USP22 controls necroptosis by regulating receptor‐interacting protein kinase 3 ubiquitination

Article28 December 2020Open Access Transparent process USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination Jens Roedig Jens Roedig Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Lisa Kowald Lisa Kowald Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Thomas Juretschke Thomas Juretschke Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Rebekka Karlowitz Rebekka Karlowitz Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Behnaz Ahangarian Abhari Behnaz Ahangarian Abhari Lighthouse Core Facility, Zentrum für Translationale Zellforschung, Universitaetsklinikum Freiburg, Klinik für Innere Medizin I, Freiburg, Germany Search for more papers by this author Heiko Roedig Heiko Roedig Pharmazentrum Frankfurt, Institut für Allgemeine Pharmakologie und Toxikologie, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Simone Fulda Simone Fulda orcid.org/0000-0002-0459-6417 Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Petra Beli Petra Beli orcid.org/0000-0001-9507-9820 Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Sjoerd JL van Wijk Corresponding Author Sjoerd JL van Wijk [email protected] orcid.org/0000-0001-6532-7651 Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Jens Roedig Jens Roedig Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Lisa Kowald Lisa Kowald Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Thomas Juretschke Thomas Juretschke Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Rebekka Karlowitz Rebekka Karlowitz Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Behnaz Ahangarian Abhari Behnaz Ahangarian Abhari Lighthouse Core Facility, Zentrum für Translationale Zellforschung, Universitaetsklinikum Freiburg, Klinik für Innere Medizin I, Freiburg, Germany Search for more papers by this author Heiko Roedig Heiko Roedig Pharmazentrum Frankfurt, Institut für Allgemeine Pharmakologie und Toxikologie, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Simone Fulda Simone Fulda orcid.org/0000-0002-0459-6417 Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Petra Beli Petra Beli orcid.org/0000-0001-9507-9820 Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Sjoerd JL van Wijk Corresponding Author Sjoerd JL van Wijk [email protected] orcid.org/0000-0001-6532-7651 Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Author Information Jens Roedig1, Lisa Kowald1, Thomas Juretschke2, Rebekka Karlowitz1, Behnaz Ahangarian Abhari3, Heiko Roedig4, Simone Fulda1, Petra Beli2 and Sjoerd JL Wijk *,1 1Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt am Main, Germany 2Institute of Molecular Biology (IMB), Mainz, Germany 3Lighthouse Core Facility, Zentrum für Translationale Zellforschung, Universitaetsklinikum Freiburg, Klinik für Innere Medizin I, Freiburg, Germany 4Pharmazentrum Frankfurt, Institut für Allgemeine Pharmakologie und Toxikologie, Goethe-University, Frankfurt am Main, Germany *Corresponding author. Tel: +49 69 67866574; Fax: +49 69 6786659158, E-mail: [email protected] EMBO Reports (2021)22:e50163https://doi.org/10.15252/embr.202050163 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death. SYNOPSIS Ubiquitin-specific protease 22 (USP22) controls necroptotic cell death by regulating RIPK3 phosphorylation and RIPK3 K518 ubiquitination in human tumor cell lines. USP22 affects RIPK3 phosphorylation and ubiquitination. Identification of novel, USP22-regulated RIPK3 ubiquitin sites at K42, K351 and K518 upon necroptosis progression. USP22-regulated RIPK3 K518 ubiquitination controls necroptosis. Introduction Ubiquitination, i.e., the covalent post-translational modification of substrates with one or multiple ubiquitin molecules, controls protein degradation, cell signaling, and other cellular processes, affecting almost all cellular proteins (Komander & Rape, 2012; Swatek & Komander, 2016; Yau & Rape, 2016). Ubiquitination can occur as single modification (mono-ubiquitination) or through linkage into poly-ubiquitin chains via internal lysine (K) residues or through the initiator methionine (M1 or linear chains) (Kirisako et al, 2006; Komander & Rape, 2012). The deposition of ubiquitin, in general, is catalyzed by an enzymatic cascade involving E3 ubiquitin ligases (Buetow & Huang, 2016), while various deubiquitinating enzymes (DUBs) hydrolyze ubiquitin signals from substrates, creating dynamically balanced systems (Reyes-Turcu et al, 2009; Clague et al, 2013). Ubiquitin-specific peptidase 22 (USP22) is a conserved DUB belonging to the ubiquitin-specific protease (USP) superfamily. USP22 is, together with ATXN7L3, ATXN7, and ENY2, part of the deubiquitinating module (DUBm) of the human Spt-Ada-Gcn5-acetyltransferase (SAGA) complex (Zhang et al, 2008b). Within this complex, the primary function of USP22 consists in deubiquitinating histone H2B K120 and histone H2A K119, thereby promoting transcriptional activation (Zhao et al, 2008; Zhang et al, 2008a). Apart from enhancing transcriptional regulation, USP22 controls additional biological processes, like cell growth and differentiation, tumor development, and cell death (Zhang et al, 2008b; Lv et al, 2011; Lin et al, 2012; Xu et al, 2012; Li et al, 2013; Sussman et al, 2013). Importantly, increased USP22 expression is closely associated with neurodegenerative diseases, carcinogenesis, and poor patient survival in a wide range of tumor types (Glinsky et al, 2005; Liu et al, 2010; Liu et al, 2011; Yang et al, 2011; Zhang et al, 2011; Piao et al, 2012; Li et al, 2012b; Wang et al, 2013; Liang et al, 2014; Ning et al, 2014; Ji et al, 2015; Tang et al, 2015; Wang et al, 2015). USP22 controls cell death regulation via deubiquitination and stabilization of sirtuin 1 (SIRT1), leading to TP53 deacetylation and transcriptional activation of TP53 target genes or deacetylation-dependent c-Myc stabilization, thereby inhibiting apoptosis (Lin et al, 2012; Li et al, 2014). Other studies suggest that USP22 overexpression induces enhanced resistance to apoptosis and treatment resistance in multiple cancer cell lines (Lin et al, 2012; Xu et al, 2012; Armour et al, 2013; Li et al, 2013; Li et al, 2014; Xiong et al, 2014). A crucial role of dynamically regulated ubiquitination underlies tumor necrosis factor (TNF)α-mediated cell fate signaling. Here, E3 ligases and DUBs critically control the signaling outcome, determining between cell survival and programmed cell death, like apoptosis or necroptosis pathway (Park et al, 2004; Haas et al, 2009; Dynek et al, 2010; Ikeda et al, 2011; Vanlangenakker et al, 2011; Moquin et al, 2013; Draber et al, 2015; Onizawa et al, 2015; Choi et al, 2018; Heger et al, 2018; Lee et al, 2019). Necroptosis is a caspase-independent form of programmed cell death, characterized by a regulated, phosphorylation-dependent interplay between RIPK1, RIPK3, and the mixed lineage kinase domain-like (MLKL), ultimately resulting in MLKL-mediated plasma membrane rupture (Cho et al, 2009; He et al, 2009; Vandenabeele et al, 2010; Mocarski et al, 2011; Sun et al, 2012; Li et al, 2012a; Morgan & Liu, 2013; Murphy et al, 2013; Sun & Wang, 2014; Petrie et al, 2019). Necroptosis is involved in ischemic injury (Degterev et al, 2005), infectious diseases (Kaiser et al, 2013; Mocarski et al, 2015; Pearson et al, 2017; Upton & Kaiser, 2017), cancer (Seifert et al, 2016; Najafov et al, 2017), and multiple sclerosis (Ofengeim et al, 2015; Alvarez-Diaz et al, 2016). Upon inhibition or inactivation of caspase-8 and cellular Inhibitor of apoptosis protein 1/2 (cIAP1/2), activation of TNF receptor-1 (TNFR1) by TNFα induces the formation of TNFR1 signaling complexes, leading to phosphorylation-dependent RIPK1 activation (Weinlich et al, 2017). Activated RIPK1 initiates the formation of the necrosome, a hetero-amyloid complex composed of kinase-activated RIPK1-RIPK3, that associate via their RIPK homotypic interaction motifs (RHIMs) (Cho et al, 2009; He et al, 2009; Zhang et al, 2009; Zhao et al, 2012; Mompean et al, 2018). RIPK3 induces phosphorylation and recruitment of the pseudokinase MLKL into the necrosome (Li et al, 2012a; Cai et al, 2014), leading to MLKL oligomerization and translocation to biological membranes, where MLKL triggers necroptosis through pore formation and membrane rupture (Wang et al, 2014; Quarato et al, 2016). Apart from phosphorylation, the necroptotic key players RIPK1 and RIPK3 are also modified by ubiquitin chains, thereby influencing necroptotic signaling (de Almagro et al, 2015; Onizawa et al, 2015; Seo et al, 2016; Choi et al, 2018). For example, it has been shown that RIPK3 ubiquitination at K5 promotes RIPK1-RIPK3 complex formation and enhances necroptosis, which is restricted by the DUB A20 (Onizawa et al, 2015). Furthermore, the E3 ubiquitin-protein ligase pellino homolog 1 (PELI1) mediates K48-linked poly-ubiquitination of kinase-active RIPK3, leading to proteasomal degradation of RIPK3 (Choi et al, 2018). Despite the relevance of (de)ubiquitination in fine-tuning necroptotic cell death signaling, the potential involvement of additional DUBs in the regulation of necroptosis still remains unclear. Here, we identify USP22 as a novel positive regulator of necroptotic cell death. Loss of USP22 expression delays TNFα/carbobenzoxyvalyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD.fmk)/Smac mimetic-induced necroptosis in several human tumor cells, without affecting TNFα-induced nuclear factor-kappa B (NF-κB) signaling or TNFα-mediated extrinsic apoptosis. We demonstrate that USP22 controls RIPK3 phosphorylation and ubiquitination and identify three novel USP22-regulated RIPK3 ubiquitination sites (K42, K351, and K518). Importantly, a central role for USP22-regulated RIPK3 K518 deubiquitination was confirmed in the control of TNFα-induced necroptotic cell death. In our study, we discover a novel role for USP22 in regulating RIPK3 ubiquitination and necroptosis and further elucidate the prominent roles of DUBs and ubiquitination in the regulation of programmed cell death. Results USP22 regulates necroptosis in HT-29 cells USP22 is a ~ 58 kDa ubiquitin hydrolase that contains a C-terminal ubiquitin-specific protease (USP) domain, well known for its role as SAGA complex-associated DUB that regulates deubiquitination of H2A and H2B. USP22 is highly expressed in various colon carcinoma cell lines and mediates apoptosis resistance (Liu et al, 2011; Lin et al, 2012; Xu et al, 2012). Since necroptosis represents an attractive alternative for overcoming apoptosis resistance in colon cancer (Chromik et al, 2014), we therefore aimed to investigate the functional role of USP22 during necroptosis. To this end, USP22 expression was reduced using siRNA in the HT-29 human colon carcinoma cell line, followed by induction of TNFα (T)/Smac mimetic (BV6; B)/zVAD.fmk (Z) (TBZ)-mediated necroptosis and quantification of propidium iodide (PI) uptake, as marker for cell death. Knockdown of USP22 expression significantly reduced TBZ-induced necroptotic cell death (Fig 1A and B). In addition, USP22 knockout (KO) HT-29 cell lines were generated using CRISPR/Cas9 and single clones were isolated. Based on Western blot analysis of USP22 expression, HT-29 clones were selected that exhibit either undetectable USP22 levels (clone #1, #2, and #3), intermediate (clone #4), or normal USP22 expression, compared to control CRISPR/Cas9 (clone #5) and wild-type (WT) HT-29 cell lines (Figs 1C and EV1A). Clone #5 was chosen to exclude possible side effects mediated by puromycin resistance or CRISPR/Cas9 artifacts. As expected, all HT-29 cell lines subjected to CRISPR/Cas9 editing expressed the Cas9 protein, which was absent in WT parental HT-29 (Fig EV1A). Furthermore, USP22-deficient HT-29 clones displayed increased levels of ubiquitinated histone H2B at K120, whereas total H2B levels remained largely unchanged (Fig EV1A). Complete lack of USP22 expression in HT-29 clones #1, #2, and #3 significantly reduced necroptotic cell death, compared to control CRISPR/Cas9 HT-29 cells (Figs 1D, and EV1B and C). Intriguingly, HT-29 clone #4, expressing intermediate USP22 levels, was only partially rescued upon TBZ treatment, suggesting that USP22 expression quantitatively corresponds with necroptosis progression (Fig 1D). Furthermore, HT-29 cells that were subjected to CRISPR/Cas9 editing while expressing near-normal USP22 expression levels underwent TBZ-induced necroptosis to the same extent as WT HT-29 (Fig EV1C), suggesting that loss of USP22 expression, and not puromycin resistance, clonal effects or potentially other CRISPR/Cas9 artifacts, is indeed the sole determinant for the delay in necroptosis sensitivity. Regardless of the cellular USP22 expression status, TBZ-induced cell death could be completely blocked by inhibiting RIPK1 with necrostatin-1s (Nec-1s), RIPK3 using GSK’872 and Dabrafenib (Dab), or necrosulfonamide (NSA) to inhibit MLKL (Figs 1D and EV1C), confirming necroptotic cell death. Figure 1. USP22 knockout (KO) decreases TBZ-induced necroptotic cell death in HT-29 cells A, B. HT-29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control. C. HT-29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control. D. HT-29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα after pre-incubation with 30 µM Nec-1s and 20 µM GSK’872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells. E. HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S), or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control. F. HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S), or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining. Data information: Data represent mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001, NS: not significant, by unpaired 2-tailed Student's t-test. Three independent experiments performed in triplicate are shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of USP22 in HT-29 cells does not affect TB-induced apoptosis or NF-κB activation upstream of IκBα Whole cell lysates of HT-29 parental, CRISPR/Cas9 control (ctr), and USP22 KO cells were analyzed by Western blotting for the indicated proteins. β-Actin was used as a loading control. HT-29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 6 h, and cell death was determined by analysis of PI-positive nuclei. HT-29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 18 h. Cells were additionally treated with 30 µM Nec-1s, 20 µM GSK’872, 20 µM Dab, and 10 µM NSA, as indicated. Cell death was determined by analysis of PI-positive nuclei. HT-29 control and USP22 KO cells were stimulated with 10 ng/ml TNFα for 5 and 15 min. Protein expression of IκBα, phosphorylated IκBα, and USP22 were examined by Western blotting. Vinculin was used as a loading control. HT-29 control and USP22 KO cells were treated for 48 h, as indicated, with 0.5 µM BV6 and 1 ng/ml TNFα. Cell death was determined by analysis of PI-positive nuclei. HT-29 control cells and USP22 KO cells, generated with three (USP22 KO #1) or with two (USP22 KO (2g)) USP22 gRNAs, were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα, and 30 µM Nec-1s for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining. Jurkat CRISPR/Cas9 control (Ctr) control and USP22 KO cells were analyzed for USP22 expression by Western blotting. β-Actin served as loading control. Jurkat control and USP22 KO cells were pre-treated with 10 µM Nec-1s before stimulation with 1 µM BV6 and 10 ng/ml TNFα. Cell death was measured after 18 h by analysis of PI-positive nuclei. Jurkat control and USP22 KO cells were pre-treated with 10 µM Nec-1s or 20 µM Dabrafenib before stimulation with 1 µM BV6 and 10 ng/ml TNFα for 8 h. Cell death was determined by analysis of PI-positive nuclei. Data information: Data represent mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001, NS: not significant, by unpaired 2-tailed Student's t-test. Three independent experiments performed in triplicate are shown. Download figure Download PowerPoint Since TBZ-mediated necroptosis requires TNFR1 activation by TNFα, we first determined the role of USP22 in TNFα-mediated NF-κB activation. Stimulating control and USP22 KO HT-29 cells with TNFα revealed no differences in the levels of NF-kappa-B inhibitor