TLR4 Acetylation And Methylation On Inflammatory Immunity Regulation

Toll like receptor 4 (TLR4) plays a crucial role in anti-infection, especially antiviral responses. Upon LPS binding, TLR4 is activated, forms a complex with CD 14, MD2, and then triggers downstream signal pathways. One of them is dependent on myeloid differentiation factor 88(MyD88). Through binding to TIR domain containing adaptor protein(TIRAP) and MyD88, TLR4 activates the nuclear factor kappa B(NF-?B), which translocates to the nucleus and induces the expressions of a number of anti-inflammatory genes such as interleukin 6 (IL6), IL12, IL17, and tumor necrosis factor a(TNF-a). The other pathway is MyD88-independent. Through binding to TIR domain containing adaptor protein inducing IFN?(TRIF) and TRIF related adaptor molecule(TRAM), TLR4 activates IFN-regulated factor-3(IRF3) and IRF7, which induce the expressions of a series of antiviral genes including interferon beta (IFN(3).Posttranslational modifications, including acetylation and methylation, are crucial for gene transcription and signal transduction. However, the posttranslational modifications of TLR4 are largely unknown. Here we explored TLR4 acetylation and methylation in TLR4 activation.I. The role of TLR4 acetylation in inflammatory signal transduction activated by LPS1. Western blot analysis using a pan-acetylation antibody showed that TLR4 was acetylated after LPS stimulation in Raw264.7 cells. Immunofluorescence (IF) analysis showed that creb-binding protein (CBP) translocated to the cytoplasm upon LPS stimulation. Co-immunoprecipitation (Co-IP) experiments showed that LPS stimulation induced the binding of CBP to TLR4. TLR4 acetylation was reduced when CBP expression was down-regulated, indicating that CBP mediated TLR4 acetylation. The acetylation of TLR4 significantly decreased when lysine 732 (K732) or lysine 813 (K813) was mutated to arginine (R). Mass spectrometry analysis recovered K813 as the acetylation site. In addition, LPS-stimulated TLR4 acetylation was detected by specific antibodies targeting acetyl-K732 and acetyl-K813. Altogether, our data strongly indicated that LPS induced TLR4 K813 and K732 acetylation which was mediated by CBP.2. K732R mutation interrupted the interaction between TLR4 with TRIF and MyD88, while K813R mutation interrupted the interaction between TLR4 and TRAM. Luciferase analysis demonstrated that enhancement of TLR4 acetylation by overexpression of CBP increased the activities of NF-?B-and IRF3-responsive elements. Both K732R and K813R mutations decreased the IRF3-responsive element activity, besides K732R mutation decreased the NF-KB-responsive element activity. Reverse transcription PCR analysis revealed that both K732R and K813R mutations inhibited the production of IFN(3 in bone marrow-derived macrophage (BMDM) from TLR4-/-mouse, while only K732R mutation inhibited the production of IL6. The above results indicated that the acetylation of lysine 732 and 813 differentially regulated downstream signaling pathways.3. Western blot analysis using a pan-acetylation antibody showed that IRF3 was acetylated after LPS stimulation in Raw264.7 cells. IRF3 acetylation was reduced when CBP expression was down-regulated, indicating that CBP mediated IRF3 acetylation. Mass spectrometry analysis recovered K77 as the acetylation site. The acetylation of IRF3 significantly decreased when lysine 77 was mutated to arginine. IRF3 acetylation was significantly reduced when histone deacetylase 6 (HDAC6) was overexpressed, indicating that HDAC6 mediated IRF3 deacetylation. Co-IP analysis showed that K77R mutation interrupted IRF3 dimerization. The inhibitors of deacetylase trichostatin A (TSA) and nicotinamide (NAM) increased IRF3 dimerization. Both down-regulating CBP expression and K77R mutation prevented IRF3 from translocating to the nucleus. The above results indicated that acetylation at K77 induced by LPS promoted IRF3 dimerization and transloction to the nucleus. Main conclusions:(1)LPS induced TLR4 K732 and K813 acetylation mediated by CBP.(2) Acetylation at K732 is essential for both MyD88-and TRAM/TRIF-mediated signaling pathways activation, while acetylation at K813 is essential for TRAM/TRIF-mediated signaling activation. (3)Acetylation at K77 induced by LPS promoted IRF3 dimerization and transloction to the nucleus.?. The role of TLR4 methylation in inflammatory signal transduction activated by LPS1. Co-IP analysis showed that TLR4 bound to IRF3 directly. Western blot analysis showed that TLR4 was methylated after LPS stimulation. Overexpression of PRMT2 increased TLR4 methylation. The methylation significantly decreased when R731 or R812 was mutated to K, indicating that LPS induced TLR4 methylation at R731 and R812.2. Co-IP analysis showed that R812K mutation interrupted the interaction between TLR4 and IRF3. Luciferase analysis showed that enhancement of TLR4 methylation by overexpression of PRMT2 increased the activities of IRF3-responsive elements, while R812K mutation decreased it. RT-PCR analysis revealed that TLR4 R812K mutation inhibited the production of IFN?in BMDM from TLR4-/-mouse. These data indicated that R812 methylation is essential for TLR4 binding to IRF3 and IRF3 activation.3. Western blot analysis also showed that IRF3 was methylated under LPS stimulation. The methylation of IRF3 significantly decreased when R285 was mutated to K. Co-IP analysis showed R285K mutation in IRF3 interrupted its dimerization and prevented IRF3 from translocating to the nucleus. The above results indicated that methylation induced by LPS promoted IRF3 dimerization and transloction to nucleus.Main conclusions:(1)LPS induced TLR4 R731 and R812 methylation mediated by PRMT2. (2)TLR4 R812 methylation mediated the direct interaction between TLR4 and IRF3 and IRF3 activation. (3)Methylation at R285 induced by LPS promoted IRF3 dimerization and transloction to the nucleus.