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. 2021 Feb 3;22(2):e50163.
doi: 10.15252/embr.202050163. Epub 2020 Dec 28.

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

Jens Roedig  1 , Lisa Kowald  1 , Thomas Juretschke  2 , Rebekka Karlowitz  1 , Behnaz Ahangarian Abhari  3 , Heiko Roedig  4 , Simone Fulda  1 , Petra Beli  2 , Sjoerd Jl van Wijk  1
Affiliations

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

Jens Roedig et al. EMBO Rep. .

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.

Keywords: RIPK1; cancer; mixed lineage kinase domain-like; post-translational modifications; ubiquitin hydrolase.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. USP22 knockout (KO) decreases TBZ‐induced necroptotic cell death in HT‐29 cells
  1. 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.

  2. C

    HT‐29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control.

  3. 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.

  4. 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.

  5. 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.
Figure EV1
Figure EV1. Loss of USP22 in HT‐29 cells does not affect TB‐induced apoptosis or NF‐κB activation upstream of IκBα

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Jurkat CRISPR/Cas9 control (Ctr) control and USP22 KO cells were analyzed for USP22 expression by Western blotting. β‐Actin served as loading control.

  8. 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.

  9. 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.
Figure 2
Figure 2. USP22 KO leads to increased TBZ‐induced RIPK3 phosphorylation in HT‐29 cells

  1. 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.

  2. 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.

  3. 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.
Figure 3
Figure 3. USP22 knockdown in RIPK3‐expressing HeLa cells induces resistance to TBZ‐induced necroptotic cell death

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.
Figure EV2
Figure EV2. USP22 KO in HeLa TRex RIPK3 does not affect TB‐induced apoptosis or complex I and II formation

  1. HeLa TRex RIPK3 CRISPR/Cas9 control (Ctr) and USP22 KO cells were treated with 5 µM BV6 and 10 ng/ml TNFα for 24 h. Cell death was determined by analysis of PI‐positive nuclei.

  2. HeLa TRex RIPK3 CRISPR/Cas9 control (Ctr) and USP22 KO cells were pre‐treated with 5 µM BV6 for 1 h before being stimulated with 10 ng/ml TNFα for 1 and 2 h, after which caspase‐8 was immunoprecipitated, followed by analysis by Western blotting with the indicated antibodies. β‐Actin served as loading control.

  3. HeLa TRex RIPK3 CRISPR/Cas9 control (Ctr) and USP22 KO cells were starved in serum‐free DMEM for 2 h. Cells were stimulated with 1 μg/ml FLAG‐hTNF for 5 and 15 min as indicated in the Materials and Methods section. FLAG‐hTNF was immunoprecipitated and analyzed by Western blotting with the indicated antibodies. β‐Actin served as loading control.

  4. RIPK3 expression in HeLa TRex CRISPR/Cas9 control and USP22 KO cells was induced by Dox treatment. For quantification, phosphorylated MLKL levels of both cell lines were first normalized to β‐Actin levels followed by normalization to the 0 h time point of HeLa TRex CRISPR/Cas9 control cells.

Data information: Data represent mean ± SD; **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. In panel D, quantification of blots from three independent experiments.
Figure 4
Figure 4. Increased RIPK3 ubiquitination in USP22 KO HT‐29 cells during TBZ‐induced necroptosis

  1. HT‐29 control, USP22 KO, and USP22 KO cells re‐expressing PAM‐mutated 3xFLAG‐HA‐USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 4 h. Poly‐ubiquitinated proteins were enriched by GST‐TUBE pull‐down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β‐Actin served as loading control. Ponceau staining was used to confirm equal loading of GST‐TUBE.

  2. HT‐29 control and USP22 KO cells were transfected with His‐ubiquitin for 24 h, as indicated. Cells were pre‐stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre‐treatment, 1 ng/ml TNFα was added for 4 h. His‐ubiquitin was immunoprecipitated using Ni‐NTA beads, and detection of indicated proteins was performed by Western blotting. β‐Actin served as loading control for the input, whereas His‐ubiquitin levels served as loading control for immunoprecipitated ubiquitin.

