Browsing by Subject "Nucleotidyltransferases"
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Item Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress(2015-04-14) Woolery, Andrew Ryan; Liu, Qinghua; Orth, Kim; Cobb, Melanie H.; Sternweis, Paul C.The post-translational modification AMPylation is emerging as a significant regulatory mechanism in both prokaryotic and eukaryotic biology. This process involves the covalent addition of an adenosine monophosphate to a protein resulting in a modified protein with altered activity. Proteins capable of catalyzing AMPylation, termed AMPylators, are comparable to kinases in that they both hydrolyze ATP and reversibly transfer a part of this primary metabolite to a hydroxyl side chain of the protein substrate. To date, all AMPylators discovered contain one of two domains: the Fic domain or the adenylyl transferase domain. All currently characterized AMPylators are bacterial in origin and are primarily Type III or Type IV secreted effector proteins, which are injected into a host cell to manipulate host signaling to the microbe's advantage. Examples of these are VopS (Vibrio parahaemolyticus), IbpA (Histophilus somni) and DrrA (Legionella pneumophila). The discovery of SidD, a deAMPylator also from L. pneumophila, shows that this modification is dynamic and could likely have a regulatory role in eukaryotic biology. Supporting this idea is the presence of a single copy of the Fic domain in most metazoans, including humans. The substrates, localization, and function of Fic proteins and other AMPylators in eukaryotic biology are perhaps the largest open questions in this rapidly expanding field. The goal of my dissertation work was to expand the understanding of the effects of AMPylation in eukaryotic signaling. I approached this goal in three ways: by examining the effects of an AMPylator (VopS) with known targets (Rho GTPases) on different aspects of cell signaling, developing screening tools for AMPylation and attempting to elucidate some of the functions of the human AMPylator, FicD, in which the targets are unclear. I found that VopS, in addition to collapsing the host actin cytoskeleton, also inhibits many aspects of host defense signaling including NFB, MAP kinases and the phagocytic NADPH oxidase system. I explored the possibility of other potential substrates of VopS by collaborating on an extensive protein microarray screen for AMPylation, determining that the entire Rho GTPase family is AMPylated. I also discovered that the human AMPylator FicD is induced during the unfolded protein response, is localized to the endoplasmic reticulum and is capable of AMPylating the ER chaperone BiP/GRP78. The progress made in these studies will contribute to understanding the role of this enigmatic modification in mammalian cell signaling.Item Genetic Analysis of Fic-Mediated BiP AMPylation in Photoreceptors(2019-04-10) Moehlman, Andrew Terry; Smith, Dean P.; Orth, Kim; Cobb, Melanie H.; Thomas, Philip J.; Krämer, HelmutIn response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. The Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or deAMPylation of BiP, the major ER chaperone. This work elucidates a critical role of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to constant light-induced stress. In response to extended light exposure, flies mutant for fic display loss of synaptic function and disintegration of rhabdomeres, closely stacked plasma membrane microvilli that house the proteins of the phototransduction cascade. These phenotypes are replicated in BiP mutants lacking the Thr366 AMPylation site. Strikingly, these degeneration-like phenotypes are reversible: photoreceptors in both fic and bip mutants regain their structure and function within 72 hours once returned to a standard light:dark cycle. In fic mutants, these phenotypes are preceded by a dysregulation of the IREI and PERK-mediated ER stress responses detected with specific reporters. These findings indicate that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands, and that this process may require proper activation of the Unfolded Protein Response. Furthermore, I have helped identify multiple cell membrane transporters involved in synaptic signaling between photoreceptors and lamina neurons. These transporters are involved directly and indirectly in the recycling of the neurotransmitter histamine in the synaptic cleft.Item Innate Immune Sensing and Signaling of Cytosolic DNA(2015-04-10) Wu, Jiaxi; Mendell, Joshua T.; Chen, Zhijian J.; Olson, Eric N.; Cobb, Melanie H.In eukaryotic cells, DNA is normally confined within the nucleus and mitochondria. DNA exposed in the cytosol