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LRRC59 modulates type I interferon signaling by restraining the SQSTM1/p62-mediated autophagic degradation of pattern recognition receptor DDX58/RIG-I.

ABSTRACT: DDX58/RIG-I, is a critical pattern recognition receptor for viral RNA, which plays an essential role in antiviral immunity. Its posttranslational modifications and stability are tightly regulated to mediate the moderate production of type I IFN to maintain the immune homeostasis. Recently, we reported that macroautophagy/autophagy balances type I IFN signaling through selective degradation of ISG15-associated DDX58 via LRRC25. However, the regulatory mechanism about the autophagic degradation of DDX58 remains largely undefined. Here, we identified LRRC59 as a vital positive regulator of DDX58-mediated type I IFN signaling. Upon virus infection, LRRC59 specifically interacted with ISG15-associated DDX58 and blocked its association with LRRC25, the secondary receptor to deliver DDX58 to autophagosomes for SQSTM1/p62-dependent degradation, leading to the stronger antiviral immune responses. Thus, our study reveals a novel regulatory role of selective autophagy in innate antiviral responses mediated by the cross-regulation of LRRC family members. These data further provide insights into the crosstalk between autophagy and innate immune responses.Abbreviations: ATG: Autophagy-related; Baf A₁: Bafilomycin A₁; DDX58/RIG-I: DEAD [Asp-Glu-Ala-Asp] box polypeptide 58; EV: Empty vector; IC poly[I:C]: Intracellular polyriboinosinic polyribocytidylic acid; IFIH1/MDA5: Interferon induced with helicase C domain 1; IFN: Interferon; ISG15: ISG15 ubiquitin like modifier; IKBKE: Inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: Interferon regulatory factor 3; KO: Knockout; LRRC: Leucine rich repeat containing; MAVS: Mitochondrial antiviral signaling protein; CGAS/MB21D1: Cyclic GMP-AMP synthase; SeV: Sendai virus; siRNA: small interfering RNA; SQSTM1/p62: Sequestosome 1; TBK1: TANK binding kinase 1; TLR: Toll like receptor; TMEM173/STING: Transmembrane protein 173; VSV: Vesicular stomatitis virus; WT: Wild type.

SUBMITTER: Xian H

PROVIDER: S-EPMC6999607 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMultiple host factors are involved in modulating type I interferon expression induced by viruses; however, the mechanism is not fully elucidated. Influenza A virus infection causes severe respiratory symptoms and triggers a series of signaling cascades and host innate immune responses, including interferon production. The co-IP/MS technology was used to screen several antiviral factors in the early stage. Among these factors, ariadne-1 homolog (ARIH1) caught our attention.MethodsWestern blot assay was performed to detect the level of proteins and software ImageJ was used to analyze the band intensities. Polymerase activity assay was conducted to evaluate the polymerase activity of influenza A virus. Tissue culture infective dose (TCID50) assay was performed to measure influenza A virus titers, and quantitative RT-PCR assay was applied to test the mRNA level of IFN-β, ISG56, and CXCL10. Luciferase reporter assay was used to confirm the target of ARIH1 in RIG-I signaling. Immunoprecipitation assay was performed to detect the interaction and the ubiquitination of the proteins. All data were analyzed by biostatistical methods and presented as means ± standard deviation from three independent experiments. Statistical significance was determined using two-tailed student's t test. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered highly significant (ns, P ≥ 0.05; *, P < 0.05; and **, P < 0.01).ResultsWe found that ARIH1, a member of E3 ubiquitin ligases, enhanced cellular antiviral responses. Subsequent study showed that ARIH1 was up-regulated during influenza A virus infection. Further analysis showed that ARIH1 enhanced IFN-β and downstream gene expression by affecting the degradation of RIG-I through the SQSTM1/p62 signaling pathway.ConclusionThis newly revealed mechanism shows that cellular response increases of ARIH1 and promotes IFN-β expression to boost host survival during viral infection.

Project description:Lipotoxicity, induced by saturated fatty acid (SFA)-mediated cell death, plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). The KEAP1 (kelch like ECH associated protein 1)-NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) pathway is a pivotal defense mechanism against lipotoxicity. We previously reported that SQSTM1/p62 has a cytoprotective role against lipotoxicity through activation of the noncanonical KEAP1- NFE2L2 pathway in hepatocytes. However, the underlying mechanisms and physiological relevance of this pathway have not been clearly defined. Here, we demonstrate that NFE2L2-mediated induction of SQSTM1 activates the noncanonical KEAP1-NFE2L2 pathway under lipotoxic conditions. Furthermore, we identified that SQSTM1 induces ULK1 (unc-51 like autophagy activating kinase 1) phosphorylation by facilitating the interaction between AMPK (AMP-activated protein kinase) and ULK1, leading to macroautophagy/autophagy induction, followed by KEAP1 degradation and NFE2L2 activation. Accordingly, the activity of this SQSTM1-mediated noncanonical KEAP1-NFE2L2 pathway conferred hepatoprotection against lipotoxicity in the livers of conventional sqstm1- and liver-specific sqstm1-knockout mice. Moreover, this pathway activity was evident in the livers of patients with nonalcoholic fatty liver. This axis could thus represent a novel target for NAFLD treatment. Abbreviations: ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; BafA1: bafilomycin A1; CM-H2DCFDA:5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; FASN: fatty acid synthase; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-L-cysteine; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; RBX1: ring-box 1; ROS: reactive oxygen species; SESN2: sestrin 2; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; TBK1: TANK binding kinase 1; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase.

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LRRC59 modulates type I interferon signaling by restraining the SQSTM1/p62-mediated autophagic degradation of pattern recognition receptor DDX58/RIG-I.

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