Kirchhoff, Alexander: Characterization of a novel multiprotein complex downstream of the cytosolic nucleic acid sensors cGAS and RIG-I. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Alexander Kirchhoff}},
title = {Characterization of a novel multiprotein complex downstream of the cytosolic nucleic acid sensors cGAS and RIG-I},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = aug,

note = {Detection of viral nucleic acids by specialized PRRs of the innate immune system triggers the expression of type I IFNs, a hallmark of antiviral defense. In vertebrates, the cGAS-STING pathway and the RLR-MAVS pathway have emerged as the major cytosolic type I IFN-inducing nucleic acid sensing systems. Although both systems sense the genetic material of different virus classes, signaling downstream of the initial receptor-ligand contact involves common kinases (i.e., TBK1, IKKε) and transcription factors (i.e., IRF3, NF-κB p65).
This thesis has characterized the role of hnRNPM, a predominantly nuclear protein, in cytosolic nucleic acid sensing in the monocytic cell line THP-1. hnRNPM positively regulated both the cGAS-STING- and RIG-I-dependent type I IFN induction by promoting the phosphorylation of TBK1 and/or IRF3. A combined AP-MS/RNAi screen identified ELAVL1 and SON as hnRNPM interactors that also promote cGAS and RIG-I signaling by enhancing TBK1 and/or IRF3 phosphorylation. IGF2BP2, an RNA-dependent hnRNPM interactor, amplified the cGAS- and RIG-I-induced type I IFN response in a TBK1 phosphorylation-independent manner.
To further evaluate the function of ELAVL1, the ELAVL1 gene locus was deleted in THP-1 cells. A more detailed investigation revealed that ELAVL1 promoted both the expression of type I IFNs and activation of NF-κB after stimulation of cGAS or RIG-I. The type I IFN response was downregulated by approximately 6-fold to 8-fold in ELAVL1 KO cells and was dependent on the N- and C-terminal RRMs of ELAVL1. Overexpression of ELAVL1-FLAG in ELAVL1-deficient THP-1 dual cells rescued the cGAS- or RIG-I-induced phosphorylation of TBK1/IRF3 and the subsequent ISG response. By contrast, the TLR1/TLR2-mediated phosphorylation of TBK1 was ELAVL1-independent, indicating that ELAVL1 might regulate the activation of different TBK1 subpopulations in a PRR-dependent manner. Analyzing the global mRNA expression profile further demonstrated that ELAVL1 does not regulate the expression of genes associated with cGAS and RIG-I signaling.
A comparative interactome analysis identified common interactors of hnRNPM and ELAVL1, including RNA-binding proteins, ribosomal proteins, splicing factors, and also type I IFN-inducing proteins (i.e., DDX3X). The interactions with SON were restricted to hnRNPM, suggesting that hnRNPM forms distinct complexes with ELAVL1 and SON. In addition, hnRNPM also interacted with TBK1, IKKε, IKKβ, and NF-κB p65. PLAs confirmed that the interactions between hnRNPM, ELAVL1, and TBK1 phosphorylated at Ser172 occur in cellulo and primarily in the cytoplasm, indicating that the minor cytoplasmic rather than the predominant nuclear portions of hnRNPM and ELAVL1 mediate TBK1 phosphorylation. Furthermore, the antibody-based purification of the multiprotein complex assembled by hnRNPM enhanced the phosphorylation of recombinant TBK1 in the activation loop.
In summary, this thesis provides new insights into the signaling cascades downstream of cGAS and RIG-I and has identified a novel multiprotein complex that positively regulates both pathways at a common point of convergence, TBK1 phosphorylation. A better understanding of the newly identified multiprotein complex might contribute to the development of new therapies for the treatment of inflammatory diseases resulting from dysregulation of cGAS-STING or RIG-I-MAVS signaling (i.e., AGS, SMS, or SAVI).},

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