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www.nature.com/cddiscovery ARTICLE OPEN 1 2 1 2 1 3 Chanchal Chauhan , Ana Martinez-Val , Rainer Niedenthal , Jesper Velgaard Olsen , Alexey Kotlyarov , Simon Bekker-Jensen , ✉ ✉ 1 4 Matthias Gaestel and Manoj B. Menon © The Author(s) 2023 The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context-dependent manner. Using a mass spectrometry-based screen to ﬁnd new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found to be a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identiﬁed as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5- mediated RIPK3 methylation to act as an efﬁcient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling. Cell Death Discovery (2023) 9:14 ; https://doi.org/10.1038/s41420-023-01299-z INTRODUCTION at residue S166, mechanisms regulating the activity of RIPK3 are less TNF receptor-interacting protein kinases (RIPK)-1 and -3 are the understood [17–19]. Apart from being a substrate of RIPK1, RIPK3 master regulators of programmed cell death signaling. The also undergoes auto-phosphorylation at residues T182, S199, and concerted action of the RIP kinases downstream to the death S227 [20–22]. Members of the casein kinase 1 family phosphorylate receptors, interferon-alpha receptor (IFNαR) and Toll like receptor RIPK3 at serine 227 and regulate its ability to recruit MLKL . In (TLR) regulate cell death/survival decisions during development addition, CHIP E3 ubiquitin ligase-mediated K48 ubiquitylation of and inﬂammation (reviewed in ref. ). In addition, viral infection RIPK3 at residues K55, K89, K363, and K501 and PELI1-mediated K48 and genotoxic stress also activate RIPK1 and RIPK3. Recruitment to ubiquitylation at K363 enhances RIPK3 turnover via proteasomal and the receptor and subsequent ubiquitination of RIPK1 in the lysosomal dependent degradation, respectively [20, 24]. Apart from receptor-associated complex I is crucial for the activation of MAP this, O-GlcNAc transferase targets RIPK3 for O-linked GlcNAcylation kinases and NFkB-dependent transcription, independent of RIPK1 and hinders necroptotic signaling . kinase activity [2–5]. Activated RIPK1 associates with CASP8 and Protein methylation is known to inﬂuence various physiological FADD to assemble a cytosolic death-inducing complex termed processes including RNA processing, transcriptional regulation, “ripoptosome” (complex IIb) resulting in apoptotic cell death [6, 7]. DNA damage response, and cell cycle progression [26–28]. During In the absence of caspase activity, active RIPK1 can also associate the methylation reaction, a methyl group from S-adenosyl-L- with RIPK3 via their RIP homotypic interaction motifs (RHIM) to methionine is transferred to lysine or arginine residues in target form a functional hetero-amyloidal structure called necrosome proteins by highly speciﬁc methyltransferases. Protein arginine [8, 9]. Subsequent RIPK3-mediated phosphorylation of MLKL leads methyltransferases (PRMTs) catalyze transfer of one or two methyl to oligomerisation of MLKL followed by its translocation to the groups to arginine residues, giving rise to monomethyl-arginine, plasma membrane causing loss of membrane integrity and cell asymmetric dimethyl-arginine (aDMA) or symmetric dimethylargi- death deﬁned as necroptosis. In addition to its canonical role as a nine (sDMA). The family of mammalian PRMTs consists of nine pro-necroptotic kinase, RIPK3 also serves as an adapter that drives members sorted into three groups: Type I methyltransferases CASP8-mediated apoptosis [10, 11]. Independent of the cell death (PRMT 1, 3, 4, 6, and 8) generate aDMA by adding two methyl events, RIPK3 also manifests pro-survival functions by regulating groups to the same terminal nitrogen of the arginine residue, Type transcriptional responses [12, 13]. II (PRMT5 and 9) mark each of the terminal nitrogen atoms of A whole lexicon of post-translational modiﬁcations regulates the arginine with one methyl group, hence generating sDMA and the pro-death and pro-survival functions of RIP kinases [14–16]. While type III member PRMT7 catalyzes mono-methylation of arginine RIPK1 phosphorylation by IKK1/2, MK2, and TBK1 was shown to act residues (reviewed in ref. ). Tight regulation of PRMTs is a as checkpoints preventing RIPK1 activation and autophosphorylation prerequisite for regulated protein arginine methylation as there is 1 2 Institute of Cell Biochemistry, Hannover Medical School, Hannover 30625, Germany. Mass Spectrometry for Quantitative Proteomics, Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen N, Denmark. Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark. Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi 110016, India. email: firstname.lastname@example.org; email@example.com Received: 11 November 2022 Revised: 19 December 2022 Accepted: 3 January 2023 Ofﬁcial journal of CDDpress 1234567890();,: C. Chauhan et al. no conclusive proof for the presence of speciﬁc arginine SILAC-based mass spectrometry (MS) analysis, we identiﬁed demethylases. PRMT activity is usually regulated by association PRMT5 as a RIPK1 interaction partner. Subsequently, our studies with regulatory proteins, post-translational modiﬁcations of PRMTs reveal PRMT5-mediated symmetric dimethylation of RIPK3 as a as well as masking of their target sites by other modiﬁcations . novel mechanism regulating necrosome-mediated signaling. Interestingly, the association of PRMT5 with WDR77/MEP50 is a prerequisite for PRMT5 activity in mammalian cells . Other interaction partners like RIOK1 and pICLN/CLNS1A are shown to RESULTS regulate substrate speciﬁcity by docking the methylosome A screen for ripoptosome interactors by MS analysis identiﬁed complex to speciﬁc substrates . PRMT5 activity and localization PRMT5 as a RIPK1 interaction partner are also regulated by its phosphorylation and methylation. While To identify RIPK1 