PELI1 functions as a dual modulator of and by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP

Huibing Wanga,b, Huyan Mengb, Xingyan Lib, Kezhou Zhub, Kangyun Dongb, Adnan K. Mookhtiara, Huiting Weia, Ying Lib, Shao-Cong Sunc, and Junying Yuana,b,1

aDepartment of Cell , , Boston, MA 02115; bInterdisciplinary Research Center on Biology and Chemistry, Institute of Organic Chemistry, Chinese Academy of Sciences, PuDong District, Shanghai, 201203, China; and cDepartment of Immunology, MD Anderson Cancer Center, The University of Texas, Houston, TX 77030

Contributed by , October 2, 2017 (sent for review September 11, 2017; reviewed by Douglas R. Green and Zheng-Gang Liu) Apoptosis and necroptosis are two distinct cell mechanisms complex IIb, which includes FADD, -8, and RIPK3. The that may be activated in cells on stimulation by TNFα. It is still activated RIPK3 in turn mediates the phosphorylation and unclear, however, how apoptosis and necroptosis may be differ- oligomerization of MLKL to promote the execution of nec- entially regulated. Here we screened for E3 ubiquitin ligases that roptosis. Necroptosis induced by TNFα is effectively inhibited by could mediate necroptosis. We found that deficiency of Pellino 1 RIPK1 inhibitor R-7-Cl-O-Nec-1 (Nec-1s), which keeps RIPK1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that in an inactive conformation (6). The activation of RIPK1 has PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase- been implicated in human diseases by mediating and dependent manner during necroptosis. Ubiquitination of RIPK1 by , as Nec-1s has shown efficacy in ameliorating tis- PELI1 promotes the formation of necrosome and execution of nec- sue injuries in animal models of diseases ranging from ischemic roptosis. Although PELI1 is not directly involved in mediating brain, kidney, and heart injuries to multiple sclerosis, amyo- the activation of RIPK1, it is indispensable for promoting the bind- trophic lateral sclerosis, and Alzheimer’s disease (7–11). It is ing of activated RIPK1 with its downstream mediator RIPK3 unclear, however, how inhibition of RIPK1 by Nec-1s prevents to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 the interaction of RIPK1 and RIPK3, a decisive signaling event kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in in necroptosis. necroptosis. However, we show that PELI1 deficiency sensitizes Pellino 1 (PELI1), a member of Pellino family, is an E3 cells to both RIPK1-dependent and RIPK1-independent apoptosis ubiquitin ligase known to be involved in mediating TLR3/ as a result of down-regulated expression of c-FLIP, an inhibitor TLR4 signaling (12, 13). PELI1 has been shown to interact of caspase-8. Finally, we show that Peli1−/− mice are sensitized with RIPK1 and mediate K63 ubiquitination of RIPK1 to to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of regulate the activation of NF-κB on stimulation of TLR4 in a RIPK1 that differentially controls the activation of necroptosis TRIF-dependent manner. The role of PELI1 in TNFα signal- and apoptosis. ing has not been investigated. Here we report that PELI1 is an E3 ubiquitin ligase that necroptosis | apoptosis | Peli1 | ubiquitination | TNF mediates K63 ubiquitination of RIPK1 on K115 residue in a RIPK1 kinase activity-dependent manner to promote the for- ecroptosis and apoptosis are two distinct regulated cell mation of complex IIb and transduction of necroptotic signaling Ndeath mechanisms involved in development, adult tissue downstream of RIPK1 in cells stimulated by TNFα. PELI1- homeostasis, and diseases (1, 2). Apoptosis is mediated by the deficient cells are protected against necroptosis induced by activation of , which controls the signal transduction and TNFα under apoptosis-deficient conditions. In contrast, PELI1- execution of apoptotic cell death (3). Under apoptotic-deficient deficient cells are sensitized to apoptosis through up-regulation conditions, for example, on inactivation of caspase-8 or the loss of c-Myc expression and the suppression of c-FLIP expression. of its adaptor FADD (Fas-associated protein with death do- main), necroptosis may be activated to lead to cell death and Significance embryonic lethality. It is still not clear, however, how necroptosis and apoptosis might be differentially regulated under physio- This study demonstrates Pellino 1 (PELI1) as an important logical or pathological conditions in cells. modulator that exerts opposite regulatory functions on apo- TNFα, an important proinflammatory involved in ptosis and necroptosis, two distinct forms of regulated cell mediating myriads of human diseases, can activate necroptosis death mechanisms. K63 ubiquitination of RIPK1 mediated by or apoptosis in different conditions and cell types. Stimulation PELI1 depends on the kinase activity of RIPK1, which provides a of TNFα promotes the rapid activation of TNFR1 to recruit mechanism to explain why inhibition of RIPK1 kinase activity RIPK1, TRADD, TRAF2, and cIAP1/2 to form a TNFR1 sig- by Nec-1s can also reduce its ubiquitination during necroptosis. naling complex (TNF-RSC, or complex I). cIAP1/2 is known to mediate K63 ubiquitination of RIPK1 to further recruit Author contributions: H. Wang and J.Y. designed experiments; H. Wang conducted the majority of the experiments; H.M., X.L., K.Z., K.D., A.K.M., H. Wei, and Y.L. assisted with TAK1 and TAB1/TAB2 to promote the activation of TAK1 and − − the experiments; S.-C.S. contributed Peli1 / mice; H. Wang and J.Y. analyzed data; and subsequent phosphorylation of the IKK complex to induce the H. Wang and J.Y. wrote the paper. activation of NF-κB. TAK1 can also phosphorylate RIPK1 to Reviewers: D.R.G., St. Jude Children’s Research Hospital; and Z.-G.L., National inhibit its kinase activation; as a result, inhibition of TAK1 by Cancer Institute. α 5z-7-oxozeaenol (5z7) combined with TNF stimulation promotes Conflict of interest statement: J.Y. and D.R.G. were coauthors on a 2016 nomenclature RIPK1 kinase activity-dependent apoptosis (4, 5). In contrast, paper. There was no collaboration between them. inhibition of protein synthesis by cycloheximide (CHX) to block Published under the PNAS license. NF-κB-mediated transcriptional response induced by TNFα 1To whom correspondence should be addressed. Email: [email protected]. leads to RIPK1-independent apoptosis. In the absence of cas- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pase activity, RIPK1 is activated to promote the formation of 1073/pnas.1715742114/-/DCSupplemental.