is a danger signal that warns the host of invading microbial pathogens and triggers innate immune responses including the production of type-I interferons (IFNs). Endogenous DNA that is inappropriately cleared can also accumulate in cytosol and drive pathological inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). It is well known that cytosolic DNA induces IFNs through the STING-TBK1-IRF3 axis. However, how DNA is sensed in the cytosol and how this sensing event leads to the activation of STING remains elusive. Using a cell-free complementation assay, we identified cyclic GMP-AMP (cGAMP), as a novel eukaryotic second messenger generated by DNA stimulated or DNA virus infected cells. cGAMP contains a unique phosphodiester linkage combination (both 2'-5' and 3'-5'), for which we referred to it as 2'3'-cGAMP. 2'3'-cGAMP bound to STING with nanomolar affinity and induced a dramatic conformational change that led to its activation. Through biochemical purification and quantitative mass spectrometry, we identified the enzyme that synthesizes cGAMP in a DNA dependent manner. This enzyme, which we named cyclic GMP-AMP synthase (cGAS), turned out to be the long sought-after cytosolic DNA sensor. Structural and functional studies revealed that cGAS is activated by DNA-induced dimerization. We further generated and characterized a cGAS knockout mouse strain, which failed to produce IFNs and other cytokines in response to DNA stimulation and was more vulnerable to lethal infection by DNA viruses. Together, these results not only elucidate the mechanism of cytosolic DNA sensing, but also uncover a novel second messenger-mediated signaling mechanism in innate immunity.Item Liquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways(August 2021) Du, Mingjian; Liu, Yi; Chen, Zhijian J.; Beutler, Bruce; O'Donnell, Kathryn A.The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling. Beyond cGAS-DNA phase separation, we sought to determine whether protein liquid-liquid phase separation is a ubiquitous mechanism across immune signaling pathways. NF-kappa-B essential modulator (NEMO), also known as IKBKG, is essential for the activation of IκB kinase (IKK) complex in NF-κB signaling, including Interleukin-1 (IL-1β), Tumor Necrosis Factor (TNFα) and Toll-like receptors (TLR) pathways. NEMO activates IKK complex by binding to polyubiquitin chains. Here we show that Lys63(K63)-linked or linear(M1)-linked polyubiquitin chains binding to NEMO robustly induced the formation of liquidlike droplets in which IKK was activated both in vitro and in cells. Both NEMO ubiquitin binding (NUB) domain and zinc finger (ZF) domain of NEMO contributed the multivalencies for binding to polyubiquitin chains. Long polyubiquitin chains were more efficient in promoting NEMO phase separation than short polyubiquitin chains. These results demonstrated that polyubiquitin chains induced phase transition of NEMO to promote IKK complex activation and NF-κB signaling.Item Measuring Activation of the Cytosolic DNA Sensing Pathway(2019-04-15) Varnado, Nicole L.; Tu, Benjamin; Beutler, Bruce; Cobb, Melanie H.; Chen, Zhijian J.In mammalian cells, DNA is normally sequestered within the confines of the nucleus or mitochondria. Entrance of DNA into the cytosol, whether foreign or self in origin, acts as a danger signal that triggers a host innate immune response. Cytosolically localized DNA is sensed by cyclic GMP-AMP synthase (cGAS), which synthesizes a novel second messenger known as cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP, in turn, binds to and activates the ER resident adaptor Stimulator of Interferon Genes (STING), which triggers downstream signaling that culminates in the production of type-I interferons and other immune modulatory molecules. The pathway underlies the recognition of pathogenic DNA necessary to quell microbial infections, as well as the aberrant detection of self-DNA responsible for inducing certain autoimmune diseases. Such appreciation for the involvement of cGAS-cGAMP-STING signaling in numerous clinical phenotypes necessitates development of tools that can outline the extent of its contribution to various diseases. Additionally, numerous questions remain regarding the regulation of cGAS-cGAMP signaling. As 2'3'-cGAMP production is a hallmark of the pathway's activation, we sought to develop a robust method to monitor its formation in vivo, and quantify its levels in a wide variety of settings. Herein we present the development of an antibody of high sensitivity and specificity for this small molecule second messenger, capable of recognizing and quantifying 2'3'-cGAMP production in vivo. We show it can be adapted for use in a variety of techniques, to track and measure levels of 2'3'-cGAMP quantitatively, to visualize 2'3'-cGAMP produced in cells, and to quickly identify cGAMP-positive cell populations within live samples. We show this antibody to be an invaluable tool to elucidate outstanding questions in the field, and demonstrate its potential to detect patients with aberrant activation of the cGAS-STING pathway. We foresee a future in which the 2'3'-cGAMP antibody is used to quantify activation of the cGAS pathway in a variety of clinical and research settings.Item The Mechanism and Function of Autophagy Induction by Cytosolic DNA(2018-07-11) Gui, Xiang; Olson, Eric N.; Hooper, Lora V.; Wang, Zhigao; Chen, Zhijian J.Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects pathogen infections or tissue damage by binding to microbial or self-DNA in the cytoplasm. Upon binding to DNA, cGAS produces cGAMP that binds and activates the adaptor protein stimulator of interferon genes (STING), which activates the kinases IKK and TBK1 to induce interferons and other inflammatory cytokines. Here, we report that STING also activates autophagy and induces cell death through a mechanism independent of TBK1 and IRF3 activation, which canonically triggers innate immunity signaling. Upon binding to cGAMP, STING translocates to the ER-Golgi intermediate compartments (ERGIC) and the Golgi in a process that depends on the COP-II complex and ARF GTPases. The STING-containing ERGIC serves as a membrane source for LC3 lipidation, a key step in autophagosome biogenesis. Interestingly, STING lacking its C tail for interferon signaling is still capable of membrane trafficking and autophagy induction. Through endosomes or autophagosomes, STING is further degraded in the lysosome to shut down its activation. Interestingly, we determined that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Furthermore, sea anemone STING induces autophagy but not interferons in response to stimulation by cGAMP, suggesting that induction of autophagy is a primordial function of the cGAS-STING pathway.Item The Mechanism of cGAS-STING Signaling in Antiviral Immunity(2021-05-01T05:00:00.000Z) Li, Minghao; Fu, Yang-Xin; Beutler, Bruce; Chen, Zhijian J.; Hooper, Lora V.Unlike inflammatory cell death pathways, apoptosis is a highly regulated process that leads to cell death without the secretion of pro-inflammatory cytokines. The intrinsic apoptosis pathway (IAP) is triggered upon sensing cellular stress, activating pro-death B cell lymphoma 2-associated X (BAX) and B cell lymphoma 2 homologous antagonist killer (BAK) proteins. BAX and BAK induce mitochondrial outer membrane permeabilization (MOMP), spilling cytochrome C (cyt C) into the cytoplasm where it forms the apotosome complex with the apoptotic protease activating factor 1 (Apaf-1). Apaf-1 subsequently induces a cysteine-aspartic protease (caspase) activation cascade culminating in activation of caspases-3/7. Nevertheless, the purpose of the caspase activation cascade remained an interesting conundrum as BAX and BAK, not Apaf-1 and caspases, are necessary for induction of apoptosis. Recent publications seem to indicate that the main function of caspases after activation of apoptosis is to prevent the induction of type I interferons by cyclic GMP-AMP synthase (cGAS) or stimulator of interferon genes (STING) to maintain an immunologically quiescent cell death. I utilized the drug ABT-737 to inhibit the B-cell lymphoma (BCL) family of proteins to prevent the activation of BAK and BAX, leading to MOMP. MOMP causes leakage of cyt C and mitochondrial DNA (mtDNA), activating Apaf-1 and cGAS, respectively. cGAS catalyzes the creation of the cyclic dinucleotide cyclic GMP-AMP (cGAMP) to activate STING which activates downstream targets such as TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) to induce the expression of interferonβ (IFNβ) and interferon-stimulated genes (ISGs) such as C-X-C motif chemokine ligand 10 (CXCL10). However, the mechanism behind caspase-dependent inhibition of the cGAS-STING pathway is currently unknown. My central hypothesis is that caspases-3/7 activate a downstream regulator that inhibits cGAS-mediated cGAMP production. These findings will further our understanding of the regulatory mechanics behind immune responses. As STING is necessary for the production of IFNs downstream of cGAS, it plays an important role in activating the innate immune response during infections. Specifically, mice lacking cGAS or STING are highly susceptible to acute herpes simplex encephalitis (HSE). STING-induced type I IFNs and immune-priming of other cell types were suggested