alpha (IκBα) phosphorylation and degradation, thereby ruling out that USP22 affects TNFR1 activation or classical NF-κB activation upstream of IκBα (Fig EV1D). Loss of USP22 expression has also been shown to increase apoptosis in several cancer cell lines, including the colorectal cell line HCT116 (Xu et al, 2012). However, no differences in TB-induced extrinsic apoptosis could be detected between control and USP22 KO HT-29 cells (Figs 1B and D, and EV1B, C, E, Appendix Fig S1), suggesting that USP22 specifically regulates necroptosis without affecting TNFα-dependent pro-survival or apoptotic cell death. To further confirm that loss of USP22 is responsible for the effects on necroptotic cell death and not any off-target effects, we argued that stable re-expression of USP22 in HT-29 cells with CRISPR/Cas9-mediated USP22 KO should resensitize these for necroptosis induction. To this end, protospacer adjacent Motif (PAM)-mutated WT and catalytically inactive C185S or C185A USP22 mutants were stably re-expressed in USP22 KO HT-29 cells generated with two guide RNAs, displaying the same necroptotic resistance as cells generated with three guide RNAs (Fig EV1F). Stable reconstitution of PAM-mutated WT and mutant USP22 in USP22 KO HT-29 cells restored USP22 expression (Fig 1E, Appendix Fig S2A and B). Importantly, only reconstitution with PAM-mutated WT USP22 was able to re-establish necroptosis sensitivity compared to USP22 KO clones reconstituted with empty vector (EV) (Fig 1F). Of note, necroptosis could not be rescued upon re-expression of C185S or C185A USP22 (Fig 1F), suggesting that the catalytic DUB activity of USP22 is required for necroptosis. Importantly, apart from HT-29, loss of USP22 expression in the human acute lymphoblastic leukemia (ALL) Jurkat cell line also prominently reduced TBZ-induced necroptosis, compared to control Jurkat cells (Fig EV1G–I). On the other hand and in agreement with USP22 KO HT-29, USP22 KO Jurkat ALL were not sensitized for TB-induced cell death, compared to control Jurkat (Fig EV1G–I), similar as USP22 KO acute promyelocytic leukemia (APL) NB4 cells (Appendix Fig S3A and B). Interestingly, CRISPR/Cas9- or siRNA-mediated KO or knockdown of USP22 in mouse embryonic fibroblasts (MEFs) or the macrophage lines Raw264.7 or J774A1, commonly used to study necroptosis (Jouan-Lanhouet et al, 2014; Sawai, 2014; Zhou & Yuan, 2014), had no effect on TB- or TBZ-induced cell death (Appendix Fig S4A–F). Taken together, these results demonstrate that USP22 is involved in the regulation of necroptosis in human tumor cells, but likely not in murine cells, through DUB-mediated effects. To investigate how USP22 controls necroptosis, the expression levels and phosphorylation status of RIPK1, RIPK3, and MLKL were determined in HT-29 control and USP22 KO cells upon TBZ exposure for different time points. As expected, TBZ-induced differences in RIPK1 and MLKL phosphorylation could already be detected after 2–3 h of treatment (Fig 2A). Interestingly, increased levels of RIPK1 Ser166 phosphorylation could be observed in USP22 KO cells compared to control HT-29 cells, already after 2 h of TBZ treatment, suggesting that loss of USP22 expression might facilitate RIPK1 activation. Strikingly, USP22 KO HT-29 cells exhibit a prominent “smear” of slower-migrating RIPK3 bands, which was almost absent in control cells. These slower-migrating RIPK3 signals in USP22 KO cells were maintained over the extended experimental time frame (Fig 2A). RIPK3 is heavily modified with multiple types of post-translational modifications, including phosphorylation and ubiquitination, upon the induction and progression of necroptosis (Cho et al, 2009; He et al, 2009; Onizawa et al, 2015). Alterations in phosphorylated RIPK3 levels could indeed be confirmed using two phospho-RIPK3-specific antibodies (recognizing RIPK3 S227 phosphorylation) upon TBZ stimulation in control and USP22 KO HT-29 cells (Fig 2B). To evaluate whether, in the absence of USP22, the slower-migrating RIPK3 bands are indeed phosphorylated forms of