interactors during TNF-induced cell death, we AKT-mediated T634 phosphorylation modulates membrane loca- ﬁrst established a mouse-embryonic ﬁbroblast (MEF) model −/− lization of PRMT5 , PKC -mediated S15 phosphorylation is a system of induced expression of epitope-tagged RIPK1. Ripk1 positive regulator of IL1-induced PRMT5 activity and NFκB- MEFs were rescued by the expression of doxycycline (dox)- activation . Moreover, CARM1/PRMT4-mediated methylation inducible FLAG-tagged RIPK1 and single clonal cell lines were of R505 residue is essential for the homodimerization and isolated and stably labeled by isotopes. Dox-induced expression of activation of PRMT5 . RIPK1 was detectable in both clones analyzed and RIPK1 was Recent evidence indicate that the switch between pro-survival efﬁciently enriched by FLAG-tag-based immunoprecipitation. and pro-death functions of RIPK1 is regulated by a panel of Immunoblots with anti-FLAG and anti-RIPK1 antibodies indicate modiﬁcations including phosphorylation and ubiquitination  that there is no detectable RIPK1 expression and enrichment in and the presence of kinase checkpoints in RIPK1 activation the non-induced cells (Dox -, Fig. 1A). A Dox-induction kinetics revealed the presence of multiple stages in the maturation of the revealed spontaneous activation of RIPK1 at 6 h (Supplementary complex IIb/ripoptosome . To understand the assembly and Fig. S1A). To avoid this and focus on TNF-induced signaling, maturation of the ripoptosome into an active death inducer, it is further studies were done with 4 h Dox-induction. Since previous important to identify novel ripoptosome interactors. Through studies have shown that caspase inhibition can stabilize the Fig. 1 A screen for ripoptosome interactors identiﬁed PRMT5 as an interaction partner of RIPK1. A Two clonal cell lines generated by retroviral transduction and single-cell cloning of RIPK1-deﬁcient ﬁbroblasts with doxycycline (Dox) inducible FLAG-tagged mRIPK1 expression vector were left untreated or treated with Dox for 9 h. RIPK1 was enriched by FLAG-immunoprecipitation and detected in immunoblots with antibodies against FLAG tag and RIPK1. B The cells were treated as indicated (30 min pre-treatment with inhibitors followed by 120 min TNF) and subjected to FLAG-IPs as in panel A. Blots were probed with CASP8 antibodies to monitor assembly of the ripoptosome. C Consistent RIPK1 interaction partners identiﬁed in both clonal lines by mass spectrometry screen are represented with the fold enrichment between TNF + SM + zVAD treated and untreated conditions. D GST-pulldown assay shows speciﬁc interaction of RIPK1 with PRMT5, independent of death domain (DD) and RHIM motif of RIPK1. Cell Death Discovery (2023) 9:14 C. Chauhan et al. complex IIb/ripoptosome , we performed ripoptosome enrich- HEK293T cells (Fig. 2B). A strong interaction between RIPK3 and ment of cells treated with human TNFα in the presence of smac- PRMT5 was also evident as GST-RIPK3 clearly co-puriﬁed co- mimetics (SM) and caspase inhibitor zVAD-fmk (zVAD). Monitoring expressed PRMT5 (Fig. 2B). To verify that RIPK3 methylation is of RIPK1 autophosphorylation in TNF + SM + zVAD treated cells mediated by PRMT5, we monitored methylation in the presence revealed saturation of active RIPK1 signals between 90 and and absence of the small molecule PRMT5 inhibitors GSK591 and 150 minutes in both clonal cell lines (Supplementary Fig. S1,B). LLY283. These PRMT5 inhibitors completely abrogated the Hence, ripoptosome complexes were enriched after 120 min of methylation signals detectable by enrichment of GST-RIPK3 stimulation to correlate with maximal RIPK1 activity, but early followed by immunoblotting with symmetric dimethylation- enough to avoid too much cell loss. Efﬁcient enrichment of speciﬁc antibodies (Fig. 2C). Moreover, while the co-expression ripoptosome-like complexes was veriﬁed by the stimulus-induced of wild-type PRMT5 enhanced the methylation of RIPK3, a catalytic association of CASP8 and its cleaved form with FLAG-RIPK1 (Fig. deﬁcient mutant of PRMT5 (PRMT5-R368A) [39, 40] failed to show 1B). This enrichment is independent of RIPK1 phosphorylation by any effect (Fig. 2D). As a conclusive proof for PRMT5-mediated MK2, since it is detectable in the presence and absence of the MK2 dimethylation of RIPK3, we performed siRNA experiments target- inhibitor PF364402 (Fig. 1B). Immunoblot analyses revealed that ing endogenous PRMT5. Consistent with the previous ﬁndings, both clones of rescued cells lacked detectable MLKL expression siRNA-mediated depletion of PRMT5 completely inhibited the excluding the possibility of necroptotic cell death in this model symmetric dimethylation of RIPK3 (Fig. 2E). These ﬁndings support (Supplementary Fig. S1B). However, RIPK3 immunoblotting a model where RIPK1 and RIPK3 both interact with PRMT5, while revealed stimulus-induced enrichment of RIPK3 to the RIPK1/ only RIPK3 acts as a methylation target of PRMT5 (Fig. 2F). CASP8 complexes (Supplementary Fig. S2). To identify ripoptosome interactors by a non-biased screen, we RIPK3 is methylated at a conserved arginine residue in the applied SILAC-based MS analysis. After the enrichment of FLAG- C-terminal tail RIPK1-associated proteins from differentially SILAC-labeled control Despite the conservation of the cell death signaling pathways and TNF + SM + zVAD (Fig. 1C) treated cells, the beads were across species and high sequence similarity between human and pooled and eluted with FLAG peptides. The eluted proteins were mouse RIPK1, RIPK3 shows more divergence between human and digested and analyzed by LC-MS/MS in technical duplicates. The mouse (Fig. 3A) with only 59% sequence identity (NP_006862.2 proteins which were identiﬁed in duplicate and consistent and NP_064339.2, respectively). Accordingly, RIPK3 and MLKL between the two independent clonal lines were considered as interactions display species-speciﬁc