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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01j098zf47k
Title: REGULATORY MECHANISMS OF INNATE IMMUNITY AND THEIR ROLES IN CELL STATE MAINTENANCE
Authors: Liu, Dawei
Advisors: Cristea, Ileana M.
Contributors: Molecular Biology Department
Keywords: DNA sensing
immune signaling
innate immunity
liquid-liquid phase separation
mesenchymal-to-epithelial transition
virus infection
Subjects: Molecular biology
Virology
Biochemistry
Issue Date: 2024
Publisher: Princeton, NJ : Princeton University
Abstract: Throughout evolution, mammalian cells have developed a complex intrinsic and innate immune system for defense against pathogens by using pattern recognition receptors (PRRs) to detect pathogen associated molecular patterns (PAMPs). During viral infection, the nucleic acid that comprises the viral genome is a prototypic PAMP that can be recognized by host PRR sensors of viral DNA or RNA . Upon detection and direct binding, these sensors initiate rapid and extensive signaling cascades that can promote the expression of type I interferons (IFN), pro-inflammatory cytokines, and interferon-stimulated genes. The functions of these signaling molecules during an infection span a broad range of immune responses, which culminate in the intra- and inter-cellular restriction of virus replication and spread. To exert such functions, a paradigm has emerged supporting the universal formation of higher-order PRR assemblies and supramolecular complexes of nanometer to micrometer scales for innate immune signal amplification.A prominent nuclear sensor of pathogenic DNA is the adaptively evolved interferon-inducible protein 16 (IFI16). IFI16 has been shown to bind to the DNA genomes of herpesviruses, including herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV), Kaposi’s sarcoma-associated herpesvirus (KSHV), as well as to DNA intermediates of retrovirus human immunodeficiency virus 1 (HIV-1). Upon binding to viral DNA via its two HIN200 domains, IFI16 undergoes cooperative oligomerization mediated by the N-terminal PYRIN domain (PY) and induces two types of antiviral responses—the induction of antiviral cytokines and suppression of viral gene expression. Despite the advances in the understanding of the innate immune functions of IFI16, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. In addition, significant gaps remain in our understanding of its functions outside of infection contexts, particularly in relation to cellular homeostasis and cell state maintenance. While its ability to modulate cytokine signaling links IFI16 to broader cellular processes, the specific mechanism of how it influences cellular behavior under non-infectious conditions remain largely unexplored. This study first explores the how the biophysical properties of IFI16 serve to initiate and control its immune activation. To accomplish this, we used an integrative approach that combined proteomics with virology, biochemistry, microscopy, genetics and classical molecular biology. We established that the innate immune functions of IFI16 are controlled by the liquid-liquid phase separation (LLPS), mediated by combinatorial phosphorylation within an intrinsically disordered region (IDR). Key phosphorylation sites within IDR are modified by CDK2, DNA-PK and GSK3β, which provide a toggle for active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. The second part of this study focuses on the role of IFI16 outside the context of infection. Specifically, we discovered that IFI16 functions to maintain the mesenchymal state of human fibroblasts. We found that mutations of the key phosphorylation residues of IFI16, or a complete knockout of IFI16, led to a mesenchymal-to-epithelial transition (MET) phenotype of fibroblasts over multiple rounds of passaging. This process is dependent upon the tumor suppressor protein p53, which was upregulated in these cells. Finally, our single nucleotide polymorphism (SNP) analysis of IFI16 supports that charge mutations within IFI16 PY-IDR are potential disease risk factors, highlighting the link between innate immune signaling and cell state maintenance.
URI: http://arks.princeton.edu/ark:/88435/dsp01j098zf47k
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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