Liquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways

dc.contributor.advisorLiu, Yien
dc.contributor.committeeMemberChen, Zhijian J.en
dc.contributor.committeeMemberBeutler, Bruceen
dc.contributor.committeeMemberO'Donnell, Kathryn A.en
dc.creatorDu, Mingjianen
dc.creator.orcid0000-0002-9464-4098
dc.date.accessioned2023-09-14T22:29:05Z
dc.date.available2023-09-14T22:29:05Z
dc.date.created2021-08
dc.date.issuedAugust 2021
dc.date.submittedAugust 2021
dc.date.updated2023-09-14T22:29:05Z
dc.description.abstractThe 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.en
dc.format.mimetypeapplication/pdfen
dc.identifier.urihttps://hdl.handle.net/2152.5/10199
dc.language.isoenen
dc.subjectDNAen
dc.subjectImmunity, Innateen
dc.subjectNucleotides, Cyclicen
dc.subjectNucleotidyltransferasesen
dc.titleLiquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathwaysen
dc.typeThesisen
dc.type.materialtexten
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.disciplineGenetics, Development and Diseaseen
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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