  3. HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA‐ubiquitin for 24 h, as indicated. Cells were pre‐stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre‐treatment, 10 ng/ml TNFα were added for 3 h. HA‐ubiquitin was immunoprecipitated using anti‐HA‐beads, and detection of indicated proteins was performed by Western blotting. β‐Actin served as loading control for the input, whereas HA‐levels served as loading control for immunoprecipitated ubiquitin.

Figure EV3
Figure EV3. USP22 KO in HT‐29 cells does not alter TBZ‐induced RIPK1 ubiquitination
HT‐29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 3 and 6 h. Ubiquitinated proteins were enriched by GST‐TUBE pull‐down. Protein expression of RIPK1, RIPK3, USP22, and ubiquitinated RIPK1, RIPK3, and USP22 was monitored using Western blotting with the indicated antibodies. GAPDH served as a loading control. Ponceau staining was used to confirm equal loading of GST‐TUBE.
Figure 5
Figure 5. Identification of novel, USP22‐regulated RIPK3 ubiquitination sites

  1. Schematic representation of the experimental strategy for quantitative analysis of ubiquitination sites in USP22 KO HT‐29 cells vs. HT‐29 CRISPR/Cas9 control (ctr) cells under untreated and necroptosis‐induced conditions. Control cells were grown in SILAC medium containing Arg0/Lys0 ("Light") labeled, whereas USP22 KO cells were labeled in SILAC medium containing Arg10/Lys8 (“Heavy”) labeled. In the treated conditions, cells were incubated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 6 h. Cells were lysed, and equal amounts of proteins extracted from “Light” or “Heavy” were pooled and digested in‐solution with trypsin. Ubiquitin remnant peptides were enriched using di‐glycine‐lysine‐specific antibodies, fractionated by Micro‐SCX, and analyzed by LC‐MS/MS.

  2. Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT‐29 cells vs. control under TBZ treatment.

  3. Schematic representation of the domain structure of RIPK3. Indicated are the two major protein domains, the kinase domain (KD), the RHIM, and the three different potential ubiquitination sites identified by ubiquitin remnant profiling.

  4. HeLa cells expressing Dox‐inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR, or 3xKR were pre‐incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre‐treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI‐positive nuclei.

  5. HeLa cells expressing Dox‐inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.

Data information: Data represent mean ± SD; **P < 0.01; ***P < 0.001, by unpaired 2‐tailed Student's t‐test. Three independent experiments performed in triplicate are shown.
Figure EV4
Figure EV4. Ubiquitin remnant profiling identifies RIPK3 K518 as USP22‐dependent ubiquitin target during necroptosis

  1. RIPK3 ubiquitin sites detected by ubiquitin remnant profiling upon USP22 KO and TBZ treatment. The mean of two biological replicates is depicted. Only RIPK3 sites are shown that were identified in both replicates.

  2. Fragment spectrum of TBZ‐treated RIPK3 di‐glycine‐modified peptide corresponding to K518. The b‐ and y‐ions detected are highlighted.

  3. SILAC‐labeled HT‐29 CRISPR/Cas9 control (ctr) and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 6 h. The percentage of PI‐positive cells was assessed by fluorescence‐based PI staining.

  4. SILAC‐labeled 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 analyzed by Western blotting with the indicated antibodies. GAPDH served as a loading control. The arrow marks specific phosphorylated MLKL bands.

  5. HeLa cells expressing Dox‐inducible RIPK3 WT or 3xKR were incubated with 30 µM Nec‐1s or 20 µM Dab and 1 µg/ml Dox overnight, as indicated. Cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 2 h. 100 μg of each lysate was incubated with 400 U/μl λ‐phosphatase for 30 min at 30°C. RIPK3 protein expression was monitored by Western blotting. β‐Actin was used as loading control.

  6. HeLa cells expressing Dox‐inducible RIPK3 WT or K518R were incubated with 1 µg/ml Dox overnight. Cells were pre‐treated with 20 µM zVAD.fmk and 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα was added for 2 h. Levels of inducible RIPK3 expression were analyzed by Western blotting under reducing and non‐reducing conditions. β‐Actin served as loading control. The asterisk marks putative RIPK3 dimers.