to be critical for protecting mice from HSE. Recent work from our lab has identified an additional primordial downstream pathway of STING: autophagy; in addition, these results indicate that the interferon- and autophagy-inducing function of STING can be uncoupled. To better understand how STING-induced autophagy is controlled, I investigated the necessity of certain canonical autophagy-related genes. To also understand the exact mechanisms downstream of STING activation required for antiviral immunity, I utilized several different STING mutant mice generated in our lab and assessed their response to HSV-1 infection. My results indicate that a novel interferon-independent function of STING was essential to protect against infection and improve the overall survival rates of infected mice; however, STING-induced autophagy was not sufficient to protect against viral challenge.Item The Pivotal Role of cGAS in Host Defense Against Mycobacterium Tuberculosis and in Particle-Induced Immunity(2017-11-02) Cai, Haocheng; Fu, Yang-Xin; Hooper, Lora V.; Yan, Nan; Chen, Zhijian J.The cGAS-cGAMP-STING pathway is a major DNA sensing pathway in the cytosol. The DNA sensing mechanism is crucial for host defense against bacteria, viruses, and parasites. In the previous study from our lab, the cGAS-STING pathway has been identified as being critical for antiviral activities. However, anti-bacterial activity is more complicated due to the complexity of the invading agents. Mycobacterium tuberculosis (Mtb) is a pandemic pathogen that triggers millions of deaths every year. It is discovered that Mtb ejects its DNA component into host cytosol. We have uncovered the cGAS pathway, which elicits type I interferons and autophagy in macrophages and other cell types, as a defending mechanism against Mtb infection. cGAS is critical for mouse survival against chronic Mtb infection. In conclusion, cGAS is critical for the host defense against Mtb, especially in the induction of type I interferon and autophagy. Previous studies revealed that some fusogenic liposomes and enveloped virus-like particles can activate type I interferons. Here we have shown that fusogenic liposomes and nonenveloped virus-like particles can activate the cGAS pathway and trigger interferon production, which is associated with the amount of cytosolic DNA. Moreover, cGAS and STING are critical for the humoral immune response elicited by virus-like particles. Overall, we have delineated the significant role of cGAS in mediating the immune response induced by fusogenic liposomes and virus-like particles. Additionally, we have proven that cGAS heterozygous mice have largely retained the antiviral activity in herpes simplex virus infection, compared with wildtype mice. This has shed light on the development of cGAS inhibition therapies against autoimmune diseases. Overall, this dissertation has identified the pivotal roles of the cGAS-STING DNA sensing pathway in Mycobacterium tuberculosis infection and particle-induced immunity, as well as the effect of cGAS heterozygosity on antiviral activities.Item Regulation of the cGAS-STING Pathway in Health and Disease(2018-11-27) Pokatayev, Vladislav Andreyevich; van Oers, Nicolai S. C.; Conrad, Nicholas; Chen, Zhijian J.; Yan, NanThe innate immune system senses non-self or altered-self molecular structures through pattern recognition receptors in order to eliminate pathogens or damaged cells, and restore an organism to its basal physiology. Nearly all nucleated cell types can sense intracellular viral nucleic acids. These sensors detect either viral RNA through RIG-I like receptors or DNA through the cGAS-STING signaling pathway. Antiviral immune pathways are vital for resolution of viral infections; however, their dysregulation may give rise to various immune-mediated diseases. The neuro-inflammatory autoimmune disease Aicardi-Goutières Syndrome (AGS) develops from mutations in genes encoding several nucleic acid processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species which trigger chronic inflammation leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), the most severe Rnaseh2a mutation categorized as it abolishes nuclease activity to less than 10% of WT RNase H2, to understand disease pathology. Importantly, I found that the G37S mutation induces a cellular anti-viral state, and an increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. G37S homozygotes are perinatal lethal, and ablation of STING in G37S mice results in partial rescue of the perinatal lethality and complete rescue of the immune phenotype. This study motivates inhibitors of the cGAS-STING pathway in the goal of resolving