preferences  and major interactors (Fig. 1C). In general, after normalization to enriched differences in the structural determinants of RIPK3-MLKL interac- RIPK1 levels, clone B showed better enrichment for most identiﬁed tion and regulation between human and mouse proteins exist proteins (c.f. Fig. 1C), but clone A also displayed signiﬁcant . To test whether human RIPK3 is also a target of PRMT5 and enrichment. One of the consistent factors enriched was PRMT5, a whether this modiﬁcation is conserved across species, we type II methyl transferase. Interestingly, the PRMT5 methylosome monitored symmetric dimethylation of hRIPK3 in the presence consists of a hetero-octameric complex consisting of a central and absence of the two PRMT5-speciﬁc inhibitors GSK591 and PRMT5 tetramer decorated with four WDR77 molecules. Remark- LLY283. Human RIPK3 underwent strong arginine dimethylation in ably, WDR77 was also amongst the most enriched proteins in our HEK 293T cells, which was completely lost upon PRMT5 inhibitor screen (Fig. 1C) indicating enrichment of the entire PRMT5 treatment (Fig. 3B) or PRMT5 knockdown (Supplementary Fig. S4). methylosome. To verify the identiﬁed interaction between PRMT5 After demonstrating that the PRMT5-RIPK3 axis is conserved in and RIPK1, we performed GST-pulldown experiments in humans, we looked at large-scale proteomic datasets for evidence HEK293T cells transfected with FLAG-PRMT5 and GST-tagged for RIPK3 methylation. The PhosphoSite database (http:// mouse RIPK1 and mutants thereof. The results clearly showed www.phosphosite.org) documents the existence of arginine strong enrichment of PRMT5 with GST-tagged full-length mRIPK1 methylation sites in both human and mouse RIPK3 outside the (1-656 aa) as well as C-terminal truncations of RIPK1 lacking the kinase domain (cf. Fig. 3A). While the residues R264, R332, R413, death domain (ΔDD, mRIPK1-1-588 aa) or both death domain and and R415 are targets of methylation in mRIPK3, R422 and R486 are RHIM motif (ΔDD/ΔRHIM, mRIPK1-1- 500 aa) (Fig. 1D). Similar methylation sites on hRIPK3. Interestingly, R486 (R415 in mRIPK3) results were obtained when a dimerization deﬁcient mutant of is the only conserved arginine methylation site between human RIPK1 was used (ΔDD, RHIMmut). Despite the moderate enrich- and mouse RIPK3 (Fig. 3C). We mutated this conserved arginine ment of PRMT5 on RIPK1 upon activation of the ripoptosome residue in human RIPK3 to alanine (R486A) or lysine (R486K) and assembly, there was signiﬁcant association of PRMT5 with RIPK1 in monitored dimethylation of the mutants. Both mutations almost untreated cells. This led us to the question whether RIPK1 completely abrogated the symmetric dimethylation of RIPK3, activation or activity is important for recruiting PRMT5 to RIPK1? establishing R486 as the predominant methylation site on RIPK3 Interestingly, neither RIPK1 inhibitor necrostatin-1 (Nec-1), nor the (Fig. 3D). catalytic inactivating D138N mutation abrogated the interaction of GST-RIPK1 with co-expressed PRMT5, suggesting that this inter- RIPK3 R486 modiﬁcation provides feedback to RIPK1-RIPK3 action is not completely dependent on RIPK1 activation (Supple- signaling mentary Fig. S3). To monitor RIPK3 functions and to investigate the signiﬁcance of RIPK3 methylation, we established a rescue model of RIPK3 activity RIPK3 is a target of PRMT5-mediated symmetric arginine using the PANC1 human pancreatic cancer cell line. PANC1 cells dimethylation do not express endogenous RIPK3 and, hence, cannot undergo Since the RIPK1 interactors included the core methylosome necroptosis . We introduced RIPK3 into the PANC1 cells by components PRMT5 and WDR77, which induce symmetric lentiviral transduction and monitored necroptotic signaling (Fig. dimethylation of arginine residues, we monitored arginine 4A). RIPK3 expression was detected only in cells exogenously methylation of RIPK1 after co-expression of PRMT5 by using a expressing RIPK3, while MLKL expression was visible in both cell symmetric di-methyl arginine motif (SdmArg) antibody. Despite lines. Upon treatment with the necroptotic stimuli (TNF + SM + clear association of RIPK1 and PRMT5 in the pulldown, there was zVAD), signiﬁcant necroptosis-associated MLKL phosphorylation no symmetric dimethylation detectable for GST-RIPK1 enriched and strong downstream ERK1/2 activation were visible only in the using glutathione beads (Fig. 2A). We then performed this analysis RIPK3-rescued cells. Interestingly, RIPK1 autophosphorylation as with RIPK3 and identiﬁed strong signals for symmetric dimethyla- indicated by the pS166 antibody signal was signiﬁcantly tion of RIPK3, which was enhanced upon PRMT5 co-expression in suppressed only in the cells expressing RIPK3, indicating a Cell Death Discovery (2023) 9:14 C. Chauhan et al. Fig. 2 RIPK3 is a target of PRMT5-mediated symmetric arginine dimethylation. GST-enrichment and probing with antibodies against symmetric dimethyl arginine (αSdmArg) to monitor PRMT5-mediated dimethylation of mRIPK1 (A) and mRIPK3 (B). C Effect of PRMT5 inhibitors GSK591 and LLY283 on RIPK3 methylation monitored by GST-enrichment and immunoblotting (inhibitor treatment: last 6 h before cell lysis). D FLAG-tagged wild-type PRMT5 but not the catalytic-deﬁcient PRMT5 mutant (PRMT5mut, R368A) is capable of enhancing RIPK3 arginine methylation. E siRNA mediated depletion of PRMT5 suppresses symmetric dimethylation of mRIPK3 in HEK293T cells. Efﬁcient depletion of PRMT5 is shown by PRMT5 immunoblots and detection of methylated proteins in the total cell lysates F While both RIPK1 and RIPK3 interact with PRMT5, only RIPK3 is a substrate of symmetric arginine dimethylation. RIPK3-mediated feedback control of RIPK1 autophosphorylation. A (Supplementary Fig. S5B). When necroptotic signaling was pre-treatment of the cells with the RIPK3 inhibitor GSK'872 compared between the four cell lines, we