14 results on '"Hannah E, Volkman"'
Search Results
2. Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Hannah E Volkman, Stephanie Cambier, Elizabeth E Gray, and Daniel B Stetson
- Subjects
nucleic acid detection ,innate Immunity ,antiviral immunity ,cGAS ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.
- Published
- 2019
- Full Text
- View/download PDF
3. Computational design of constitutively active cGAS
- Author
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Quinton M, Dowling, Hannah E, Volkman, Elizabeth E, Gray, Sergey, Ovchinnikov, Stephanie, Cambier, Asim K, Bera, Banumathi, Sankaran, Max R, Johnson, Matthew J, Bick, Alex, Kang, Daniel B, Stetson, and Neil P, King
- Abstract
Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.
- Published
- 2021
4. Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Daniel B. Stetson, Elizabeth E. Gray, Stephanie Cambier, and Hannah E. Volkman
- Subjects
0301 basic medicine ,Mouse ,medicine.disease_cause ,chemistry.chemical_compound ,0302 clinical medicine ,Immunology and Inflammation ,Cytosol ,Nuclear protein ,Biology (General) ,Cells, Cultured ,Mutation ,General Neuroscience ,Nuclear Proteins ,General Medicine ,Nucleotidyltransferases ,Cell biology ,Nuclear DNA ,antiviral immunity ,Medicine ,Research Article ,Human ,Protein Binding ,Base pair ,QH301-705.5 ,Science ,General Biochemistry, Genetics and Molecular Biology ,Cell cycle phase ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,DNA Cleavage ,innate Immunity ,Cell Nucleus ,Innate immune system ,General Immunology and Microbiology ,nucleic acid detection ,DNA ,Mice, Inbred C57BL ,030104 developmental biology ,chemistry ,030217 neurology & neurosurgery ,DNA Damage ,HeLa Cells ,cGAS - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity.
- Published
- 2019
5. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant.
- Author
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Hannah E Volkman, Hilary Clay, Dana Beery, Jennifer C W Chang, David R Sherman, and Lalita Ramakrishnan
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.
- Published
- 2004
- Full Text
- View/download PDF
6. Author response: Tight nuclear tethering of cGAS is essential for preventing autoreactivity
- Author
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Daniel B. Stetson, Hannah E. Volkman, Stephanie Cambier, and Elizabeth E. Gray
- Subjects
Tethering ,Chemistry ,Cell biology - Published
- 2019
7. cGAS is predominantly a nuclear protein
- Author
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Hannah E. Volkman, Elizabeth E. Gray, Daniel B. Stetson, and Stephanie Cambier
- Subjects
0303 health sciences ,Innate immune system ,Chemistry ,Base pair ,3. Good health ,Nuclear DNA ,Cell cycle phase ,Cell biology ,03 medical and health sciences ,genomic DNA ,Cytosol ,chemistry.chemical_compound ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Nuclear protein ,DNA ,030304 developmental biology - Abstract
cGAS is an intracellular innate immune sensor that detects double-stranded DNA. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. Here, we demonstrate that cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We propose that tethering prevents activation of cGAS by genomic DNA, and that it might enable cGAS to distinguish between self DNA and foreign DNA within the nucleus.