11944–11949 | PNAS | November 7, 2017 | vol. 114 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1715742114 Downloaded by guest on September 30, 2021 Thus, we conclude that PELI1 is a key modulator of TNFα- PELI1 Is Indispensable for the Formation of RIPK1-RIPK3 Necrosome. mediated cell death pathways that can promote necroptosis and In WT MEFs, RIPK1 is activated after treatment of TNFα/ inhibit apoptosis. SM164/zVAD to lead to the formation of complex IIb, also called necrosome, which includes FADD, caspase-8, and RIPK3, Results which mediates the activation of RIPK3 and the subsequent PELI1 Deficiency Protects Against Necroptosis. Because RIPK1 is phosphorylation and activation of MLKL (14–18). The activated subject to extensive ubiquitination modifications in TNF-RSC MLKL then oligomerizes to execute cell death by disrupting the α integrity of plasma membranes (17, 19–21). (complex I) and complex IIb in response to TNF (14), we con- −/− sidered the possibility that RIPK1 might be modulated by addi- In Peli1 MEFs stimulated by TNFα/SM164/zVAD to induce tional E3 ubiquitin ligases beyond cIAP1/2, the only E3 ubiquitin necroptosis, the phosphorylation of S166 RIPK1, a marker for its ligase characterized as involved in regulating the activation of activation (6, 8, 22), occurred on schedule as that of WT, sug- RIPK1. We conducted a targeted screen of E3 ubiquitin ligases gesting PELI1 deficiency does not affect the activation of A B reported to interact with RIPK1 (Fig. S1A). In this screen, we RIPK1 in MEFs (Fig. 2 and ). In contrast, the phosphory- identified PELI1 as an E3 ubiquitin ligase whose knockdown lation of S232 RIPK3 and S345 MLKL, the markers for the activation of RIPK3 and MLKL, respectively, were blocked in protected cells against necroptosis. Peli1−/− To confirm the role of PELI1 in mediating necroptosis, we MEFs. Furthermore, the oligomerization of MLKL, −/− detectable by Western blotting using nonreducing conditions, generated WT and Peli1 MEFs (mouse embryonic fibroblasts −/− −/− was also blocked in Peli1 MEFs (Fig. 2C). Thus, although cells) derived from WT and Peli1 mice (13) and tested their −/− PELI1 is important for the activation of RIPK3 and MLKL to sensitivity to necroptosis. We found that Peli1 MEFs were promote necroptosis, it is not directly involved in mediating the highly resistant to necroptosis induced by TNFα/SM164/zVAD, activation of RIPK1. TNFα/5z7/zVAD, or TNFα/CHX/zVAD (Fig. 1 A and B and − − We next analyzed the formation of complex IIb by immu- Fig. S1B). The resistance of Peli1 / MEFs to necroptosis in- α α noprecipitation, using an anti-FADD. We found that the duced by TNF /SM164/zVAD and TNF /5z7/zVAD was com- binding of RIPK1, including p-S166 RIPK1, with FADD in cells plete and could not be further enhanced by the treatment of stimulated by TNFα/SM164/zVAD was not affected by PELI1 C–E Nec-1s (Fig. 1 ). We also tested the role of PELI1 in hu- deficiency (Fig. 2B). In contrast, the binding of RIPK3 with man HT29 cells and mouse RGC5 cells, using lentiviral vector- RIPK1andFADDwasblockedbyPELI1deficiency(Fig.2A Peli1 Peli1 mediated shRNA targeting . Knockdown of in and C). HT29 protected against TNFα/SM164/zVAD-induced or TNFα/ Because the complex IIb/necrosome is known to be present in CHX/zVAD-induced necroptosis (Fig. 1 F and G). Furthermore, an insoluble amyloid-like conformation (23), we next analyzed knockdown of Peli1 in RGC5 cells protected against necroptosis the translocation of RIPK1 and RIPK3 into the insoluble frac- induced by TNFα/SM164/zVAD (Fig. S1C). In addition, we tion on induction of necroptosis. We found that although the checked the effect of knocking down Peli1 in murine fibrosar- translocation of modified RIPK1 was largely normal at early − coma L929 cells and found that knockdown of Peli1 protected points (15 min 1·h) after induction of necroptosis, the presence L929 cells from necroptosis induced by zVAD (Fig. S1D). of highly modified RIPK1 in insoluble fraction at 2 h, the time at

−/− CELL BIOLOGY Taken together, these data suggest that PELI1 deficiency which complex IIb formed, was reduced in Peli1 MEFs (Fig. protects against necroptosis. 2C). Furthermore, the translocation of RIPK3 into the insoluble