observed clear rescued RIPK1 autophosphorylation and suppressed necroptotic suppression of RIPK1 autophosphorylation by WT-RIPK3 again, signaling, suggesting that the RIPK3-mediated feedback control however, the R486A and R486K mutant cell lines consistently on RIPK1 activation requires RIPK3 kinase activity. Moreover, displayed a stronger signal for pS166-RIPK1 similar to those inhibitors of oligomerization of both RIPK3 (PP2) and MLKL (NSA) observed in RIPK3-deﬁcient empty vector transduced cells (Fig. also inhibited necroptotic signaling as well as RIPK3-mediated 4B). The necroptotic downstream signaling indicated by MLKL suppression of RIPK1-S166 phosphorylation (Fig. 4A). phosphorylation and ERK1/2 activation was enhanced in the cells To further investigate the role of PRMT5-mediated methylation expressing the K486 mutated RIPK3, indicating that methylation of RIPK3 at residue R486, we generated PANC1 cells rescued with acts as negative effector of overall necroptotic signaling. However, wild-type (WT) and methylation-deﬁcient mutants (R486K or phosphorylation of Akt/PKB in response to necroptotic stimulus R486A) of RIPK3. After transduction and selection of cell lines for was enhanced in RIPK3 or mutant-expressing cells, obviously uniform transduction, the selected empty vector and RIPK3 independent of RIPK3 methylation (Fig. 4B and Supplementary expression lines showed similar expression levels of GFP Fig. S6). In contrast to the negative effect of RIPK3 on necroptotic expressed from the bidirectional promotor demonstrating uniform RIPK1 autophosphorylation, S166 phosphorylation of RIPK1 in transduction and similar expression levels of WT and mutant RIPK3 response to a pro-apoptotic stimulus (TNF + SM) was not expression (Supplementary Fig. S5A). Moreover, necroptosis- suppressed by RIPK3 expression. Interestingly, in this case a resistant PANC1 cells were sensitized to RIPK1/RIPK3/MLKL- dose-dependent increase of S166 phosphorylation was observed dependent necroptosis upon lentiviral rescue of RIPK3 expression (Supplementary Fig. S7). Cell Death Discovery (2023) 9:14 C. Chauhan et al. and/or ripoptosome assembly. Moreover, GST-RIPK3 was consti- tutively methylated by PRMT5 and this modiﬁcation did not require RIPK1 and/or RIPK3 kinase activity (Supplementary Fig. S8). It is important to note that one of the earliest reports on PRMT5 function was its interaction with death receptors and consequen- tial inhibition of apoptosis by promoting NFkB activation . This study also revealed that while PRMT5 interacted with the TRAIL receptors, there was no association with the TNFR1 and PRMT5- regulated TRAIL sensitivity in cancer cells. However, more recent evidence indicates a role for PRMT5 downstream to TNF receptors in mounting an NFkB-dependent inﬂammatory response. PRMT5 has been shown to enhance NFkB activation by methylating R30 and R174 residues on NFkB p65 subunit [45, 46]. PRMT5-mediated YBX1-R205 methylation was also shown to affect NFkB-dependent gene expression promoting colon cancer cell proliferation and anchorage-independent growth . CARM1/PRMT4-mediated methylation of PRMT5 was shown to be essential for its homodimerisation and activation leading to histone methylation and suppression of gene expression , while S15 phosphoryla- tion of PRMT5 by PKCι acts as a positive modulator of PRMT5 activity and PRMT5-dependent NFκB activation . Interestingly, PRMT4 is known to regulate a subset of NFκB-target genes by CBP300 methylation downstream to TNF and TLR4 signaling . Fig. 3 Identiﬁcation of conserved arginine methylation sites on CFLAR (CASP8 and FADD-like apoptosis regulator), also known as RIPK3. A Schematic representation of human and mouse RIPK3 c-FLIP is another TNF receptor downstream molecule which is a proteins with domain organization and arginine (R) methylation target of arginine methylation . A role for PRMTs including sites. B Human RIPK3 is a substrate of PRMT5-mediated symmetric arginine methylation (inhibitor treatment: last 6 h before cell lysis). PRMT5 in inﬂammatory gene expression is gaining prominence C Alignment of mRIPK3 and hRIPK3 sequences reveal R486 in hRIPK3 and incidentally, we also observed a role for PRMT5-mediated (R415 in mRIPK3) as the only evolutionarily conserved methylation RIPK3 methylation in necrosome downstream MAPK activation. site. K/A denotes mutagenesis of the site to lysine (K) and alanine (A) The methylation-deﬁcient mutants consistently gave enhanced residues to generate methylation-deﬁcient mutants. D Mutagenesis RIPK3-dependent ERK1/2 phosphorylation signals in response to of R486 residue (R486K or R486A) abrogates symmetric arginine necroptotic stimulus (Fig. 4). RIPK1 and RIPK3 mediated inﬂam- dimethylation of hRIPK3 as shown by FLAG-IP enrichment and matory response is mediated by ERK1/2 kinases, independent of analysis with SdmArg antibodies. the cell death . We also observed a detectable increase in the methylation- DISCUSSION deﬁcient RIPK3 mutant protein levels compared to the wild-type The ripoptosome/complex IIb formed during death receptor- RIPK3 in the rescued PANC1 cells. The reason for such an effect is dependent and -independent cell death consists of the core not clear. However, despite moderately elevated levels, RIPK3 components RIPK1, FADD, and CASP8. Inhibition of CASP8 activity mutants were not effective in providing feedback suppression on leads to the association of RIPK3 with this complex, leading to the RIPK1 autophosphorylation. Ubiquitination and subsequent lyso- formation of a RIPK1-RIPK3 containing necrosome capable of somal degradation of RIPK3 by the CHIP-E3 ubiquitin ligase were phosphorylating MLKL and inducing necroptosis. In the last years, shown as a mechanism of necroptosis suppression . Interest- many interaction partners and post-transcriptional modiﬁcations ingly, PRMT5 is also a proven substrate of CHIP-mediated of RIPK1 