Rnaseh2a-mediated AGS. As my previous work implicates STING in the development of AGS, I performed a genetic screen to identify novel regulators of this protein. I discovered that TOLLIP, a protein previously identified as a regulator of extracellular Toll-like receptor pathways, can function as a positive regulator of the cGAS-STING pathway. TOLLIP antagonizes STING protein degradation through a regulatory pathway controlled by the protein IRE1α. In Tollip-/- cells, IRE1α is activated and induces lysomal-mediated degradation of STING. Chronic activation of this degradative pathway blunts the cellular response to cGAS or STING agonists. These findings have implications in vivo, as deleting Tollip in a mouse model for AGS, the Trex1-/- mouse, can rescue symptoms of the disease. These findings have clinical importance, as novel therapeutics against TOLLIP can be developed to treat auto-inflammation caused by dysregulation of the cGAS-STING signaling pathway.Item Regulatory Mechanisms in Innate Immunity(2018-07-16) Ren, Junyao; Olson, Eric N.; Chen, Zhijian J.; Hooper, Lora V.; Beutler, BruceInnate immunity is the frontline for the host to defend against infections. This process entails the cooperation among pathogen recognition receptors, adaptor proteins, kinases, and transcription factors that elicit the production of effector cytokines. As an important transcription factor, IRF5 was known to be essential for the host cytokine production in response to various ligands and SNPs in IRF5 have been closely related to autoimmune diseases. However, the mechanism by which IRF5 is activated is not well understood. In the first part of this dissertation, I presented evidence that the kinase IKK2 phosphorylates IRF5 on Serine 445, leading to its dimerization and nuclear translocation. cGAMP is the first cyclic di-nucleotide discovered in metazoan. It is produced by the cytosolic DNA sensor cGAS in response to pathogen or self DNA as a second messenger to activate STING. cGAMP has been proven to be very important in anti-viral response and anti-tumor process. In the second part of the dissertation, I used next-generation sequencing techniques and presented that STING is the predominant receptor for cGAMP and innate immune response. As the essential and general DNA sensor, cGAS was purified and identified from cellular cytosols. Upon DNA binding, cGAS utilize ATP and GTP to synthesize cGAMP. However, the regulation of cGAS activity in cells is still poorly understood. Here I presented that certain RNA species that is interferon inducible could inhibit cGAS catalytic activity in vitro and probably regulate cGAS mediated immune response in cells. Besides, I have discovered that during the cell cycle, cGAS is recruited and co-localize with chromosome and in actively dividing cells, cGAS remains in the nucleus. I further presented evidence that certain protein(s) in the nucleus can inhibit cGAS activity thus prevent cGAS from being activated by host DNA in the nucleus.Item The Role of the cGAS-Sting Pathway in DNA Vaccination and Autoimmune Disease(2017-07-10) Cheng, Philip R.; Hooper, Lora V.; Zinn, Andrew R.; Wakeland, Edward K.; Chen, Zhijian J.The innate immune system recognizes certain molecular patterns expressed by pathogens via pattern recognition receptors (PRR). As a PRR, cyclic GMP-AMP synthase (cGAS) functions as a cytosolic DNA sensor. Stimulator of Interferon Genes (STING) functions as the downstream adaptor protein. Activation of the cGAS-STING pathway results in proinflammatory cytokine production. Here I show that the cGAS-STING pathway plays dual roles in mediating DNA adjuvant activity and the use of 2'3'-cyclic GMP-AMP (cGAMP) as a vaccine adjuvant in mice, in addition to promoting autoantibody production and autoimmune inflammatory cell accumulation in lupus-prone mice. It is unclear which DNA sensor is responsible for mediating the adjuvant effects of plasmid DNA during the course of DNA vaccination. I show that the cGAS-STING pathway is required for generation of antigen-specific immune responses following DNA-adjuvanted vaccination. Mice vaccinated with influenza antigens co-administered with 2'3'-cGAMP develop robust neutralizing antibody titers, enhanced antigen-specific CD8+ T-cell responses, and are protected against lethal influenza virus challenge. The efficacy of 2'3'-cGAMP as a vaccine adjuvant can be enhanced by liposome-assisted delivery, the use of non-hydrolyzable analogs, or co-administration with CpG-C DNA. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. The exact etiology of SLE is unclear, but work utilizing mouse models of SLE have shown that the PRRs of the innate immune system contribute to disease pathogenesis. My results show that in C57BL/6J-Faslpr/Faslpr mice, genetic ablation of cGAS or STING significantly decreases antinuclear autoantibody titers as well as a number of autoimmune inflammatory cell populations. These results are dependent on the genetic background of the mice, as genetic ablation of cGAS or STING in B6.MRL/Mp-Faslpr/Faslpr mice or B6.Sle1-Faslpr/Faslpr mice does not recapitulate the phenotype of cGAS-/- or STING gt/gt.C57BL/6J-Faslpr/Faslpr mice. My results provide more insight into the innate immune mechanisms involved in DNA vaccination and show that 2'3'-cGAMP promotes the enhanced development of protective immune responses, thereby demonstrating the potential utility of 2'3'-cGAMP as a molecular adjuvant for vaccines. Furthermore, my results demonstrate that the cGAS-STING pathway contributes to autoimmune disease development in C57BL/6J-Faslpr/Faslpr mice and implicates cGAS or STING as potential therapeutic targets for the treatment of SLE.Item Roles of Cyclic GMP-AMP Synthase in Immune Defense Against Retroviruses and Autoimmunity(2015-12-10) Gao, Daxing; Yan, Nan; Chen, Zhijian J.; Hooper, Lora V.; Pasare, ChandrashekharThe presence of DNA in the cytosol is known to trigger robust innate immunity. Cyclic GMP-AMP synthase (cGAS) is the sensor of cytosolic DNA and activation of cGAS initiates cytokine production. Here we show cGAS plays essential roles in immune defense against retroviruses as well as in autoimmune diseases caused by self-DNA. HIV infection abrogates adaptive immunity by the depletion of CD4 T cells. However, innate immune defense mechanisms against HIV are largely unknown. We show that pseudotyped HIV can infect human and mouse cell lines, leading to the production of interferons (IFN) and other antiviral cytokines. Activation of innate immunity by HIV requires viral cDNA synthesis but not cDNA integration. Furthermore, retrotranscribed HIV cDNA is sensed by the cytosolic DNA sensor cGAS, which then produces the second messenger 2'3'-cyclic GMP-AMP (cGAMP) to activate the adaptor STING. Importantly, wild-type HIV also triggers cGAMP production in human primary macrophages, underscoring the key role of cGAS in HIV sensing. Moreover, cytosolic sensing of other retroviruses such as murine leukemia virus and simian immunodeficiency virus also depends on cGAS. cGAS is important for the immune response against retroviruses, however, overactive cGAS causes autoimmunity. TREX1 is a cytosolic DNase which clears mislocalized DNA in the cytosol. Loss-of-function mutations in TREX1 cause the human disease Aicardi-Goutières syndrome (AGS). AGS manifests with abnormal type I IFN production and inflammation in multiple organs. Trex1-/- mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of IFN-induced genes (ISGs). Here we show that genetic ablation of cGAS in Trex1-/- mice eliminates all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Similarly, deletion of cGAS in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescues the lethal autoimmune phenotype of the DNaseII-/- mice. Also, polyarthritis in DNaseII-/- Ifnar1-/- mice is dependent on cGAS. These results improve our understanding of immune detection of HIV and provide cGAMP as a new adjuvant for developing HIV vaccines. Moreover, our results suggest that inhibition of cGAS may lead to new treatments of some human autoimmune diseases caused by self-DNA.Item Structural and Kinetic Characterization of Protein Ampylation by VopS Fic Domain(2012-07-20) Luong, Phi Hoang; Orth, KimThe bacterial pathogen Vibrio parahaemolyticus manipulates host signaling pathways by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylating a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. We describe features of the VopS crystal structure, including a hairpin element that is responsible for protein–protein interaction and residues involved in ATP binding. Steady-state analyses of VopS point mutants provide kinetic understanding on the functions of conserved residues for the AMPylation activity. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. The structure of VopS and its ternary reaction mechanism provide critical groundwork for future studies on AMPylators, a novel family of enzymes that modify hydroxyl-containing residues with AMP. We also developed molecular tools to facilitate the study of protein AMPylation in collaboration with Howard Hang at The Rockefeller University. An ATP analogue, N6pATP, was developed that utilizes click chemistry to allow for the detection of AMPylated proteins by fluorescent or biotin tags. N6pATP can be utilized in in vitro AMPylation reactions catalyzed by known AMPylators including Fic domain and adenylyltransferase domain proteins. Further, we showed that N6pATP can be used for the detection and purification of endogenous AMPylated proteins. Preliminary studies were performed on another effector protein of unknown function, VopQ from Vibrio parahaemolyticus. The protein sequence of VopQ does not resemble any known protein domains. Various constructs were made for VopQ, and here I describe the purification and crystallization of VopQ.Item Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase(2010-05-14) Huerta, Carlos, Jr.; Zhang, HongFlavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Extensive biochemical studies from endogenous bacterial and mammalian FMNATs using FMN analogs and various cations have suggested that the architectures of the substrate binding and catalytic sites are different. Sequence comparison reveals that eukaryotic FMNAT is related to the PAPS reductase-like family, which belongs to the adenine nucleotide α hydrolase-like superfamily. Despite the classification of eukaryotic FMNAT, the residues involved in substrate binding and catalysis are not completely known, as eukaryotic FMNAT has no sequence similarity to other known flavin binding proteins. To determine the unique flavin binding site, and to investigate the residues involved in substrate binding and the mechanism of catalysis, we utilized X-ray crystallography and biochemical methods. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata (CgFMNAT). Four crystal structures of CgFMNAT in different complexed forms were determined at resolutions between 1.20-1.95 Å, capturing the enzyme active site states prior to and after catalysis. These structures reveal a novel flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNAT provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. The different complexed forms of CgFMNAT allowed a structure-based investigation into the kinetic properties of eukaryotic FMNAT, whereby two "supermutants" were identified from mutagenic analysis. The steady-state kinetics and product inhibition properties of the two "supermutants" provided a basis for understanding the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.Item Uncovering Reversible AMPylation of BiP Mediated by dFic During ER Homeostasis(2015-01-16) Ham, Hyeilin; Tu, Benjamin; Orth, Kim; Krämer, Helmut; Liu, QinghuaAMPylation is a posttranslational modification involving a covalent attachment of an AMP moiety from ATP to hydroxyl side chains of target substrates. Fic domain which mediates AMPylation is highly conserved across species, including higher eukaryotes, implicating an essential role of this modification in cellular function. Despite the recent discoveries and characterization of a number of bacterial AMPylators and their targets during pathogenesis, the knowledge of AMPylation in eukaryotic system is still elusive. Therefore, the goal of my thesis is to determine the eukaryotic function of AMPylation and identifying the endogenous substrates of this novel modification. In an attempt to understand the physiological function of AMPylation in eukaryotes, we used Drosophila melanogaster as our genetic model organism and created mutant flies lacking functional Drosophila Fic (dFic). We found that the flies without enzymatic function of dFic exhibit blind phenotype due to impaired synaptic transmission. dFic enzymatic activity is required in glial cells for the normal visual neurotransmission. This suggests that a target of dFic may be a component of the visual signaling pathway. dFic was observed in the cell surface of the glial cells particularly enriched in capitate projections. However, dFic is localized to the ER in a number of fly tissues and also in the S2 cells, indicating that there may be another target of dFic in the ER that plays a more general role in the cellular function. In this study, we identified an ER molecular chaperone BiP/GRP78 as a novel substrate for dFic-mediated AMPylation. BiP was predominantly labeled with AMP by dFic in S2 cell lysate. AMPylation of BiP decreases during ER stress but increases upon the reduction of unfolded proteins. Both dFic and BiP are transcriptionally activated upon ER stress induction, implicating a role for dFic in the UPR. We identified a conserved threonine residue, Thr366, as the AMPylation site, which is in close proximity to the ATP binding site of BiP's ATPase domain. Our study presents the first substrate of AMPylation by a eukaryotic protein and proposes a new mode of posttranslational regulation of BiP, which is likely to serve a crucial role in maintaining ER protein homeostasis.