The Discovery Of The Formation Of A Nuclear Translocation Necroptotic Complex RIP3K-tAIF-MIF And The Nuclear Envelope Translocation Of MLKL

Objective:Necroptosis is a type of programmed cell death with great significance in many pathological processes.It is well known that receptor-interacting protein 1 kinase(RIP1K),RIP3K and mixed lineage kinase domain-like protein(MLKL)are three key components in the necroptotic pathway.The nuclear translocation of RIP3K and MLKL are involved in necroptosis.However,the molecular mechanisms underlying nuclear translocation of RIP3K and MLKL are largely unknown.Here,we investigate the underlying mechanisms of the nuclear translocation of RIP3K and MLKL.Methods:TNFa+zVAD was used to induce necroptosis in L929 cells,the final concentrations of TNFa and zVAD used in this study were lOng/ml and 20mM,respectively.The distribution of necrosis-related proteins(RIP1K,RIP3K and MLKL),apoptosis-inducing fator(AIF),and macrophage migration inhibitory factor(MIF)were observed in cytoplasmic,nuclear and nuclear envelope fraction with western blotting and immunofluorescence methods in TNFa+zVAD-induced L929 cells.At the same time,immunoprecipitation was used to detect the interaction of these proteins.AIF-KO L929 cells and AIF-NLS-KO L929 cells line were constructed by CRISPR-Cas9 system,and then the cell necrosis was observed with ATP level detection and PI staining in TNFa+zVAD-induced L929 cells.The effects of AIF-KO and AIF-KO on RIP3K and MLKL nuclear localization were also observed with western blotting analysis.Finally,pharmacological intervention was used to observe the relationship between RIP3K nuclear localization and MLKL nuclear localization and the effect of RIP1K on them.In vivo,we established a rat model of cerebral middle artery occlusion and reperfusion(MCAO/R).PI staining and western blotting were used to observe whether ischemic stroke can cause cerebral tissue necrotic-like death,and the expression and activation of necrosis-related protein.Finally,western blotting and immunoprecipitation were used to verify whether ischemic stroke can induce the same intracellular events in brain tissue cells to TNF?+zVAD-induced L929 cells.Results:? Cytoplasm,nucleus and nuclear envelope fractions were extracted and quantified by western blotting.The results showed that,in cytoplasm,TNFa+zVAD stimuli up-regulated the protein levels of RIP1K,RIP3M,MLKL,tAIF and MIF,and promoted the phosphorylation of RIPIK,RIP3K and MLKL in L929 cells.RIP3K,MLKL,tAIF and MIF were translocated to nucleus in TNFa+zVAD-induced L929 cells,but RIP1K didn't.Western blotting results also showed that the nuclear protein level of MLKL and nuclear envelope protein level of MLKL was indeed increased greatly.? Immunoprecipitation assays showed that RIP3K-tAIF-MIF complex was detected both in the cytoplasm and the nucleus in TNFa-stimulated cells.Immunofluorescent double labeling revealed the formation of RIP3K-tAIF-MIF complex both in the cytoplasm and the nucleus,consistent with the immunoprecipitation assays.? The CRISPR-Cas9 system was used to deplete AIF or AIF-NLS in L929 cells.The data obtained by using cell death assays(cell ATP level measurement and propidium iodide staining assay)demonstrated that depletion of AIF or AIF-NLS significantly blocked TNFa-induced necroptosis.Western blotting analysis showed the depletion of AIF or AIF-NLS led to a loss of RIP3K translocation to the nucleus.These results suggested that AIF recruited RIP3K and MIF to form the RIP3K-tAIF-MIF complex,and translocated to nucleus via an AIF-NLS-dependent manner.? We found that MLKL inhibition by pharmacological intervention blocked the cleavage of mAIF and reduced the protein level of tAIF in cytoplasm and nucleus.Inhibition of RIP3 kinase activity also blocked the cleavage of mAIF,reduced the protein level of tAIF in cytoplasm and nucleus,and also blocked the nuclear envelope localization of MLKL.Genetic study demonstrates that the depletion of AIF has no effect on the interaction of RIP3K-MLKL in cytoplasm,and the the depletion of AIF-NLS has no effects on the nuclear envelope localization of MLKL during necroptosis in cells.? L929 cells were treated with TNFa+zVAD in the presence of nec-1,and then quantified by western blotting.We observed that nec-1 treatment didn't only block the nuclear translocation of RIP3K,but also blocked the cleavage of mAIF.Immunoprecipitation assays showed that nec-1 treatment inhibited the nuclear envelope of MLKL.? In vivo,we established a rat model of middle cerebral artery occlusion reperfusion(MCAO/R)to simulate the pathological process of human cerebral ischemic injury.PI staining and western blotting results showed that ischemic stroke can cause cerebral tissue necrotic-like death,and up-regulated the expression and activation of necrosis-related protein.Immunoprecipitation assays showed the formation of the nuclear translocation necroptotic complex RIP3K-tAIF-MIF and the nuclear envelope translocation of MLKL in ischemia cerebral cortex of rat model.Conclusion:?The nuclear translocation complex,RIP3K-tAIF-MIF,is formed in a MLKL-independent and AIF-NLS-independent manner during necroptosis.? Activated MLKL doesn't only translocate to nuclear envelope,but also regulates the cleavage of mAIF into tAIF.? The formation of the nuclear translocation necroptotic complex RIP3-tAIF-MIF and the nuclear envelope translocation of MLKL are RIP1 kinase-dependently.? Necroptosis is involved in the pathological process of ischemic stroke,and cerebral ischemia-reperfusion increased the cleavage of mAIF and MLKL-nuclear envelope translocation.?Cerebral ischemia-reperfusion causes the formation and nuclear translocation of the RIP3K-tAIF-MIF complex in ischemic area.