Browsing by Subject "Nucleotides, Cyclic"
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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 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.