Functional And Mechanistic Study Of Inflammasome-associated Viral Sensor

The inflammasome plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine.NLRP6 and NLRP9 are members of the NOD-like receptor(NLR)family.It recognizes microbe-associated molecular patterns(MAMPs)and damage-associated molecular patterns(DAMPs)as a cytosolic innate immune sensor.Upon receiving activation signal,NLRP6 and NLRP9 recruits the adaptor apoptosis-associated speck-like protein(ASC)and caspase-1 or caspase-11 to form an inflammasome,which mediates the maturation and secretion of the proinflammatory cytokines IL-18 and IL-1β,as well as GSDMD-mediated pyroptosis.On the one hand,tight regulation of the NLRP6 inflammasome is essential to maintain tissue homeostasis.On the other hand,improper inflammasome activation may contribute to the development of multiple diseases.In intestinal epithelial cells,the NLRP6 inflammasome plays a key role in regulating gut microbiome composition,goblet cell function,gastrointestinal inflammation,viral infection and susceptibility to tumor.However,the molecular mechanisms underlying the regulation of NLRP6 inflammasome to maintain homeostasis and prevent excessive inflammation remain unclear.Meanwhile,NLRP9 relies on DHX9 to recognize viral RNA.The mechanism by which DHX9 antiviral infection is unclear.In summary,although NLRP6 and DHX9 are both RNA virus sensors,the specific mechanism to regulate viral infection needs to be further explored.Post-translational modifications(PTMs)play key roles in regulating the function of individual protein molecules and large complexes.Emerging roles have been identified for many intracellular molecules in regulating inflammasome activation.Various modifying enzymes regulate inflammasome activation by binding to the components of inflammasome to change PTMs,such as acetylation,sumoylation,ubiquitination and phosphorylation.To date,NLRP6 has only been reported to be deubiquitinated by CYLD,but not any specific modification site was identified.The deubiquitinase CYLD regulates the function of the NLRP6 inflammasome and prevents excessive inflammation via the production of IL-18.Mechanistically,CYLD cleaves the K63-linked ubiquitination of NLRP6,thereby inhibiting NLRP6 ability to interact with ASC.However,how NLRP6 inflammasome maintains homeostasis and whether other PTMs exist in NLRP6 remains largely unknown.More importantly,the key questions of NLRP6 inflammasome about recognition,assembly,activation,and regulation at the molecular level remain unclear.Therefore,considering the subsequent precise targeted intervention,the specific modification site of NLRP6 needs to be identified.Moreover,although viral sensor DHX9 are mostly localized in the nucleus,it is not clear whether DHX9 in the nucleus perform antiviral functions through NLRP9independent pathways.In our work,as for NLRP6 inflammasome,we identified the monoubiquitination modification of NLRP6 in vivo for the first time.Moreover,we found the ubiquitin ligase UBE2O monoubiquitinates NLRP6.In detail,We first identified the NLRP6 binding protein UBE2O in mouse intestinal epithelial cells by GST-pulldown.We demonstrated in vitro and in vivo that UBE2O specifically and directly binds to NLRP6,but not NLRP3 or NLRP9.And both are co-localized in the cytoplasm.Subsequently,we found that UBE2O modifies NLRP6 in an enzyme-dependent manner.And results from different methods demonstrate that UBE2O is a ubiquitin ligase of NLRP6,capable of monoubiquitinating NLRP6 at multiple sites.Furthermore,we demonstrated that UBE2O modifies the PYD,NATCH and LRR domains of NLRP6 respectively with biochemical methods.Then,after constructing a mutant library and screening,we identified the modified lysine sites of NLRP6:K115-K130,K365-K455 and K680K687.And we demonstrated that these modification sites are sufficient and necessary for multi-monoubiquitination of NLRP6.Then we found that UBE2O promotes the cytoplasmic sequestration,oligomerization,ASC recruitment and RNA binding of NLRP6 in an enzyme-dependent manner in vitro.Subsequently,we identified the nuclear localization signals K115-K130 of NLRP6.Finally,we found that NLRP6 exhibit significant nucleocytoplasmic colocalization in vivo and identified NLRP6 monoubiquitination for the first time.Morever,intestinal epithelial cell UBE2O conditional knockout mice show a protective role of UBE2O against DSS-induced colitis.Finally,intestinal epithelial cell UBE2O conditional knockout mice significantly show a protective role against rotavirus infection.As for NLRP9 inflammasome viral sensor DHX9,we constructed myeloidspecific and liver-specific knockout mice to demonstrate that DHX9 acts as a transcriptional co-activator in the nucleus in addition to traditional double-stranded RNA induction in the cytoplasm by mitochondrial antiviral signaling protein MAVS or transcriptional activator STAT1 knockout in DHX9 deficient mice.Stimulated by interferon,DHX9 binds directly to STAT1 in the nucleus and recruits RNA polymeraseⅡ to the interferon-stimulating gene promoter region.Then DHX9 participates in STAT1-mediated interferon-stimulated gene transcription Sand promotes interferonstimulated gene expression to antagonize RNA viruses.In conclusion,we first reported the monoubiquitination modification of NLRP6 and demonstrated that the intestinal UBE2O-NLRP6 axis is critical for maintaining intestinal homeostasis.In the future,we will further explore the pathogenic mechanism and physiological significance of the UBE2O-NLRP6 axis at different tissues and levels in order to explain the relevant host defense mechanisms and develop effective therapeutic strategies for chronic diseases.At the same time,we reported for the first time the mechanism by which DHX9 exerts antiviral function in the nucleus in inflammasome-independent pathway,which also provides important ideas for understanding the regulatory strategy of interferon-stimulating genes.