TNFα+SM164+zVAD AB150 WT MEFs ns ns WT MEFs **** **** **** **** 100 **** **** Peli1-/- MEFs 100 Peli1-/- MEFs WT MEFs 50 +Nec-1s 50 Survival / % / Survival

Peli1-/- MEFs Survival / % +Nec-1s 0 0 03456 Z Fig. 1. PELI1 deficiency protects against necroptosis. O SZ 7 Z+ −/− Time / hours S T M 5z7Z 5z TS (A) WT and Peli1 MEFs were pretreated with Nec- D T T ec-1s Nec-1s α + + N 1s or DMSO for 1 h and then treated with TNF SM164 + zVAD for indicated periods of time. (B)WT − − CDTNFα+5z7+zVAD TNFα+SM164+zVAD and Peli1 / MEFs were pretreated with Nec-1s or 150 150 DMSO for 1 h and then treated with TNFα + 5z7 + WT MEFs WT MEFs zVAD (T5z7Z) or TNFα 10 ng/mL + SM164 + zVAD Peli1-/- MEFs Peli1-/- MEFs − − 100 100 (TSZ) for 6 h. (C) WT and Peli1 / MEFs were pre- WT MEFs WT MEFs treated with Nec-1s or DMSO for 1 h and then +Nec-1s +Nec-1s 50 Peli1-/- MEFs 50 Peli1-/- MEFs treated with TNFα + 5z7 + zVAD for 6, 12, or 24 h. − − Survival / % / Survival Survival / % Survival +Nec-1s +Nec-1s (D)WTandPeli1 / MEFs were pretreated with Nec-1s 0 0 or DMSO for 1 h and then treated with TNFα + SM164 + 061224 061224 zVAD for 6, 12, or 24 h. (F and G) HT29 cells were Time / hours Time / hours transfected with shRNA targeting Peli1 or Scrambled - -/ α + + i1 shRNA for 7 d and then treated with TNF SM164 E el F T P sh-NC zVAD 25 (TSZ) or TNFα + CHX + zVAD (TCZ) for 24 h. W 100 PELI1 50 sh-Peli1 The cell survival was measured with CellTiterGlo. (E *** *** −/− Tubulin and G) WT and Peli1 MEFs (E) or the control and 50 Peli1 knockdown HT29 cells (G) were lysed with SDS li1 50 C e buffer and the samples were analyzed by Western G N P - Survival / % - h sh s blotting with indicated antibodies (G). Concentrations μ α PELI1 50 0 of compounds used Nec-1s, 20 M; TNF , 10 ng/mL; DMSO TSZ TCZ SM164, 50 nM (A, B, D,andF), 500 nM (C); zVAD.fmk, Tubulin 50 25 μM; CHX, 1 μg/mL.

Wang et al. PNAS | November 7, 2017 | vol. 114 | no. 45 | 11945 Downloaded by guest on September 30, 2021 A WT Peli1-/- B WT Peli1-/- M1-ubiquitin chain-specific antibodies (24), we found that K63 TSZ ubiquitination of RIPK1 in complex IIb was inhibited, whereas 0 30m 1h 2h 0 1h 30m 2h

TSZ 0 30m 1h 2h 0 30m 1h 2h −/− RIPK1 75 M1 ubiquitination of RIPK1 increased in Peli1 MEFs after 2 h 170 α D 130 of treatment of TNF /SM164/zVAD (Fig. 2 ). In contrast, we RIPK1 found that PELI1 deficiency resulted in no difference in K63 or Long exp IP: RIPK3 MLKL M1 ubiquitination of RIPK1 in the TNF-RSC (complex I) (Fig. 75 E RIPK3 50 2 ). These results suggest that PELI1 is involved in regulating 50 Peli1 50 p-RIPK1S166 75 RIPK1 ubiquitination in complex IIb. p-RIPK3S232 50 D WT Peli1-/- p-MLKLS345 50 TSZ 2h -+-+ RIPK1 75 wcl 170 IP: K63 130 RIPK3 50 IB: RIPK1 100 A WCL IP: FLAG D Tradd 75 FLAG-TNFα WT -/- Peli1 50 FLAG-TNFα 0 5m 30m 1h 1h Time/ 0 5m 30m 0 5m 30m 0 Tubulin 50 IP: M1 170 Time/ 5m 30m WT Peli1-/- IB: RIPK1 130 170 PELI1 50 C 100 130 RIPK1 75 RIPK1 100 30m 15m 30m 1h 2h 0 15m 1h 2h TSZ 0 IB: RIPK1 75 wcl 75 IP: FLAG RIPK1 170 S166 WT Peli1-/- PELI1 50 130 p-RIPK1 E 100 FLAG-TNFα Tubulin 75 50 75 0 5m 15m 0 Time/ 5m 15m PELI1 50 wcl IP: Nec-1s pre 1h ---+++ 170 B FLAG-TNFα RIPK1 FADD 1st IP: FLAG 130 E SM164 pre 4h ---+++ Time/ 0 5m 30m 0 5m 30m 75 2nd IP: K63 100 FLAG-TNFα 5m 30m 30m 0 5m 75 Time/ 0 RIPK3 50 IB: RIPK1 170 PELI1 50 50 RIPK1 IP: FLAG FADD 25 130 170 100 130 IP: FLAG S166 1st IP: FLAG 75 RIPK1 100 p-RIPK1 75 130 75 NP40 2nd IP: M1 100 PELI1 50 RIPK1 75 PELI1 50 cIAP1/2 75 soluble IB: RIPK1 75 wcl FADD Tubulin 50 PELI1 50 25 50 RIPK1 75 wcl p-RIPK1S166 75 WT Ripk1-/- Tubulin C FLAG-TNFα 50 RIPK3 50 IP: FLAG 170 WT Hoip-/-

