| Innate immune system is the first line for host to fight against pathogens infection.During the long-time struggle against pathogens,the innate immune system has evolved various pattern recognition receptors?PRRs?to recognize pathogen-associated molecular patterns?PAMPs?and promptly activate innate immune responses.cGAS?Cyclic GMP-AMP synthase?is one of the key pattern recognition receptors that could recognize DNA as a special type of“non-self”signals.DNA is strictly limited in the subcellular structures of nucleus or mitochondria under steady state.However,upon pathogen infection or host damage,it will be released into cytosol,which will be regarded as“non-self”signals and trigger downstream immune responses.Upon recognition and binding of DNA,cGAS is activated through conformational change and then catalyze the synthesis of a dinucleotide cGAMP from ATP and GTP.The engagement of the second messenger,cGAMP,to STING leads to activation of downstream signaling,including induction of type I IFN by transcription factor IRF3,which helps host for immune defense.cGAS could binds to DNA that derived from different types of pathogens,independently of specific DNA sequence.Such a wide range recognition of DNAs may account for the activation of cGAS by self-DNA and consequently leads to a variety of diseases onset and progression,including autoimmune diseases.Therefore,the precise regulation of cGAS activity is essential for maintaining homeostasis under non-infectious conditions and mounting adequate immune responses against pathogen infection.Our study mainly identified G3BP1?GTPase-activating protein-?SH3domain?-binding protein 1?as a pivotal regulator for DNA sensor cGAS and revealed the underlying mechanism of the regulation.In this study,we first identified G3BP1 as one of the cGAS-interacting proteins through liquid chromatography–mass spectrometry?LC–MS?.To study the role of G3BP1 in cGAS mediated type I IFN production,we established G3BP1-/-U937 cells?human monocytic cell line?by employing CRISPR/Cas9 system.Upon challenge with different types of intracellular DNAs,we found that the deletion of G3BP1 significantly suppressed DNA-induced signaling transduction and the production of type I IFN,which indicated that G3BP1 plays a critical role in the positive regulation of DNA-induced type I IFN signaling.We obtained similar results in G3bp1-/-primary mouse embryo fibroblasts?MEFs?.We infected the cells with herpes simplex virus-1?HSV-1?as DNA virus or human immunodeficiency virus?HIV?as retrovirus,to study the role of G3BP1 in antiviral immune responses.Through detecting IFN production and examining the abundance of amplified viral RNA,we demonstrated that G3BP1 promotes cGAS-mediated antiviral defense against both DNA virus and retrovirus.As the amount of cGAMP could indicate the enzymatic activity of cGAS,we establish a LC–MS/multiple reaction monitoring?LC–MS/MRM?-based approach to directly determine cGAMP production.Knockout of G3BP1 greatly reduced the cGAMP production in response to cytosolic DNA,while it did not inhibit cGAMP-induced IFN signaling.Another STING activator,c-di-GMP,further confirmed that G3BP1 does not appreciably affect the activation of STING and downstream signaling.These results suggested that G3BP1 may act on cGAS to regulate type I IFN pathway.We also test the potential effect of G3BP1 on TLR-mediated NF?B signaling,we found G3BP1 specifically regulate cGAS-mediated type I IFN signaling,but not NF?B signaling.Thus,we found that G3BP1promotes cGAS-mediated type I IFN signaling in response to different types of cytosolic DNAs and even DNAs derived from viruses,regardless of DNA length.Previous studies showed that G3BP1 is a critical component of stress granule?SG?and required for SG assembly when cell is under stress.Accordingly,we investigated whether SGs is involved in cGAS-mediated DNA-sensing signaling.We observed that DNA transfection led to the aggregation of cGAS and the co-localization of cGAS-DNA aggregation,but not SGs formation;while poly?I:C?induced the formation of SGs where there was no cGAS localization.We further disrupted the formation of SGs by knockdown of T cell–restricted intracellular antigen-1?TIA1?,which is another critical SG component,and proved that SGs is not involved in cGAS-mediated activation of IFN responses.On the other hand,cGAS deficiency showed no obvious effect on SGs formation triggered by different cellular stress.Theses results together suggested that G3BP1 regulates cGAS activation independently of SGs formation.To understand the mechanism by which G3BP1 regulates cGAS,we tested the interaction between cGAS and G3BP1.We found that G3BP1 bound to cGAS when cell was under steady state and gradually dissociated from cGAS under DNA stimulation.We next investigated the interacting-domain of these two proteins respectively by generating truncated cGAS or G3BP1 mutants.We found that any deletion of G3BP1 affect its interaction with cGAS.Only the full-length G3BP1,but not any of the mutants,could bound to cGAS and rescued IFN expression in G3BP1-deficient cells.In addition,G3BP1 was shown to bind to cGAS through N terminus of cGAS.Since N-terminal region of cGAS is required for efficient DNA binding of cGAS,we proposed that G3BP1 may regulate the DNA-binding capacity of cGAS.We therefore employed different DNA-binding assays to verify this hypothesis.We found that deletion of G3BP1 severely reduced the binding of DNA to cGAS.Consistently,DNA-induced cGAS aggregation was markedly decreased in G3BP1-deficient cells.To directly assessing the binding between cGAS and DNA,we carried out EMSAs using purified cGAS and G3BP1 recombinant proteins.The results showed that the addition of G3BP1 produced DNA-ladders and high-molecular-weight cGAS-DNA complexes at a lower cGAS concentration,which means a lower dissociation constant between cGAS and DNA and higher capacity for DNA-binding.However,different G3BP1 truncations did not show such effect.Together,we demonstrated that G3BP1 binds to N terminus of cGAS and increases the DNA-binding affinity of cGAS.Recently,a study revealed that N terminus of cGAS enhances DNA-induced phase separation of cGAS and promote the activation of cGAS.Considering the binding of G3BP1 to N terminus of cGAS,we further test the effect of G3BP1 on cGAS phase condensation.We found that G3BP1 could form a large complex with cGAS?larger than 300k Da?and promote the oligomerization of cGAS.By incubating cGAS with G3BP1 in the absence of DNA,the formation of liquid-like droplets was observed under bright-field microscope;while in the presence of DNA,G3BP1 could further promote the cGAS liquid phase separation.Consistently,different G3BP1 truncated mutants showed no obvious effect.These preliminary data suggest the potential role of G3BP1 in cGAS phase condensation,which may contribute to DNA-sensing and activation of cGAS.Additionally,in vitro cGAMP synthesis assays were performed to determine the effect of G3BP1 on cGAS activity directly.Full-length G3BP1,but not any other mutants,significantly promoted cGAMP production by enhancing cGAS activity.Since G3BP1 bound to cGAS through N terminal region,G3BP1 was unable to enhance the activity of C-terminal of cGAS.Collectively,these data support the findings that regulation of cGAS by G3BP1 depends on the binding of G3BP1 to N terminus of cGAS,which in turn promotes the DNA-binding and activation of cGAS.In conclusion,our study identified that G3BP1,a interacting-protein of DNA sensor cGAS,plays a pivotal role in the positive regulation of cGAS-mediated type I IFN signaling.It was further demonstrated that G3BP1 promotes cGAS activation and consequently activation of IFN signaling through assistance in cGAS aggregation and its binding to DNA.This mechanism offers a new perspective to illustrate the role of cGAS in various physiological and pathological conditions.This also provided a new molecular target along with its theoretical basis for seeking new strategies to control cGAS activity. |
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