Data information: Data represent mean ± SD; **P < 0.01, by unpaired 2‐tailed Student's t‐test. Three independent experiments are shown.
Figure 6
Figure 6. USP22‐regulated modification of RIPK3 lysine residue 518 affects necroptosis in RIPK3‐reconstituted HeLa cells

  1. HeLa cells expressing Dox‐inducible RIPK3 WT, K518R, or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β‐Actin was used as a loading control.

  2. HeLa cells expressing Dox‐inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre‐treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα were added for 1, 2, 3, and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β‐Actin served as loading control.

  3. HeLa cells expressing Dox‐inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre‐treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β‐Actin served as loading control.

  4. HeLa cells expressing Dox‐inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre‐treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β‐Actin served as loading control.

  5. HeLa cells expressing Dox‐inducible RIPK3 WT, D160N, K518R, or 3xKR were incubated overnight with 1 µg/ml Dox and pre‐treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre‐treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep‐RIPK3 was immunoprecipitated using anti‐Strep‐beads. Co‐immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β‐Actin served as a loading control.

  6. HeLa cells expressing Dox‐inducible RIPK3 WT, K518R, or 3xKR were incubated with 1 µg/ml Dox and transfected with HA‐ubiquitin for 24 h, as indicated. 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. HA‐ubiquitin was immunoprecipitated using anti‐HA‐beads and detection of indicated proteins was performed by Western blotting. β‐Actin served as loading control for the input, whereas HA‐ubiquitin levels served as loading control for immunoprecipitated ubiquitin.

Figure EV5
Figure EV5. HT‐29 RIPK3 KO cells, reconstituted with RIPK3 mutants, show homogenous RIPK3 expression upon Dox treatment

  1. HT‐29 CRISPR/Cas9 control (ctr) and RIPK3 KO cells were analyzed by Western blotting for RIPK3 expression. β‐Actin served as loading control.

  2. HT‐29 control and RIPK3 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, and 1 ng/ml TNFα for 18 h. The percentage of PI‐positive cells was assessed by fluorescence‐based PI staining.

  3. HT‐29 cells expressing EV and Dox‐inducible RIPK3 WT, D160N, K518R, and 3xKR were treated with 1 µg/ml Dox overnight. Strep‐RIPK3 expression was imaged using anti‐RIPK3 immunofluorescence staining. Scale bars represent 100 µm.

  4. Quantification of FITC‐positive cells after RIPK3 immunofluorescence staining of HT‐29 cells expressing Dox‐inducible RIPK3 WT, D160N, K518R, and 3xKR.

Data information: Data represent mean ± SD; ***P < 0.001, NS: not significant, by unpaired 2‐tailed Student's t‐test. Three independent experiments are shown.
Figure 7
Figure 7. RIPK3 K518R hypersensitizes HT‐29 cells for TBZ‐induced necroptosis

  1. HT‐29 RIPK3 KO cells re‐expressing Dox‐inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6, and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI‐positive nuclei.

  2. HT‐29 RIPK3 KO cells re‐expressing Dox‐inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6, and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β‐Actin served as a loading control.

  3. Schematic overview of the putative mechanistic roles of USP22 in necroptosis. Activation of TNFR1 by TNFα, upon caspase‐8 inhibition by zVAD.fmk and cIAP1/2 inactivation by BV6, induces RIPK1/3 activation, necrosome formation, and execution of necroptosis. In the absence of USP22 KO, TBZ‐induced necroptosis is delayed, accompanied by increased RIPK3 phosphorylation and ubiquitination of RIPK3 at lysine 518. This USP22‐mediated necroptotic signaling could be caused either by direct USP22‐mediated (de)ubiquitination of RIPK3 and/or indirectly by, for example, regulating RIPK3 autophosphorylation or the activity of RIPK3‐associated E3 ligases or kinases. Additionally, USP22‐mediated retrograde effects on RIPK1 phosphorylation and RIPK3 oligomerization might be involved as well.

Data information: Data represent mean ± SD; **P < 0.01, by unpaired 2‐tailed Student's t‐test. Three independent experiments performed in triplicate are shown.
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