Time/ 0 5m 30m 0 5m 30m F Urea IB: RIPK1 130 FLAG-TNFα

100 5m 0 15m 0 5m fracon 170 Time/ 15m RIPK1 75 130 170 WCL RIPK1 IP: FLAG 130 50 100 100 IB: RIPK1 75 RIPK1 75 PELI1 50 75 50 PELI1 wcl PELI1 50 Fig. 2. PELI1 is indispensable for the formation of RIPK1-RIPK3 necrosome. Tubulin SHARPIN 50 IP: FLAG −/− 50 (A and B)WTorPeli1 MEFs were pretreated with zVAD and SM164 for WT Abin-1-/- A20 100 α G 75 30 min and then treated with TNF (TSZ) for indicated periods of time. The FLAG-TNFα TNFR1 50 cells were lysed with Nonidet P-40 buffer. The cell lysates were immuno- 0 5m 30m Time/ 30m 0 5m NEMO 50 precipitated with anti-RIPK3 antibody (A) or anti-FADD antibody (B). (B) The 170 RIPK1 75 Nonidet P-40 insoluble fraction was further extracted with 6M urea buffer. 130 100 PELI1 50 The whole-cell lysates and precipitated proteins were analyzed by Western RIPK1 75 100 IP: FLAG SHARPIN 50 blotting with indicated antibodies. (C) The cells were treated with same A20 75 A20 100 condition as A. The cells were lysed with Nonidet P-40 buffer without NEM PELI1 50 75 wcl TNFR1 50 and analyzed by Western blotting with anti-MLKL in nonreducing SDS/PAGE. PELI1 50 −/− wcl NEMO 50 (D)WTorPeli1 MEFs were pretreated with zVAD and SM164 for 30 min ABIN 75 Tubulin 50 and then treated with TNFα 10 ng/mL (TSZ) for another 2 h. The cells were lysed with 3M urea buffer and immunoprecipitated with anti-K63 ubiquitin Fig. 3. PELI1 is recruited into TNF-RSC in a RIPK1-dependent manner. ab, or with 6M urea buffer and immunoprecipitated with anti-M1 ubiquitin (A) WT MEFs were treated with FLAG-TNFα for indicated periods of time and −/− α antibody. (E) WT and Peli1 MEFs were treated with FLAG-TNF 50 ng/mL then lysed with Nonidet P-40 buffer. The cell lysates were immunoprecipi- for indicated periods of time and then lysed with Nonidet P-40 buffer. The tated with anti-FLAG antibody-conjugated agarose. (B) WT MEFs were pre- cell lysates were immunoprecipitated with anti-FLAG antibody-conjugated treated with Nec-1s or vehicle control (0.01% DMSO) for 1 h and then agarose (first IP). The immunoprecipitated proteins were eluted with 6M treated with FLAG-TNFα for indicated periods of time. The cells were lysed urea buffer. The secondary immunoprecipitation (second IP) of eluted pro- with Nonidet P-40 buffer, and cell lysates were immunoprecipitated with − − teins with anti-M1 ubiquitin ab was performed in 6M urea; alternatively, the anti-FLAG antibody-conjugated agarose. (C and D) WT and Ripk1 / MEFs − − eluted proteins were diluted into 3M urea buffer and immunoprecipitated (C), or WT and Tradd / MEFs (D) were treated with FLAG-TNFα for indicated with anti-K63 ubiquitin antibody. All immunoprecipitated proteins and periods of time and then lysed with Nonidet P-40 buffer. The cell lysates whole-cell lysates (WCL) were analyzed by Western blotting with indicated were immunoprecipitated with anti-FLAG antibody-conjugated agarose. μ α antibodies. Concentrations of compounds used: Nec-1s, 20 M; TNF , 10 ng/mL; (E) WT MEFs were pretreated with SM164 or DMSO for 4 h and then treated μ SM164, 50 nM; zVAD.fmk, 25 M. with FLAG-TNFα for indicated periods of time. The cells were then lysed with Nonidet P-40 buffer, and cell lysates were immunoprecipitated with anti- − − FLAG antibody-conjugated agarose. (F) WT and Hoip / MEFs were treated fraction was blocked by PELI1 deficiency. These data suggest with FLAG-TNFα for indicated periods of time and then lysed with Nonidet PELI1 is involved in promoting the binding of RIPK1 to P-40 buffer. The cell lysates were immunoprecipitated with anti-FLAG anti- RIPK3 to mediate the formation of complex IIb. body-conjugated agarose. (G) WT and Abin-1−/− MEFs were treated with We next analyzed the mechanism that might explain the in- FLAG-TNFα for indicated periods of time and then lysed with Nonidet ability of RIPK1 to bind to RIPK3 in TNFα/SM164/zVAD- P-40 buffer. The cell lysates were immunoprecipitated with anti-FLAG anti- Peli1−/− body-conjugated agarose. The whole cell lysates and immunoprecipitated treated MEFs. Because PELI1 is an E3 ubiquitin li- proteins (A–F) were analyzed by Western blotting with indicated antibodies. gase, we compared the ubiquitination patterns of RIPK1 in Concentrations of compounds used: FLAG-TNFα, 50 ng/mL; Nec-1s, 20 μM; −/− complex IIb in WT and Peli1 MEFs. Using anti-K63 and TNFα, 10 ng/mL; SM164, 50 nM.