and RIPK3 were identiﬁed as modulators of these cell ubiquitination and proteasomal degradation . In lung cancer death pathways. In the present study, we provide the ﬁrst proof patients, an expression signature with low CHIP expression and for an arginine dimethylation of RIPK3 as such a modulatory high RIPK3 expression is associated with bad prognosis . mechanism. Using the mass spectrometry approach, we have Regulation of RIPK3 levels could indeed be a mechanism relevant identiﬁed the protein arginine methyl transferase PRMT5 as a to inﬂammation and cancer. RIPK1 interactor which mediates a symmetric arginine dimethyla- Despite clear reduction in the feedback control on RIPK1 tion of RIPK3 C-terminus. Using a RIPK3 rescue model in a RIPK3- phosphorylation and enhanced signaling to MLKL in PANC1 cells deﬁcient pancreatic cancer cell line which normally cannot rescued with the methylation-resistant mutants of RIPK3, we could undergo necroptosis, we demonstrate that the evolutionary not observe differences in necroptotic cell death between WT and conserved dimethylated arginine residue R486 is important for mutant cells (Supplementary Fig. S5C and Supplementary Table RIPK3-mediated feedback control on RIPK1 autophosphorylation. S3). The mutual regulation of RIPK1 and RIPK3 is still poorly Hence, we established a link between PRMT5 and the necroptosis understood. RIPK1 is the upstream activator of RIPK3. However, it regulator RIPK3. A recent study also corroborates our ﬁnding by is also known as a factor preventing spontaneous RIPK3 showing that PRMT5 exhibits anti-tumor effects via regulation of oligomerisation and activation . While RIPK1-mediated necrop- necroptosis . PRMT5-depletion was shown to sensitize tosis, but not CASP8-dependent apoptosis, requires RIPK3 and glioblastoma cells to the antitumor effects of the protein MLKL, artiﬁcial oligomerization of RIPK3 can result in both phosphatase 2A inhibitor LB100 by facilitating necroptosis. apoptosis and necroptosis. In the absence of RIPK1, oligomerised Interestingly, the ripoptosome interactome detected in our MS RIPK3 predominantly induces MLKL-dependent necroptosis, but screen included several methylosome components including also apoptosis in MLKL-deﬁcient cells . Studies using genetic RIOK1 and WDR77 in addition to PRMT5 (Fig. 1 and Supplemen- models of RIPK1 deﬁciency has shown a kinase activity- tary Table S1). This indicates a potentially more general role for independent scaffolding role for RIPK1 in preventing epithelial protein arginine methylation in the regulation and/or maturation cell apoptosis and necroptosis . Cells from the RIPK3-RHIM of the ripoptosome. The MS screen, as well as our further studies, mutant mice were protected from necroptosis as well as apoptosis also suggest signiﬁcant association of PRMT5 and RIPK1/RIPK3 in further conﬁrming the complex signaling interplay of these co-expressed cells which may be independent of TNF stimulation protein kinases in the regulation of different pathways of cell Cell Death Discovery (2023) 9:14 C. Chauhan et al. Fig. 4 RIPK3-mediated feedback control on RIPK1 activity is dependent on RIPK3 methylation. A The necroptosis-incompetent PANC1 cell line, which lacks RIPK3, was rescued by lentiviral transduction of FLAG-tagged WT-RIPK3 or empty vector. Necroptotic signaling was monitored by stimulating cells with TNFα, Smac-mimetics (SM) and zVAD-fmk for 90 min in the absence or presence of indicated kinase inhibitors (Nec-1, GSK'872) or PP2 (RIPK3 oligomerisation inhibitor) or necrosulfonamide (NSA), an inhibitor of human MLKL oligomerisation (inhibitor pre- treatment for 30 minutes before TNF stimulation). The right panel shows the schematic summary of the observations. RIPK3 expression facilitates necroptotic signaling in PANC1 cells (MLKL and ERK1/2 phosphorylation) but suppresses RIPK1 autophosphorylation (RIPK1-S166). The feedback control of RIPK3 on RIPK1 was abrogated by RIPK3 and MLKL inhibitors. B PANC1 rescue model as in A comparing the necroptotic signaling in cells transduced with WT and methylation site mutants (R486K and R486A) of FLAG-tagged RIPK3 (*indicates remnants of pMLKL bands when reprobed with RIPK3). The corresponding panel to the right summarizes the conclusions from the immunoblots. Top right panel depicts the signaling effects and the processes targeted by the inhibitors used. RIPK3 activation by RIPK1 leads to MLKL phosphorylation and oligomerisation, with downstream ERK1/2 signaling. The oligomerisation of MLKL also seems necessary for suppressing pS166-RIPK1 phosphorylation. The lower panel scheme depicts the possible effect of RIPK3 methylation in the same system. RIPK3-mediated suppression of RIPK1-S166 is lost upon methylation site mutation. In addition, the necroptotic signaling is also enhanced in the absence of R486 methylation. This indicates an inhibitory role for RIPK3-methylation in RIPK3 downstream signaling including the feedback control of RIPK1 activity. Cell Death Discovery (2023) 9:14 C. Chauhan et al. death . This study also revealed the presence of enhanced 36 h post-transfection. To monitor the effect of pharmacological inhibitors on methylation and interaction in transfected cells, cells were treated with RIPK1 autophosphorylation and RIPK1-dependent lymphoproli- −/− inhibitors for the last 6 h prior to cell lysis. ferative disease in the RIPK3-RHIM mutant mouse in a Fadd background suggesting a role for RIPK3-mediated feedback on RIPK1-mediated inﬂammatory response. PRMT5-mediated methy- Retroviral transduction −/− lation of RIPK3 may participate in the modulation of this feedback. Ripk1 MEFs were stably rescued with Doxycycline inducible retroviral vectors expressing mouse RIPK1. For preparation of viral supernatants, 7.5 Further studies using genetic mouse models will be necessary to million Ecopack-HEK293T cells were seeded in 10 cm plates and reveal the complex interplay of PRMT5 and RIPK3 in inﬂammation transfected overnight with 5 µg each of pCL-Eco and pSERS11 based and to identify additional substrates of PRMT5-mediated methyla- retroviral vectors using PEI, in antibiotic-free medium with 10% FCS. tion in the RIPK1/3 RHIM interactome. Medium was changed to fresh complete media supplemented with 1X non-essential amino acids. Virus-containing supernatants were harvested ﬁve times from transfected Ecopack-HEK293 cells (Clontech/Takara), over a MATERIALS AND METHODS period of three days post-transfection and ﬁltered with 0.45 µm ﬁlters. 