The Mechanism That TRAF4 Regulates Cell Death

Cell death is closely related to development,aging and disease.Apoptosis is a relatively mild way of cell death.However,necrosis occurs fast.Early studies believed that cell necrosis was uncontrollable.However,many studies now prove cell necrosis is also regulated.When cell death occurs,if the activity of caspase-8 is blocked,RIPK1 and RIPK3 will not be cleaved by Caspase-8.RIPK1 and RIPK3 bind through the RHIM domain and RIPK3 is phosphorylated.RIPK3 then recruit MLKL and phosphorylate MLKL.Phosphorylated MLKL will oligomerize and be transferred to the membrane to cause cell death.RIPK3 plays a critical role in necrosis.However,the mechanisms that regulate RIPK3 activity are not well understood.To further investigate the cell death mechnism,we analyzed ZBP1 interacting proteins with Mass Spec,and found TRAF4 is a ZBP1 binding protein.By generating TRAF4-/-MEF with CRISPR-Cas9 method,The study found that the expression of RIPK3 protein in TRAF4-/-MEF is greatly higher than that in TRAF4+/+MEF,but the expression of some other death-related proteins RIPK1 and MLKL in TRAF4-/-MEF is not different from that in TRAF4+/+ MEF.We speculate that TRAF4 has a regulatory effect on RIPK3.In response to TNF-α,Smac mimetic and caspase inhibitor z-VAD-fmk stimulation,necrosis occurs earlier in TRAF4-/-MEF than in TRAF4+/+ MEF.We believe that TRAF4 inhibits necrosis by down-regulating RIPK3 level.Our results show that TRAF4 binds to RIPK3 via the C-TRAF domain.We find that TRAF4 is the E3 ubiquitination ligase of RIPK3,which degrades RIPK3.The RING domain of TRAF4 is responsible for the ubiquitination modification of RIPK3.We also investigated the effect of TRAF4 on the activation of NF-κB,and we found that TRAF4 has basically no effect on the activation of endogenous NF-κB.We used PI staining to measure cell death and Western blot to detect the expression and activation of MLKL.We found that TRAF4 inhibits the oligomerization of RIPK3 and MLKL and the membrane translocation of MLKL.In summary,our results demonstrate that TRAF4 inhibits necrosis by degrading RIPK3.Our findings shed new light on cell necrosis study.Chemotherapy and radiotherapy for cancer treatment is mainly to kill cancer cells through apoptosis.But many cancers are resistant to chemotherapy and radiotherapy.Therefore,necrosis might be used in the treatment of cancer,and our research also provide some ideas for the development of anti-cancer drugs.