11946 | www.pnas.org/cgi/doi/10.1073/pnas.1715742114 Wang et al. Downloaded by guest on September 30, 2021 A B K63 ubiquitination of RIPK1 in complex I under ABIN-1- IP: FLAG WCL FLAG-RIPK1 +++ deficient conditions was increased (28), this result suggests that FLAG-PELI1 +++++ +++++ HA-PELI1 -++ the recruitment of PELI1 into complex I is also regulated by HA-RIPK1-FL -+--- -+--- Nec-1s --+ 75 K63 ubiquitination of RIPK1. However, the recruitment of HA-RIPK1-ΔKD --+-- --+-- IB: FLAG −/− − − IP: HA Cyld Nemo / HA-RIPK1-ΔC ---+- ---+- IB: HA 50 PELI1 into complex I was not altered in or HA-RIPK1-ΔDD ----+ ----+ 75 MEFs (Fig. S2). IB: FLAG NP40 soluble HA 75 IB: HA 50 Taken together, these results suggest that both K63 and 50 35 M1 ubiquitination of RIPK1 are involved in regulating the re- FLAG IB: FLAG NP40 Insoluble 75 cruitment of PELI1 into complex I. HA-RIPK1-FL HA KD ID DD HA-RIPK1-ΔKD HA ID DD PELI1 Mediates K63 Ubiquitination of RIPK1 in a Kinase Activity- HA-RIPK1-ΔC HA KD HA-RIPK1-ΔDD HA KD ID Dependent Manner. RIPK1 contains a kinase domain at N termi- C D nus, a death domain (DD) at its C terminus, and an intermediate WT K115R WT K115R WT S161E domain. To investigate the domain of RIPK1 involved in mediating His-K63Ub ++++++ ++++++ His-K63Ub ++++++ HA-PELI1 -++-++ -++-++ HA-PELI1 -++-++ its binding with PELI1, we overexpressed full-length and truncation SM164 ------++++++ Nec-1s --+-+- mutants of RIPK1, HA-RIPK1-ΔKD, HA-RIPK1-ΔC, and HA- Nec-1s --+--+ --+--+ Ni-NTA RIPK1-ΔDD with that of FLAG-tagged PELI1 in 293T cells, and IB: FLAG Ni-NTA IB: FLAG His 170 75 Pulldown characterized their interactions by immunoprecipitation. We found 130 His p-RIPK1S166 Δ Δ 100 Pulldown 75 that HA-RIPK1, HA-RIPK1- KD, and HA-RIPK1- DD, but not 75 75 WCL Δ A S16675 FLAG HA-RIPK1- C,couldbindtoPELI1(Fig.4 ). Thus, PELI1 in- p-RIPK1 50 FLAG 75 WCL HA teracts with the intermediate domain of RIPK1, and the interaction HA 50 of RIPK1 and PELI1 was not inhibited by Nec-1s (Fig. 4B). Because PELI1 was reported to be able to mediate K63 Fig. 4. PELI1 mediates K63 ubiquitination of RIPK1 on K115 in a kinase activity- κ dependent manner. (A) 293T cells were transfected with expression vectors for ubiquitination of RIPK1 to regulate the activation of NF- Bin FLAG-tagged PELI1 and HA-tagged full-length, ΔKD, ΔC, or ΔDD RIPK1 for 24 h response to TLR stimulation (13), and we found that K63 and then lysed with Nonidet P-40 buffer. The cell lysates were immunoprecipi- ubiquitination of RIPK1 in complex IIb in response to the tated with anti-FLAG antibody-conjugated agarose. (B) 293T cells were pre- treatment of TNFα/SM164/zVAD was strongly defective in treated with Nec-1s (20 μM) or vehicle control (0.01% DMSO) for 1 h and then PELI1-deficient cells (Fig. 2D), we next examined whether transfected with expression vectors for HA-tagged PELI1 and FLAG-tagged PELI1 was involved in mediating K63 ubiquitination of RIPK1 RIPK1 for 24 h. The cells were then lysed with Nonidet P-40 buffer, and cell lysates were immunoprecipitated with anti-HA antibody conjugated agarose. during necroptosis. Furthermore, as the K115 residue of RIPK1 The Nonidet P-40 insoluble fraction was then lysed with 6M urea buffer. (C) 293T cells were transfected with expression vectors for His-K63 ubiquitin and WT or K115R FLAG-RIPK1 with or without that of HA-PELI1 for 24 h and then treated with SM164 or DMSO for 4 h before being lysed with 6M urea. His- A TNFα+5z7 D 150

tagged proteins were pulled down with Ni-NTA. (D) 293T cells were pretreated WT MEFs WT MEFs CELL BIOLOGY ns **** **** with Nec-1s or DMSO for 1 h and then transfected with expression vectors for 100 Peli1-/- MEFs 100 Peli1 -/- MEFs ** WT MEFs **** WT MEFs His-K63 ubiquitin and WT or S161E RIPK1 with or without that of HA-PELI1 for **** +Nec-1s 50 +Nec-1s 50 Peli1-/- MEFs Survival / % 24 h. The cells were lysed with 6M urea buffer, and His-tagged proteins were Survival / % Peli1-/- MEFs * +Nec-1s +Nec-1s pulleddownwithNi-NTA.Allcelllysates and immunoprecipitated or pulled 0 0 03456 DMSO T5z7 TC down proteins were analyzed by Western blotting with indicated antibodies. Time / hours μ Concentrations of compounds used: Nec-1s, 20 M; SM164, 50 nM. BETNFα+SM164 TNFα+CHX 150 WT MEFs 150 Peli1-/- MEFs WT MEFs 100 WT MEFs 100 **** Peli1-/- MEFs PELI1 Is Recruited into TNF-RSC in a RIPK1-Dependent Manner. We **** **** **** **** +Nec-1s **************** **** WT MEFs 50 Peli1-/- MEFs **** 50 +Nec-1s next determined whether PELI1 might be recruited into TNF- Survival / % +Nec-1s