5 −/− Cell culture 1.2 × 10 Ripk1 MEF cells were seeded per well in a six well plate and 3T3-immortalized Ripk1−/− MEFs were kindly provided by Prof. K. were cultured for 4 days with viral supernatants in the presence of 8 µg/ml Ruckdeschel, (Universitätsklinikum Hamburg-Eppendorf, Germany). polybrene. RIPK1 expression was monitored by intracellular staining and Ripk1−/− MEFs, HEK293T cells, and PANC-1 cells were cultured in DMEM ﬂow cytometry analysis using an Accuri-C6 cytometer (BD Biosciences). supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin. The cell lines were maintained at 37 °C and with Lentiviral transduction 5% CO in a humidiﬁed atmosphere. To generate a rescue model for RIPK3, PANC-1 cells (RIPK3-deﬁcient) were transduced with lentiviral vectors expressing WT/mutant human RIPK3 or Antibodies and reagents pLBID-MCS-GFP-P2A-Puro empty vector. ViraPower Lentiviral Expression MLKL (#14993), αSdmArg (#13222), RIP3 (#13526), Caspase-8 (#4927), System (Invitrogen) was used to package pLBID lentiviral vectors pS166-RIPK1 (#65746), pS358-MLKL (#91689), pS345-MLKL (#37333), expressing FLAG-hRIPK3 or methylation site mutants or empty vector. pS227-RIPK3 (#93654) phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Viral supernatants were collected 48 h post-transfection and ﬁltered (#4370) and pS473-Akt (#4060), pS536-NFkB p65 (#93H1), IkBα (#9242) through 0,45 µm ﬁlters as described previously . PANC-1 cells were antibodies were from Cell Signaling Technology (CST). Further antibodies transduced twice with the supernatant containing lentivirus and 8 µg/ml used were against GST (sc-138, Santa Cruz Biotechnology), RIPK1 (#610459, polyberene followed by positive selection of transduced cells in the BD Biosciences), GAPDH (#MAB374, Millipore), EF2 (sc-166415, Santa Cruz presence of 1–2 µg/ml puromycin. Flow cytometry analysis of GFP was Biotechnology), AKT1/2/3 (sc-8312, Santa Cruz Biotechnology), ERK2 (sc- used to monitor comparable transduction efﬁciency between the cell lines. 154, Santa Cruz Biotechnology), FLAG (#F3165, Sigma-Aldrich), PRMT5 Polyclonal antibody (#18436-1-AP; Proteintech) and GFP (sc-9996, Santa Intracellular ﬂow cytometry staining of RIPK1 Cruz Biotechnology). The secondary antibodies used were anti-rabbit IgG- Cells were suspended in PBS at a concentration of 10 cells/ml and were HRP (Conformation Speciﬁc) (#5127, CST), mouse TrueBlot® ULTRA (#18- ﬁxed with 3-volumes of 4% PFA. After 30 min at room temperature, ﬁxed 8817-33, Rockland Immunochemicals), goat anti-mouse IgG (H + L)-HRP cells were washed and permeabilized for 30 min with ice-cold 90% (#115-035-003, Dianova) and goat anti-rabbit IgG (H + L)-HRP (111-035- methanol. After 2× washes with PBS, cells were blocked with 4% BSA on 003, Dianova). ice for 30 min. After 1 h incubation at room temperature with anti-RIPK1 The following reagents at given concentrations were used for cell antibody (BD Biosciences # 610459, 1:100 diluted in 1% BSA-PBS) cells were treatments: Doxycycline (D9891, Sigma, 1 µg/ml), recombinant human incubated with 1:700 diluted secondary antibodies (anti-mouse Alexa TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/Smac Mimetics Fluor-488, Invitrogen) for 30 min. Samples were washed with PBS and (HY-16591, MedChem Express, 1 µM), pan-caspase inhibitor zVAD-fmk analyzed with Flow cytometry using an Accuri-C6 cytometer (BD (4026865.0005, Bachem, 25 μM), PF3644022 (4279, Tocris, 5 μM), GSK591 Biosciences). (Cay18354-1, Cayman, 10 µM), LLY-283 (HY-107777, MedChem Express, 10 µM), Nec-1 (BML-AP309-0020, Enzo Life Sciences, 50 µM), PP2 (HY- 13805, MedChem Express, 10 µM), NSA (5025, Tocris, 10 µM), GSK’872 (HY- SILAC-based mass spectrometry analysis of RIPK1 interactome 101872, MedChem Express, 5 µM). Post-transduction and sorting, two clonal cell lines were generated from −/− dox-inducible FLAG-RIPK1 rescued Ripk1 MEFs. These clones were metabolically labeled with light (Lys0, Arg 0) and medium (Lys4, Arg 6) Plasmids, cloning, and mutagenesis non-radioactive isotope amino acids. SILAC Protein Quantitation Kit Dox-inducible pSERS retroviral vector as described previously  was (Trypsin), DMEM (A33972, ThermoFisher Scientiﬁc) was used for generating converted to a Gateway Destination vector and a FLAG-tagged mRIPK1 light labeled cell lines and for generation of medium labeled cell lines, L- cDNA  was shuttled in to create doxycycline-inducible retroviral Lysine-2HCL,4,4,5,5-D4 (ThermoFisher Scientiﬁc) and 13C-labeled L-Argi- expression system for low-level RIPK1 expression. The human and mouse nine HCl (201203902, Silantes) were individually purchased. Cells were RIPK3 coding region (NM_006871.4 and NM_019955.2, respectively) PCR- cultured in respective medium supplemented with 200 µg/ml of L-Proline ampliﬁed from HeLa cell and MEF cell cDNA, respectively, were cloned into for at least 10 passages for achieving incorporation of each respective the pENTR-D-TOPO directional cloning vector. L-R clonase II mediated labels. Cells were seeded at a density of 3 × 10 cells in 10 cm plates and shuttling was used to generate N-terminally tagged expression vectors. were treated the next day with doxycycline (1 µg/ml) for 4 h, followed by 3xFLAG-PRMT5 and 3xFLAG-PRMT5-R368A mutant expression vectors were treatment of light-labeled cells with only DMSO