RIPK3, lysates of TBZ-treated control and USP22 KO HT-29 cells were incubated with λ-phosphatase. Importantly, phosphatase treatment almost completely reduced the TBZ-induced RIPK3 shift observed in USP22 KO HT-29 cells (Fig 2C). In addition, high molecular weight RIPK3 signals were shown to be more pronounced upon loss of USP22 expression (Fig 2C). As expected, Dab and Nec-1s almost completely blocked these slower-migrating RIPK3 signals, suggesting multiple types of necroptosis-induced modifications. These results demonstrate that USP22 specifically regulates TBZ-induced necroptosis and that USP22 influences RIPK3 post-translational modifications. Figure 2. USP22 KO leads to increased TBZ-induced RIPK3 phosphorylation in HT-29 cells HT-29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control. HT-29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control. HT-29 control and USP22 KO cells were incubated with 30 µM Nec-1s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 5 h. 100 μg of each lysate was incubated with 400 U/μl λ-phosphatase for 30 min at 30°C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 “smears” were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-actin levels. Data information: Data represent mean ± SD; *P < 0.05; by unpaired 2-tailed Student's t-test. Three independent experiments performed in triplicate are shown. In panel (C), quantification of blots from three independent experiments is shown. Download figure Download PowerPoint Loss of USP22 induces resistance to necroptotic cell death in RIPK3-expressing HeLa cells To further investigate the functional roles of USP22 during necroptosis and RIPK3 modification, HeLa TRex cells, that lack endogenous RIPK3 expression and therefore are resistant against TBZ-induced necroptotic cell death, were modified to express doxycycline (Dox)-inducible Strep-tagged RIPK3 (Fig 3A). Furthermore, these cell lines were subjected to USP22 deletion using CRISPR/Cas9 (Fig 3A). Importantly, the HeLa TRex RIPK3 USP22 KO cells express largely comparable RIPK3 expression levels upon Dox induction compared to control CRISPR/Cas9 HeLa TRex RIPK3 cells, suggesting that USP22 likely does not affect basal RIPK3 protein stability (Fig 3A). TBZ treatment considerably increased cell death in HeLa TRex RIPK3 CRISPR/Cas9 control cells, while HeLa TRex RIPK3 USP22 KO displayed significantly reduced cell death (Fig 3B), suggesting that USP22 also controls necroptotic cell death in necroptosis-sensitive HeLa cells. As expected, USP22 KO HeLa TRex cells did not die upon TB-induced apoptotic cell death (Figs 3B and EV2A). In line with this and with previous observations in HT-29, Jurkat, and NB4 cells, the formation of the apoptotic signaling complex II was not affected upon KO of USP22 (Fig EV2B). Of note, USP22 KO also did not alter TNFα-induced complex I formation (Fig EV2C), corresponding with the lack of USP22 function in TNFα-induced NF-κB signaling. Importantly, no differences in the amount of phosphorylated RIPK1 could be observed in complex I and II IPs from USP22 KO cells compared to control cells (Fig EV2B and C). Figure 3. USP22 knockdown in RIPK3-expressing HeLa cells induces resistance to TBZ-induced necroptotic cell death HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band. HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei. HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4, and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL, and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control. HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti-Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control. USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads, and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control. Data information: Data represent mean ± SD; *P < 0.05; ***P < 0.001, by unpaired 2-tailed Student's t-test. Three independent experiments performed in triplicate are shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. USP22 KO in HeLa TRex RIPK3 does not affect TB-induced ap