Survival / % Peli1-/- MEFs RSC. We found that in cells treated with FLAG-TNFα for 0 +Nec-1s − 0 6 12 24 0 1· 012141618 30 min h, but not at 5 min, immunoprecipitation by anti-FLAG Time / hours Time / hours revealed the presence of PELI1 (Fig. 3A). This recruitment of C TNFα+5z7 150 PELI1 into complex I was independent of RIPK1 kinase activity, WT BV2 ns ns Peli1-/- BV2 as the pretreatment with RIPK1 kinase inhibitor Nec-1s had no 100 WT BV2 50 B **** +Nec-1s PELI1 effect on the recruitment of PELI1 into complex I (Fig. 3 ). 50 **** Peli1-/- BV2 However, the recruitment of PELI1 into complex I was blocked Survival / % Tubulin −/− +Nec-1s 50 Ripk1 C 0 in MEFs (Fig. 3 ). Thus, the recruitment of PELI1 into 046 BV2 complex I requires RIPK1, but not its kinase activity. Time / hours The recruitment of PELI1 into complex I was also blocked in Fig. 5. PELI1 inhibits RIPK1-dependent and RIPK1-independent apoptosis. MEFs deficient for TRADD, a key adaptor protein that directly −/− D (A)WTandPeli1 MEFs were pretreated with Nec-1s or DMSO for 1 h and binds to the intracellular death domain of TNFR1 (25) (Fig. 3 ). then treated with TNFα + 5z7 for the indicated periods. (B) WT and Peli1−/− Because TRADD is involved in mediating the recruitment of MEFs were pretreated with Nec-1s or DMSO for 1 h and then treated with − − cIAP1/2, we next pretreated cells with cIAP1/2 inhibitor SM164, TNFα 10 ng/mL + SM164 for the indicated periods. (C) WT and Peli1 / which led to the degradation of cIAP1/2, and found that cIAP1/ BV2 cells were pretreated with Nec-1s (20 μM) or DMSO for 1 h and then 2 deficiency also inhibited the recruitment of PELI1 into com- treated with TNFα 10 ng/mL + 5z7 for the indicated periods. (Right)WTand − − plex I (Fig. 3E). The LUBAC complex, consisting of HOIP/ Peli1 / BV2 cells were lysed with SDS buffer, and proteins were analyzed by −/− HOIL/SHARPIN, is involved in mediating M1 ubiquitination of Western blotting with indicated antibodies. (D) WT and Peli1 MEFs were Hoip−/− pretreated with Nec-1s or DMSO for 1 h and then treated with TNFα + 5z7 or TNF-RSC, including RIPK1 (26, 27). In cells stimulated α + −/− α TNF CHX for 6 h. (E) WT and Peli1 MEFs were pretreated with Nec-1s or by TNF , no PELI1 was detected in complex I, suggesting that DMSO for 1 h and then treated with TNFα + CHX for the indicated periods. the recruitment of PELI1 into complex I requires M1 ubiquiti- The cell survival was determined using CellTiterGlo. Concentrations of nation (Fig. 3F). In contrast, the recruitment of PELI1 into compounds used: Nec-1s, 20 μM; TNFα, 10 ng/mL; 5z7, 500 nM; SM164, −/− complex I in Abin-1 MEFs was increased (Fig. 3G). Because 50 nM; zVAD.fmk, 25 μM; CHX, 1 μg/mL.

