The Regulation Of CGAS Phase Separation And Activation

Background: Phase separation(phase transition)is a ubiquitous phenomenon existed in nature,which was originally a physical concept,has been observed in numerous biological processes in recent years.The traditional perspective holds that there are amounts of membrane organelles in cells,including mitochondria and nucleus which have a single or multilayer membrane structure,sequester themselves from environment.The membrane organelles make sure that the reactions,which occur in cell,could be more efficiently.Recently years,plenty of evidences show that many membraneless organelles also exist in cells,such as nucleolus,stress granules,autophagosome and P granules,and are responsible for the inner contents exchange with external.However,the formation mechanism of membraneless organelles is still confusing and the appearance of phase separation open a new window for us to understand it.Based on the phase separation,some of specific protein or nucleic acid could condense into a new phase differed from the former,which is known as membraneless organelle.Usually,phase separation of specific molecules in cells contribute to the rational division of functional area and carry out normal physiological activities.However,the abnormal of the process is relevant with many neurodegenerative diseases,including ALS(Amyotrophic Lateral Sclerosis)、 FTD(Frontotemporal lobe dementia)and AD(Alzheimer’s disease).It is worth mentioning that phase separation is closely related to the immune system,which is not only regulates T cell receptor signaling molecule transduction in adaptive immunity,but also tightly regulates the function of cGAS,a key receptor for DNA recognition in innate immunity.In 2013,Professor Zhijian Chen of Southwest Medical Center first discovered cGAS,a critical DNA sensor in the cytoplasm.cGAS,namely cyclic guanosine phospho-adenosine synthase,which recognizes the intracellular DNA and synthesizes the second messenger 2’,3’-c GAMP with ATP and GTP,followed by activating the downstream interferon signaling pathway.In 2018,Professor Chen Zhijian found that DNA-induced phase separation of cGAS and leads to the transformation from dispersion state into droplets.This process makes the cGAS-DNA complex a highly agglutinated ‘micro-reactor’,in which the cGAS protein,DNA,ATP and GTP are highly concentrated,thus greatly promote the production of the effector molecule c GAMP.In addition,zinc ions can enhance the enzyme activity of cGAS by promoting the phase separation of cGAS-DNA.Another class of nucleic acid-associated membraneless organelles called stress granules are dynamic membraneless organelles composed of RNA and proteins,which are mainly induced by various stresses such as heat shock,oxidative stress,nutrient deficiency and viral infection.As a key protein of stress granules,G3BP1(G3BP stress granule assembly factor 1)contains a special structural domain,namely internal disorder region(IDRS),which plays an essential role in mediating the weak interaction between protein and protein or nucleic acid and the occurrence of phase separation.We previously found that G3BP1 can promote the DNA binding and activation of cGAS.In this study,we explored the molecular mechanism about how G3BP1 regulates the cGAS phase separation.Objective: Revealing the role of G3BP1 played in DNA-induced phase separation of cGAS and providing a new strategy for cGAS regulation and the related diseases treatment.Methods: Firstly,We expressed and purified cGAS protein,various truncated G3BP1 proteins in the prokaryotic expression system of E.coli.Turbidity detection,time-lapse and fluorescence bleaching recovery(FRAP)were performed to determine the role of G3BP1 in the regulation of DNA-induced cGAS phase separation.The U937 cell line with knockout of G3BP1 gene was generated by CRISPR-Cas9 technology.The effect of G3BP1 on cGAS phase separation and activation were explored based on immunofluorescence imaging,real-time quantitative PCR detection,pull-down assay and other technologies.Results: The G3BP1 promoted the pre-condensation formation of cGAS and increased the turbidity of the reaction system,and the above effects were positively correlated with the concentration of G3BP1.Time-lapse showed that the cGAS-DNA droplets were positively correlated with the concentration of double-stranded DNA,with the increasing concentration of ds DNA,the size and number of droplets formed by cGAS increased significantly.More importantly,G3BP1 promoted the droplets formation process of DNA-cGAS.Fluorescence recovery after photobleaching(FRAP)showed that the droplets formed by the co-incubation of G3BP1 and cGAS could not recover soon after fluorescence bleaching,which indicated that the G3BP1-cGAS droplet had poor internal fluidity.We then observed droplets formation by incubating various truncated G3BP1 proteins with cGAS and ds DNA(Double strand DNA),the results showed that only full-length G3BP1 enhanced DNA-induced phase separation of cGAS.In addition,by detecting the synthesis of c GAMP downstream of cGAS,we also found that only the full-length G3BP1 can significantly increase the activation of cGAS.Above findings were also validated in cells.Compared with WT(Wild-type)cells,G3BP1-deficiency cells had a significant reduction in the number of intracellular cGAS condensates under DNA stimulation.These results suggested that G3BP1 also promoted intracellular DNA-induced cGAS phase separation.Combine with that G3BP1 knockout could suppress the interferon expression induced by different concentrations of ds DNA,G3BP1 plays an critical role in the cGAS-mediated interferon signaling pathway.Conclusions:The full-length G3BP1 can promote the formation of cGAS pre-condensate state.Furthermore,full-length G3BP1 can promote DNA-induced cGAS phase separation and activation.