control and of medium- described previously . The expression vector pCR3.V62-Met-FLAG-RIP3 labeled cells with TNF+ SM + zVAD for 2 h. FLAG-RIPK1 bound complexes was reported earlier . Site-directed mutagenesis was performed using were enriched from 1.5 mg of protein lysate per sample. Pre-cleared lysates the QuikChange mutagenesis kit (Agilent) to generate FLAG-RIP3-486A and were incubated with 40 µl of 50% ANTI-FLAG® M2 afﬁnity gel (#A2220, FLAG-RIP3-486K methylation site mutants. pLBID-MCS-GFP-P2A-Puro was Sigma) on a rotor for 3 h at 4 °C. After immunoprecipitation, Anti-FLAG gifted by Dr. A. Schambach (MHH, Germany). pLBID-FLAG-hRIPK3 plasmids beads were pooled from the two differentially treated light and medium were generated by subcloning PCR products from the pCR3.V62-Met labeled samples for each clonal cell line. Proteins were eluted from pooled vector into the AgeI/XhoI sites. All other plasmids were described beads with 0.5 mg/ml FLAG peptide (#F3290, Millipore Sigma) by shaking previously . All cloning and mutagenesis primer sequences used are at 1000 rpm for 1 h at 4 °C. Samples were separated on a 10% SDS-PAGE listed in Supplementary Table S2. gel and in-gel digested overnight with Trypsin/Lys-C protease mix and resulting peptides were desalted using C18-stage tip. The puriﬁed peptides Transient transfection from each sample were analyzed by mass spectrometry in technical HEK293T cells were transfected using polyethylenimine (PEI; Sigma- duplicates. Samples were analyzed on the Evosep One system using an in- Aldrich). Transfected cells were maintained in antibiotic-free DMEM media house packed 15 cm, 150 μm i.d. capillary column with 1.9 μm Reprosil-Pur supplemented with 10% FCS for 12–16 h followed by providing with C18 beads (Dr. Maisch, Ammerbuch, Germany) using the pre-programmed complete DMEM media. Transfected cells were analyzed between 20 and gradients for 60 samples per day (SPD). The column temperature was Cell Death Discovery (2023) 9:14 C. Chauhan et al. maintained at 60°C usinganintegratedcolumnoven(PRSO-V1, DATA AVAILABILITY Sonation, Biberach, Germany) and interfaced online with the Orbitrap All data generated during this study leading to the ﬁndings presented here are Exploris 480 MS. Spray voltage was set to 2.0 kV, funnel RF level at 40, included in this published article and its supplementary data ﬁles. and heated capillary temperature at 275 °C. Full MS resolutions were set to 60,000 at m/z 200 and full MS AGC target was 300 with an IT of 22 ms. Mass range was set to 350−1400. 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Orozco SL, Daniels BP, Yatim N, Messmer MN, Quarato G, Chen-Harris H, et al. gels and immunoblotted to Nitrocellulose blotting membrane (#10600001, RIPK3 activation leads to cytokine synthesis that continues after loss of cell Amersham Biosciences). Western blots were blocked with 5% powdered membrane integrity. Cell Rep. 2019;28:2275–87.e5. skim milk in PBS with 0.1% Tween 20 for 1 h at room temperature. After 13. Moriwaki K, Chan FK. The inﬂammatory signal adaptor RIPK3: functions beyond three washes with PBS containing 0.1% Tween 20, membranes were necroptosis. Int Rev Cell Mol Biol. 2017;328:253–75. incubated overnight with the primary antibody at 4 °C followed by 1 h 14. Annibaldi A, Meier P. Checkpoints in TNF-induced cell death: implications in incubation with horseradish peroxidase-conjugated secondary antibodies inﬂammation and cancer. Trends Mol Med. 2018;24:49–65. at room temperature. Blots were developed with an ECL detection kit, 15. 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Positive and negative phosphorylation regulates beads were blocked with 1% BSA to avoid non-speciﬁc binding of PRMT5 RIP1- and RIP3-induced programmed necrosis. Biochem J. 2013;456:409–15. to Glutathione beads. For the enrichment of the fusion proteins, pre- 22. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinase domain- cleared lysates were incubated with Monoclonal ANTI-FLAG® M2 afﬁnity like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. gel (A2220, Sigma) or Protino Glutathione Agarose 4B (745500.10, 2012;148:213–27. Macherey-Nagel) for 16 h at 4 °C. The beads were then washed 4× with 23. Hanna-Addams S, Liu S, Liu H, Chen S, Wang Z. CK1alpha, CK1delta, and immunoprecipitation wash buffer containing 1X TBS, 50 mM Sodium CK1epsilon are necrosome components which phosphorylate serine 227 of ﬂuoride, 1% Triton X-100, 1 mM Na VO The beads were boiled in 2× SDS 3 4. human RIPK3 to activate necroptosis. 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RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), com- Genetic deletion of SEPT7 reveals a cell type-speciﬁc role of septins in microtubule petes with pICln for binding and modulates PRMT5 complex composition and destabilization for the completion of cytokinesis. PLoS Genet. 2014;10:e1004558. substrate speciﬁcity. J Biol Chem. 2011;286:1976–86. 58. Ronkina N, Lafera J, Kotlyarov A, Gaestel M. Stress-dependent phosphorylation of 33. Espejo AB, Gao G, Black K, Gayatri S, Veland N, Kim J, et al. PRMT5 C-terminal myocardin-related transcription factor A (MRTF-A) by the p38(MAPK)/MK2 axis. phosphorylation modulates a 14-3-3/PDZ interaction switch. J Biol Chem. Sci Rep. 2016;6:31219. 2017;292:2255–65. 34. Hartley AV, Wang B, Jiang G, Wei H, Sun M, Prabhu L, et al. Regulation of a PRMT5/NF-kappaB axis by phosphorylation of PRMT5 at serine 15 in colorectal ACKNOWLEDGEMENTS cancer. Int J Mol Sci. 2020;21:3684. We thank Dr. Klaus Ruckdeschel (UKE-Hamburg) for the gift of RIPK1 KO Mouse- 35. Nie M, Wang Y, Guo C, Li X, Wang Y, Deng Y, et al. CARM1-mediated methylation embryonic ﬁbroblasts, Dr. Peter Claus (Hannover Medical School, Germany) for the gift of of protein arginine methyltransferase 5 represses human gamma-globin gene PRMT5 expression vectors, Dr. Axel Schambach (Hannover Medical School, Germany) for expression in erythroleukemia cells. J Biol Chem. 2018;293:17454–63. sharing the pLBid lentiviral expression vector and Dr. Sven Diederichs (DKFZ-Heidelberg) 36. Wang Q, Fan D, Xia Y, Ye Q, Xi X, Zhang G, et al. The latest information on the for providing PANC1 cells. This work was supported by the Deutsche Forschungsge- RIPK1 post-translational modiﬁcations and functions. Biomed Pharmacother. meinschaft (DFG) grants ME4319/3-1 (MBM) and GA453/16-1 (MG). Work at The Novo 2021;142:112082. Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous 37. Amin P, Florez M, Najafov A, Pan H, Geng J, Ofengeim D, et al. Regulation of a donation from the Novo Nordisk Foundation (Grant number NNF14CC0001). MS-based distinct activated RIPK1 intermediate bridging complex I and complex II in proteomics work was also funded by grant EPIC-XS-823839. TNFalpha-mediated apoptosis. Proc Natl Acad Sci USA. 2018;115:E5944–E53. 38. Tenev T, Bianchi K, Darding M, Broemer M, Langlais C, Wallberg F, et al. The Ripoptosome, a signaling platform that assembles in response to genotoxic stress AUTHOR CONTRIBUTIONS and loss of IAPs. Mol cell. 2011;43:432–48. CC performed majority of the experiments and analyzed data. AMDV, JVO, and SB-J 39. Bruns AF, Grothe C, Claus P. Fibroblast growth factor 2 (FGF-2) is a novel substrate designed and performed the Mass Spectrometry analyses. RN provided vectors and for arginine methylation by PRMT5. Biol Chem. 2009;390:59–65. protocols. AK and MG designed experiments and gave conceptual insights. MG and 40. Kwak YT, Guo J, Prajapati S, Park KJ, Surabhi RM, Miller B, et al. Methylation of MBM procured funding. MBM designed experiments, analyzed data and supervised SPT5 regulates its interaction with RNA polymerase II and transcriptional elon- the study. MG, CC, and MBM prepared the manuscript. gation properties. Mol Cell. 2003;11:1055–66. 41. Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, et al. Diverse sequence determinants control human and mouse receptor-interacting protein 3 (RIP3) FUNDING and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. Open Access funding enabled and organized by Projekt DEAL. J Biol Chem. 2013;288:16247–61. 42. Meng Y, Davies KA, Fitzgibbon C, Young SN, Garnish SE, Horne CR, et al. Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis. Nat Commun. 2021;12:6783. COMPETING INTERESTS 43. Otani Y, Sur HP, Rachaiah G, Namagiri S, Chowdhury A, Lewis CT, et al. Inhibiting The authors declare no competing interests. protein phosphatase 2A increases the antitumor effect of protein arginine methyl- transferase 5 inhibition in models of glioblastoma. Neuro Oncol. 2021;23:1481–93. 44. Tanaka H, Hoshikawa Y, Oh-hara T, Koike S, Naito M, Noda T, et al. PRMT5, a novel ADDITIONAL INFORMATION TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear Supplementary information The online version contains supplementary material factor-kappaB activation. Mol Cancer Res. 2009;7:557–69. available at https://doi.org/10.1038/s41420-023-01299-z. 45. WeiH,WangB, MiyagiM,She Y, GopalanB,Huang DB,etal. PRMT5dimethylatesR30 of the p65 subunit to activate NF-kappaB. Proc Natl Acad Sci USA. 2013;110:13516–21. Correspondence and requests for materials should be addressed to Matthias Gaestel 46. Harris DP, Chandrasekharan UM, Bandyopadhyay S, Willard B, DiCorleto PE. or Manoj B. Menon. PRMT5-mediated methylation of NF-kappaB p65 at Arg174 is required for endothelial CXCL11 gene induction in response to TNF-alpha and IFN-gamma Reprints and permission information is available at http://www.nature.com/reprints costimulation. PLoS One. 2016;11:e0148905. 47. Hartley AV, Wang B, Mundade R, Jiang G, Sun M, Wei H, et al. PRMT5-mediated Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims methylation of YBX1 regulates NF-kappaB activity in colorectal cancer. Sci Rep. in published maps and institutional afﬁliations. 2020;10:15934. 48. Covic M, Hassa PO, Saccani S, Buerki C, Meier NI, Lombardi C, et al. Arginine methyltransferase CARM1 is a promoter-speciﬁc regulator of NF-kappaB- dependent gene expression. EMBO J. 2005;24:85–96. 49. Li M, An W, Xu L, Lin Y, Su L, Liu X. The arginine methyltransferase PRMT5 and Open Access This article is licensed under a Creative Commons PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLARL in Attribution 4.0 International License, which permits use, sharing, human lung cancer cells. J Exp Clin Cancer Res. 2019;38:64. adaptation, distribution and reproduction in any medium or format, as long as you give 50. Najjar M, Saleh D, Zelic M, Nogusa S, Shah S, Tai A, et al. RIPK1 and RIPK3 kinases appropriate credit to the original author(s) and the source, provide a link to the Creative promote cell-death-independent inﬂammation by toll-like receptor 4. Immunity. Commons license, and indicate if changes were made. The images or other third party 2016;45:46–59. material in this article are included in the article’s Creative Commons license, unless 51. Zhang HT, Zeng LF, He QY, Tao WA, Zha ZG, Hu CD. The E3 ubiquitin ligase CHIP indicated otherwise in a credit line to the material. If material is not included in the mediates ubiquitination and proteasomal degradation of PRMT5. Biochim Bio- article’s Creative Commons license and your intended use is not permitted by statutory phys Acta. 2016;1863:335–46. regulation or exceeds the permitted use, you will need to obtain permission directly 52. Kim J, Chung JY, Park YS, Jang SJ, Kim HR, Choi CM, et al. Prognostic signiﬁcance from the copyright holder. To view a copy of this license, visit http:// of CHIP and RIPK3 in non-small cell lung cancer. Cancers. 2020;12:1496. creativecommons.org/licenses/by/4.0/. 53. Orozco S, Yatim N, Werner MR, Tran H, Gunja SY, Tait SW, et al. RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ. 2014;21:1511–21. © The Author(s) 2023 Cell Death Discovery (2023) 9:14
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