Wang et al. PNAS | November 7, 2017 | vol. 114 | no. 45 | 11947 Downloaded by guest on September 30, 2021 resistant to necroptosis, knockout of Peli1 in both MEFs and A Peli1 B Peli1 WT -/- WT -/- BV2 cells sensitized to apoptosis induced by TNFα/SM164 or TNFα/5z7 TNFα/CHX −/− −/− 0 1h 2h 4h 0 1h 2h 4h 4h 2h 0 1h 6h 0 1h 4h 6h 2h α A–C Peli1 Peli1 c-FLIP c-FLIP TNF /5z7 (Fig. 5 ). Apoptosis of MEFs and 50 50 BV2 cells induced by TNFα/SM164 or TNFα/5z7 was fully c-Myc 50 50 −/− Caspase-8 blocked by Nec-1s (Fig. 5 A–C). In addition, Peli1 MEFs were 50 α Caspase-8 20 also sensitized to apoptosis induced by TNF /CHX, which was 20 Cleaved not inhibitable by Nec-1s (Fig. 5 D and E). Thus, PELI1 de- Cleaved Caspase-3 20 25 130 ficiency sensitizes to apoptosis mediated by both RIPK1-de- Caspase-3 20 PARP-1 Caspase-3 35 100 pendent and RIPK1-independent pathways. RIPK1 75 PARP-1 130 We characterized the expression of key involved in reg- 100 Caspase-3 35 α Peli1−/− FADD 25 FADD 25 ulating TNF -mediated apoptosis in WT and cells. The ITCH 100 ITCH 100 cleavage of caspase-8, caspase-3, and PARP-1, the important p-c-JunS63 50 p-c-JunS63 50 hallmarks of apoptosis, induced by TNFα/5z7 and TNFα/CHX −/− c-Jun 50 c-Jun 50 were elevated in Peli1 MEFs compared with that of WT (Fig. 6 −/− Peli1 50 Peli1 50 A and B), consistent with increased sensitivity of Peli1 MEFs to Tubulin 50 Tubulin 50 both RIPK1-dependent and RIPK1-independent apoptosis (Fig. C 5). In contrast, we found that the protein levels of caspase-8, WT MEFs 3 **** caspase-3, FADD, ITCH, c-Jun, and PARP-1 were not different in Peli1-/- MEFs Peli1−/− 2 MEFs compared that of WT. Interestingly, the levels of **** c-FLIP, a key regulator of TNFα-mediated caspase-8 activation, 1 Peli1−/− A mRNA level were reduced in MEFs (Fig. 6 ). Consistent with the 0 important role of c-FLIP in regulating both RIPK1-dependent and c-Myc c-FLIP RIPK1-independent apoptosis, knockdown of c-FLIP sensitized cells to apoptosis induced by TNFα/CHX and TNFα/5z7 (Fig. S4). Fig. 6. PELI1 inhibits RIPK1-dependent and RIPK1-independent apoptosis − − by regulating c-FLIP. (A) WT and Peli1 / MEFs were treated with TNFα + We next explored the mechanism by which PELI1 might 5z7 for the indicated periods and then lysed with Nonidet P-40 buffer. The regulate the levels of c-FLIP. Because the expression of cell lysates were analyzed by Western blotting with the indicated antibodies. PELI1 has been found to be positively coregulated with that of − − (B) WT and Peli1 / MEFs were treated with TNFα + CHX for the indicated c-Myc in diffuse large B-cell lymphoma (32) and c-Myc binds to periods and then lysed with Nonidet P-40 buffer. The cell lysates were an- and represses the c-FLIP promoter (33), we considered the alyzed by Western blotting with indicated antibodies. (C) The mRNA was possibility that PELI1 might regulate the levels of c-Myc. We −/− −/− extracted from WT and Peli1 MEFs and reverse-transcribed into cDNA. The found indeed that the levels of c-Myc were increased in Peli1 mRNA levels of c-Myc and c-FLIP were determined by quantitative real-time MEFs (Fig. 6A). To examine whether the regulation of c-FLIP PCR with primers specific to corresponding genes. GAPDH was used as loading control. Concentrations of compounds used: Nec-1s, 20 μM; TNFα, and c-Myc by PELI1 might occur at the transcriptional levels, we 10 ng/mL; 5z7, 500 nM; CHX, 1 μg/mL. measured and compared the mRNA levels of c-FLIP and c-Myc by qPCR. We found that, consistent with their protein levels, the levels of c-Myc mRNA were increased, whereas that of c-FLIP was reported to be a dominant ubiquitination site of RIPK1 in necroptosis (29), we considered the possibility that PELI1 might ubiquitinate RIPK1 on K115. We confirmed that overexpression of PELI1 can increase both ABWT ++--++-- 100 WT and K63 ubiquitination of RIPK1 (Fig. S3A). Furthermore, WT Mice Peli1-/- --++--++ the K63 ubiquitination of RIPK1 promoted by PELI1 was sig- Peli-/- Mice TNFα ----++++ 50 c-FLIP 50 nificantly reduced in the K115R mutant compared with that of c-Myc 50

WT, with or without the treatment of SM-164, which promotes Survival / % Caspase-8 50 the degradation of cIAP1/2 (30) (Fig. 4C). Interestingly, al- 0 Caspase-3 CL 20 0 5 10 15 20 25 Caspase-3 35 though the treatment of Nec-1s did not disrupt the binding of Time after TNFα injection (hr) PELI1 and RIPK1 (Fig. 4B), it inhibited the K63 ubiquitination PELI1 50 Tubulin of RIPK1 mediated by PELI1 (Fig. 4C). However, Nec-1s did C K63 Ubi 50 not reduce the PELI1-mediated K63 ubiquitination of S161E K115 PELI1 RIPK1, a mutation that keeps RIPK1 in constitutively active P RIPK1 conformation and resistant to the inhibition by Nec-1s (6) (Fig. K115 PELI1 RIPK3 P c-Myc P RIPK1 4D). In contrast, the expression of PELI1 did not promote the Complex IIb Transcripon ubiquitination of caspase-8, the other component of complex IIb c-FLIP that is also known to be subject to ubiquitination (31) (Fig. S3B). Nec-1s These results suggest that PELI1 is involved in mediating the MLKL P Caspases K63 ubiquitination of activated RIPK1 on K115 in necroptotic α cells induced by TNF . Furthermore, as K63 ubiquitination of Necroptosis Apoptosis WT RIPK1, but not the S161E mutant of RIPK1 by PELI1, is − − blocked by Nec-1s, these results suggest that inhibition of PELI1- Fig. 7. Peli1 / mice are sensitized to TNFα-induced SIRS. (A) Survival curves of − − mediated K63 ubiquitination of RIPK1 by Nec-1s may, at least in nine male WT and nine male Peli1 / mice (8–12 wk) injected with mTNFα i.v. (mTNFα/mouse = 360 μg/kg). P = 0.0166 according to Gehan-Breslow-Wilcoxon part, explain the resistance of Nec-1s-treated cells to necroptosis, −/− as PELI1 deficiency offers strong resistance to necroptosis. test. (B) Two WT mice and two Peli1 mice were killed 6 h after injection with mTNFα and perfused. The spleens from these four mice were lysed and ana- lyzed by Western blotting with indicated antibodies. (C) A model for the role of PELI1 Inhibits RIPK1-Dependent and RIPK1-Independent Apoptosis by PELI1 in necroptosis and apoptosis. In necroptotic cells, PELI1 mediates Regulating c-FLIP. To investigate the role of PELI1 in apoptosis, K63 ubiquitination of activated RIPK1 on K115, which can be inhibited by Nec- we next investigated the sensitivity of PELI1 knockout cells 1s. PELI1-mediated RIPK1 ubiquitination promotes the interaction of RIPK1 and −/− to the induction of apoptosis. Although Peli1 MEFs were RIPK3 to form necrosome and activation of MLKL to lead to .

