase-11–dependent, cell-autonomous immunity in response to cytoplasmic LPS. Caspase-11–dependent pyroptosis is triggered in IFN-activated macrophages infected with the Gram-negative bacterial pathogen Legionella pneumophila. The rapid induction of pyroptosis in IFN-activated macrophages required a cluster of IFN-inducible Gbp Proteins encoded on mouse chromosome 3 (Gbpchr3). Induction of pyroptosis in naive macrophages by infections with the cytosol-invading ΔsdhA L. pneumophila mutant was similarly dependent on Gbpchr3, suggesting that these Gbp Proteins play a role in the detection of bacteria accessing the cytosol. Cytoplasmic LPS derived from Salmonella ssp. or Escherichia coli has recently been shown to trigger caspase-11 activation and pyroptosis, but the cytoplasmic sensor for LPS and components of the caspase-11 inflammasome are not yet defined. We found that the induction of caspase-11–dependent pyroptosis by cytoplasmic L. pneumophila-derived LPS required Gbpchr3 Proteins. Similarly, pyroptosis induced by cytoplasmic LPS isolated from Salmonella was diminished in Gbpchr3-deficient macrophages. These data suggest a role for Gbpchr3 Proteins in the detection of cytoplasmic LPS and the activation of the noncanonical inflammasome.
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Guanylate-Binding Protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii
PLoS pathogens, 2013Co-Authors: Elizabeth M. Selleck, John D Macmicking, Daniel Degrandi, Klaus Pfeffer, Sarah J. Fentress, Wandy L. Beatty, Herbert W. Virgin, L. David SibleyAbstract:IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of Guanylate-Binding Proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy Protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1−/− cells, and decreased virulence of this mutant was compensated in Gbp1−/− mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this Protein in host defense.
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murine guanylate binding Protein 2 mgbp2 controls toxoplasma gondii replication
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Daniel Degrandi, Elisabeth Kravets, Carolin Konermann, Cornelia Beutergunia, Verena Klumpers, Sarah Lahme, Eva Wischmann, Anne K Mausberg, Sandra Beerhammer, Klaus PfefferAbstract:IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding Proteins (GBP) comprise the most abundant IFN-γ–induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the C-terminal domain is sufficient and essential for recruitment to the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.
Birgit Strodel - One of the best experts on this subject based on the ideXlab platform.
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Biochemical and structural characterization of murine GBP7, a guanylate binding Protein with an elongated C-terminal tail
The Biochemical journal, 2019Co-Authors: Larissa Legewie, Jennifer Loschwitz, Birgit Strodel, Daniel Degrandi, Nora Steffens, Martin Prescher, Xue Wang, Sander H. J. Smits, Lutz Schmitt, Klaus PfefferAbstract: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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Large-scale, dynamin-like motions of the human guanylate binding Protein 1 revealed by multi-resolution simulations.
PLoS computational biology, 2019Co-Authors: Bogdan Barz, Jennifer Loschwitz, Birgit StrodelAbstract:Guanylate binding Proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding Protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible Protein that undergoes a hinge motion similar to the movements observed for other dynamin-like Proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13–α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, revealing why GTP binding is needed for hGBP1 dimerization to occur.
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Large-scale, dynamin-like motions of the human guanylate binding Protein 1 revealed by multi-resolution simulations
2019Co-Authors: Bogdan Barz, Jennifer Loschwitz, Birgit StrodelAbstract:Abstract Guanylate binding Proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding Protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible Protein that undergoes a hinge motion similar to the movements observed for other dynamin-like Proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13–α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, disclosing why GTP binding is needed for hGBP1 dimerization to occur. Author summary Gunaylate binding Proteins are key fighters against microbial and viral pathogens. In the human body there are seven types of such Proteins, among which is the guanylate binding Protein 1 (hGBP1). This Protein is able to perform its function only once it is activated by binding and converting guanosinetriphosphat (GTP) to guanosinediphosphat and guanosinemonophosphat via hydrolysis. In concert with the conversion of GTP the dimerization of hGBP1 occurs, which can further interact with the lipid membrane of the pathogen and disrupt it. While the crystal structure of the Protein is known, the activation and dimerization steps are not well understood at molecular level as studying them experimentally is difficult. An alternative approach is given by molecular simulations, allowing us to elucidate the Protein dynamics closely connected to these steps. From our simulations applied to both the hGBP1 monomer and dimer we identified large-scale motions taking place in hGBP1 that had not been reported before. We discuss the relevance of these motions in terms of their biological function, such as possible membrane damage caused by one of the motions or locking the Protein in the dimer state.