- Published
- 2018
- Full Text
- View/download PDF
8. Intracellular Nucleic Acid Detection in Autoimmunity
- Author
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John T. Crowl, Elizabeth E. Gray, Hannah E. Volkman, Daniel B. Stetson, and Kathleen Pestal
- Subjects
0301 basic medicine ,cGAS-STING ,Immunology ,Context (language use) ,Autoimmunity ,Computational biology ,Aicardi-Goutières syndrome ,Biology ,medicine.disease_cause ,Nervous System Malformations ,Autoimmune Disease ,Basic Behavioral and Social Science ,Article ,03 medical and health sciences ,Immune system ,Autoimmune Diseases of the Nervous System ,systemic lupus erythematosus ,Aicardi-Goutieres syndrome ,Nucleic Acids ,Behavioral and Social Science ,Genetics ,medicine ,Extracellular ,Innate ,Immunology and Allergy ,Animals ,Humans ,Lupus Erythematosus, Systemic ,Innate immune system ,Lupus Erythematosus ,Prevention ,Inflammatory and immune system ,Systemic ,Toll-Like Receptors ,Immunity ,Immunity, Innate ,Infectious Diseases ,030104 developmental biology ,type I interferons ,Virus Diseases ,Interferon Type I ,RIG-I-like receptors ,Nucleic acid ,Intracellular ,Nucleic acid detection - Abstract
Protective immune responses to viral infection are initiated by innate immune sensors that survey extracellular and intracellular space for foreign nucleic acids. The existence of these sensors raises fundamental questions about self/nonself discrimination because of the abundance of self-DNA and self-RNA that occupy these same compartments. Recent advances have revealed that enzymes that metabolize or modify endogenous nucleic acids are essential for preventing inappropriate activation of the innate antiviral response. In this review, we discuss rare human diseases caused by dysregulated nucleic acid sensing, focusing primarily on intracellular sensors of nucleic acids. We summarize lessons learned from these disorders, we rationalize the existence of these diseases in the context of evolution, and we propose that this framework may also apply to a number of more common autoimmune diseases for which the underlying genetics and mechanisms are not yet fully understood.
- Published
- 2017
9. Tumor Necrosis Factor Signaling Mediates Resistance to Mycobacteria by Inhibiting Bacterial Growth and Macrophage Death
- Author
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Hilary Clay, Lalita Ramakrishnan, and Hannah E. Volkman
- Subjects
Programmed cell death ,Embryo, Nonmammalian ,Necrosis ,Immunology ,Mycobacterium Infections, Nontuberculous ,Apoptosis ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Cell Movement ,medicine ,Animals ,Immunology and Allergy ,Macrophage ,Zebrafish ,Mycobacterium marinum ,030304 developmental biology ,0303 health sciences ,Granuloma ,Innate immune system ,Cell Death ,biology ,Macrophages ,biology.organism_classification ,medicine.disease ,Immunity, Innate ,3. Good health ,Infectious Diseases ,Receptors, Tumor Necrosis Factor, Type I ,CELLIMMUNO ,Tumor Necrosis Factors ,Cytokines ,Tumor necrosis factor alpha ,medicine.symptom ,Signal Transduction ,030215 immunology - Abstract
SummaryTumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.
- Published
- 2008
10. Zebrafish and frog models of Mycobacterium marinum infection
- Author
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Hannah E. Volkman, Lalita Ramakrishnan, Laura E. Swaim, J. Muse Davis, and Christine L. Cosma
- Subjects
animal structures ,Tuberculosis ,Embryo, Nonmammalian ,Danio ,Mycobacterium Infections, Nontuberculous ,Biology ,Microbiology ,Immune system ,Virology ,Fluorescence microscope ,medicine ,Animals ,Humans ,Zebrafish ,Rana pipiens ,Embryo ,General Medicine ,medicine.disease ,biology.organism_classification ,Disease Models, Animal ,Granuloma ,Immunology ,Host-Pathogen Interactions ,Mycobacterium marinum ,Parasitology ,Mycobacterium - Abstract
Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium-host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.
