NLRC3 alleviates hypoxia/reoxygenation induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6

Background:NOD-like receptor family CARD domain containing 3(NLRC3)plays an important role in both innate and adaptive immunity.This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation(H/R)-induced inflammatory response in RAW264.7 cells.Methods:Liver ischemia-reperfusion(I/R)model in mice and H/R model in RAW264.7 cells were constructed.Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3,TRAF6,p–p65,p65,IκB–α,and the K63-linked ubiquitination level of TRAF6 in cells.The immunofluorescence assay was performed to evaluate the nuclear level of the NF–κB(p65).ELISA was conducted to measure the content of IL–1βin serum and cell supernatant.The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.Results:The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time(P<0.05).The expression of NLRC3 and IκB–αprotein in RAW264.7 was decreased gradually,while the expression of p–p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time(P<0.05).The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly.However,NLRC3 had no effect on the expression of TRAF6 protein.The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R+Lv–NLRC3 group was significantly lower than that in the H/R+negative control(NC)group(P<0.05).Moreover,the activation of NF–κB in H/R+Lv–NLRC3 group was inhibited compared with that in the H/R+NC group,and the level of the inflammatory factor IL–1βin the cell culture supernatant was also decreased accordingly(P<0.05).Conclusions:NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.

Coordinated regulation of plant immunity by poly(ADP-ribosyl)ation and K63-linked ubiquitination

Poly(ADP-ribosyl)ation(PARylation)is a posttranslational modification reversibly catalyzed by poly(ADP-ribose)polymerases(PARPs)and poly(ADP-ribose)glycohydrolases(PARGs)and plays a key role in multi-ple cellular processes.The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes.Here we show that Arabidopsis UBC13A and UBC13B,the major drivers of lysine 63(K63)-linked polyubiquitination,directly interact with PARPs/PARGs.Activation of pathogen-associated molecular pattern(PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13.Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related(PR)proteins but decreased accu-mulation of secreted PR proteins.Protein disulfide-isomerases(PDIs),essential components of endo-plasmic reticulum quality control(ERQC)that ensure proper folding and maturation of proteins destined for secretion,complex with PARPs/PARGs and are PARylated upon PAMP perception.Significantly,PARylation of UBC13 regulates K63-linked ubiquitination of PDIs,which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion.Taken together,these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.