11948 | www.pnas.org/cgi/doi/10.1073/pnas.1715742114 Wang et al. Downloaded by guest on September 30, 2021 −/− decreased, in Peli1 MEFs (Fig. 6C). These results suggest the to apoptosis induced by TNFα in both an RIPK1-dependent and possible contribution of elevated c-Myc to reduce the expression an RIPK1-independent manner because of the reduced expres- −/− of c-FLIP in Peli1 MEFs. sion levels of c-FLIP, a critical regulator of caspase-8 activation. Because the cleavage of RIPK1 during apoptosis separates its −/− Peli1 Mice Are Sensitized to TNFα-Induced SIRS. Because both kinase domain from the intermediate domain (34), PELI1 cannot necroptosis and apoptosis play important roles in TNFα injection- perform ubiquitination of RIPK1 during apoptosis, which might induced systemic inflammatory response syndrome (SIRS) in explain why increased sensitivity to apoptosis is dominant over the −/− −/− mice, we next investigated the response of Peli1 mice to SIRS. resistance to necroptosis in Peli1 mice on TNFα stimulation. Peli1−/− We found that mice were sensitized to the lethality in- We propose that increased c-Myc might suppress the expression α Peli1−/− duced by i.v. injection of mTNF . mice died more and of c-FLIP, as c-Myc can bind to and repress the activation of μ α A earlier than that of WT at 360 g/kg of TNF (Fig. 7 ). Consis- c-FLIP promoter (33). However, our study does not rule out the tently, the levels of c-Myc were higher and those of c-FLIP were −/− potential involvement of NF-κB in regulating the expression of lower in the spleens of Peli1 mice (Fig. 7B). Furthermore, we −/− c-FLIP. Because RIPK1 can also be activated by LPS (35), reg- detected increased cleavage of caspase-3 in the spleens of Peli1 α ulation of RIPK1 ubiquitination in its kinase-dependent manner mice injected with TNF compared with that of WT. Thus, in- by PELI1 may also contribute to the production of proin- creased activation of apoptosis is sufficient to promote the le- flammatory in response to certain TLR ligands. thality of PELI1-deficient mice in response to TNFα stimulation. Materials and Methods Discussion Antibodies and Reagents. The following commercial antibodies and reagents In this study, we demonstrate that PELI1 is a pronecroptosis E3 were used in this study: Pellino-1, Abcam (ab199336); TAK1, Cell Signaling Tech- ubiquitin ligase that modulates K63 ubiquitination of RIPK1 to nology (5206); RIPK1, Cell Signaling Technology (3493) and BD Biosciences (610459); promote the interaction of activated RIPK1 with RIPK3 in nec- RIPK3, Biorad/Serotec (AHP1797); c-Myc, Santa Cruz (sc-764); c-FLIP, EnZo (ADI-AAP- roptosis. We show that PELI1 mediates K63 ubiquitination of 440); NEMO, CST (2685); TBK1, Cell Signaling Technology (3504); FADD, Abcam RIPK1 in kinase domain on K115, although our study does not (ab124812) and Santa Cruz (6036); α-Tubulin, Sigma-Aldrich (T9026); β-actin, Santa rule out the involvement of Peli1 in mediating ubiquitination of Cruz (81178); FLAG, CST (2368); 5Z-7 was from Sigma-Aldrich (O9890). RIPK1 on additional Lys residues. Because the hallmark of RIPK1 activation involves autophosphorylation on the kinase Statistics. Data are expressed as the mean ± SEM. Pairwise comparisons be- tween two groups were performed using the Student’s t test. Differences domain (6, 8), ubiquitination of RIPK1 by PELI1 might involve < < < the recognition of phosphorylated RIPK1. In contrast, because were considered statistically significant if *P 0.05, **P 0.01, ***P 0.001 or not significant (n.s.). At least three independent repeats were in- PELI1 deficiency promotes strong resistance to necroptosis by cluded in each data point. Each experiment was repeated at least three times. blocking the formation of complex IIb, inhibition of PELI1-me- diated K63 ubiquitination of RIPK1 in Nec-1s-treated cells might ACKNOWLEDGMENTS. We thank Dr. of Genentech for M1 and be important to explain the inhibition of complex IIb formation K63 ubiquitin-specific antibodies. This work was supported in part by Grants and necroptosis by Nec-1s, and why inhibition of RIPK1 prevents from the National Institute on Aging (1R01AG047231) and the National Insti- not only the phosphorylation of RIPK1 but also its ubiquitination tute of Neurological Disorders and (1R01NS082257), and Grants from

the Chinese Academy of Sciences, the National Key R&D Program of China, the CELL BIOLOGY in complex IIb (29). China Ministry of Science and Technology Program (2014ZX09102001-002), and We show that PELI1 inhibits apoptosis by modulating the the China National Natural Science Foundation (31530041), and the Chinese expression levels of c-FLIP. PELI1-deficient cells are sensitized Academy of Sciences and the National Science Foundation in China (to J.Y.).

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