- Published
- 2008
11. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity
- Author
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Olivier Humbert, Hannah E. Volkman, Donald E. Born, Lalita Ramakrishnan, Lynn E. Connolly, and Laura E. Swaim
- Subjects
Author's Correction ,Tuberculosis ,animal structures ,Immunology ,Dose-Response Relationship, Immunologic ,Mycobacterium Infections, Nontuberculous ,Caseous necrosis ,Biology ,Microbiology ,Mycobacterium tuberculosis ,Necrosis ,Immunity ,medicine ,Animals ,Longitudinal Studies ,Zebrafish ,Mycobacterium marinum ,Host Response and Inflammation ,Innate immune system ,Granuloma ,medicine.disease ,Acquired immune system ,biology.organism_classification ,Infectious Diseases ,Immunity, Active ,embryonic structures ,Chronic Disease ,Disease Progression ,bacteria ,Parasitology - Abstract
The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum , a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis . We previously developed a zebrafish embryo- M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum -induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of ∼9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis , we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum ΔRD1 mutants are attenuated in adult zebrafish and extend this finding to show that ΔRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with ΔRD1 attenuation in zebrafish embryos is ongoing during adult infection.
- Published
- 2006
12. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant
- Author
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David R. Sherman, Dana Beery, Lalita Ramakrishnan, Hannah E. Volkman, Hilary Clay, and Jennifer C. Chang
- Subjects
Programmed cell death ,Time Factors ,Ranidae ,QH301-705.5 ,Immunology ,Virulence ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Mycobacterium tuberculosis ,03 medical and health sciences ,Mice ,Immune system ,Danio (Zebrafish) ,In Situ Nick-End Labeling ,Animals ,Tuberculosis ,Secretion ,Tuberculoma ,Biology (General) ,Zebrafish ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Mycobacterium Infections ,Granuloma ,Microscopy, Video ,General Immunology and Microbiology ,biology ,Cell Death ,030306 microbiology ,General Neuroscience ,Chemotaxis ,Macrophages ,Macrophage Activation ,biology.organism_classification ,Virology ,Infectious Diseases ,Mutation ,General Agricultural and Biological Sciences ,Bacteria ,Mycobacterium ,Research Article - Abstract
Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence., A zebrafish model of mycobacterium infection demonstrates that formation granulomas - immune structures composed of macrophages - requires a bacterial gene, indicating that mycobacteria exploit granuloma formation for infection
- Published
- 2004
13. Innate immunity to tuberculosis in zebrafish: roles of MMPs
- Author
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Hannah E. Volkman, Lalita Ramakrishnan, T. Pozos, Jeffrey I. Gordon, and John F. Rawls
- Subjects
Tuberculosis ,Innate immune system ,biology ,Immunology ,medicine ,Matrix metalloproteinase ,biology.organism_classification ,medicine.disease ,Molecular Biology ,Zebrafish - Published
- 2006
14. The Mycobacterium marinum RD1 locus promotes virulence and macrophage aggregation into tuberculous granulomas by enhancing induction of host matrix metalloproteinase 9 in proximal epithelial cells (133.19)
- Author
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Hannah E Volkman, Tamara Pozos, John Zheng, John F Rawls, and Lalita Ramakrishnan
- Subjects
Immunology ,Immunology and Allergy - Abstract
Granulomas are organized aggregates of differentiated macrophages and other immune cells that form in response to mycobacterial infection and other persistent stimuli. While the tuberculous granuloma has long been considered a host-protective structure, our recent work in the genetically tractable and optically transparent zebrafish embryo-Mycobacterium marinum model of tuberculosis, has suggested that the early granuloma actually benefits mycobacteria. The mycobacterial RD1 virulence locus, a specialized bacterial secretion system, enhances granuloma formation, which in turn, promotes bacterial expansion and dissemination. Here we present the molecular and cellular details of RD1-induced granuloma formation. RD1-secreted effectors induce host matrix metalloproteinase 9 (mmp9) in epithelial cells neighboring the infected macrophages. By specifically knocking down host mmp9, we have shown that its expression facilitates macrophage recruitment to form granulomas and bacterial expansion. Selective mmp9 induction in immotile epithelial cells neighboring infected macrophages may serve as a mechanism to fix and expand infection foci at optimal sites. Our results provide direct evidence that early granulomas benefit mycobacteria and suggest new tuberculosis therapeutic strategies that target granuloma-promoting host susceptibility determinants like mmp9.
- Published
- 2009
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