E3 Ubiquitin Ligase TRIM11 Regulates The Stability Of Necrotic Kinase RIPK3 And Necroptosis

Necroptosis is a programmed form of necrosis,or inflammatory cell death.Similar to apoptosis,necroptosis can be induced by diverse signals,including TNFR,TLR3,TLR4,and TCR.The initiation of necroptosis involves the activation of protein kinases RIPK1 and RIPK3,which subseqently phosphorylate the executioner MLKL to induce cell memebrane rupture.Previous studies showed that with protein synthesis inhibitor CHX treated in IECs and MEFs,RIPK3 and MLKL protein levels were decreased.RIPK3 is a serine/threonine kinase which has emerged as a key regulator of inflammation and programmed cell death signaling in maintaining immune responses and tissue homeostasis.MLKL is a downstream target of RIPK3 activity and interacts with RIPK3 in the necroptosis pathway.While RIPK3 expression can be regulated via both transcriptional and post-transcriptional mechanisms,it has been reported that a fraction of polyubiquitinated RIPK3 underwent rapid degradation by E3 ubiquitin ligases like CHIP and PELI1,as well as the modification of deubiquitinase A20.We want to know whether there are other E3 ubiquitin ligases that regulate stability of RIPK3.So we focused on TRIM family members as many of them have been shown to regulate autophagic degradation and innate immunity.By screening a panel of TRIM proteins to mediate RIPK3 degradation,we identified TRIM11 as one of the candidates capable of promoting RIPK3 degradation.We further investigated the effect of TRIM11 on RIPK3 abundance and found that increasing amounts of TRIM11 led to a considerable loss of overexpressed RIPK3 and MLKL proteins,but not RIPK1.Then,immunoprecipitation assay revealed that TRIM11 could interact with RIPK3,but not MLKL.We also found interactions between endogenous RIPK3 and HA-TRIM11 immunoprecipitated in the TRIM11 stably overexpressed L929 cells.Additionally,we generated the enzymatic inactive mutant TRIM11-C56 A in which the cysteine was replaced with alanine to determine whether the enzyme activity of TRIM11 was crucial for the degradation of RIPK3,our results showed that the C56 A enzymatic inactive mutant partially lost its ability to promote RIPK3 degradation.Next,we conducted in vivo ubiquitination assay to investigate the role of TRIM11 in RIPK3 ubiquitination.Strikingly,TRIM11 was able to promote robust ubiquitination on RIPK3 proteins.Since there are at least three pathways of protein degradation(the proteasome,the lysosome and the autophagosome dependent pathways),we used different inhibitors to block the degradation of RIPK3.We found that only the PI3K/Vps34 inhibitors 3-MA and SAR405 could rescue RIPK3 protein levels.Subsequently,we generated TRIM11 KO HT-29 cells by CRISPR-Cas9 technology,and further evaluated the role of TRIM11 in RIPK3 degradation.In TRIM11 KO HT-29 cells,while p62 degradation occurred similarly following AA starvation,the RIPK3 degradation was considerably ameliorated.The fact that SAR405 was able to block both p62 and RIPK3 degradation further supported the notion that TRIM11-mediated RIPK3 degradation may be dependent on autophagy.Meanwhile,after the TNF?,BV-6,z-VAD treatment in TRIM11 KO HT-29 cells to induce necroptosis,two KO cell lines caused more cell death compared with WT cells by ATP and LDH assay.In response to this,the results of TRIM11 stably overexpressed L929 cells showed that TRIM11 could protect the cells from TNF?,z-VAD induced necroptosis.Taken together,this work identified TRIM11 as a novel E3 ubiquitin ligases involved in RIPK3 ubiquitination and degradation.Although TRIM11 has been previously linked to the degradation of misfolded poly Q and AIM2,its role in necroptosis has not been implicated.Our data suggest that TRIM11 primarily assemble K48-type polyubiquitin chains on RIPK3,a marker could be recognized by autophagy adaptor p62 and then targeted for degradation.In summary,our results suggest selective autophagy as a novel mechanism mediating RIPK3 degradation,thus adding another twist into RIPK3 regulation.And what's more,it provided insights into the connection between necroptosis and autophagy,facilitating the application process of effective treatments in the future.