Site‐specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2019103303
ISSN1460-2075
AutoresLilian M. Fennell, Carlos Gómez-Díaz, Luiza Deszcz, Anoop Kavirayani, David Hoffmann, Kota Yanagitani, Alexander Schleiffer, Karl Mechtler, Astrid Hagelkrüys, Josef Penninger, Fumiyo Ikeda,
Tópico(s)interferon and immune responses
ResumoArticle20 November 2020free access Source DataTransparent process Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling Lilian M Fennell Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria These authors contributed equally to this works as first and second author Search for more papers by this author Carlos Gomez Diaz orcid.org/0000-0002-6416-806X Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria These authors contributed equally to this works as first and second author Search for more papers by this author Luiza Deszcz Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Anoop Kavirayani Vienna Biocenter Core Facilities (VBCF), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author David Hoffmann Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Kota Yanagitani Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan Search for more papers by this author Alexander Schleiffer orcid.org/0000-0001-6251-2747 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Karl Mechtler orcid.org/0000-0002-3392-9946 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Astrid Hagelkruys Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Josef Penninger orcid.org/0000-0002-8194-3777 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Fumiyo Ikeda Corresponding Author [email protected] orcid.org/0000-0003-0407-2768 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan Search for more papers by this author Lilian M Fennell Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria These authors contributed equally to this works as first and second author Search for more papers by this author Carlos Gomez Diaz orcid.org/0000-0002-6416-806X Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria These authors contributed equally to this works as first and second author Search for more papers by this author Luiza Deszcz Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Anoop Kavirayani Vienna Biocenter Core Facilities (VBCF), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author David Hoffmann Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Kota Yanagitani Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan Search for more papers by this author Alexander Schleiffer orcid.org/0000-0001-6251-2747 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Karl Mechtler orcid.org/0000-0002-3392-9946 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Astrid Hagelkruys Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Josef Penninger orcid.org/0000-0002-8194-3777 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Fumiyo Ikeda Corresponding Author [email protected] orcid.org/0000-0003-0407-2768 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan Search for more papers by this author Author Information Lilian M Fennell1, Carlos Gomez Diaz1, Luiza Deszcz1, Anoop Kavirayani2, David Hoffmann1, Kota Yanagitani3, Alexander Schleiffer1,4, Karl Mechtler1,4, Astrid Hagelkruys1, Josef Penninger1,5 and Fumiyo Ikeda *,1,3 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria 2Vienna Biocenter Core Facilities (VBCF), Vienna Biocenter (VBC), Vienna, Austria 3Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan 4Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria 5Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada *Corresponding author. Tel: +81 92 642 4769; E-mail: [email protected] EMBO J (2020)39:e103303https://doi.org/10.15252/embj.2019103303 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 HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling. SYNOPSIS The E3 ligase complex LUBAC generates Met1-linked linear ubiquitin chains, and regulates inflammation and cell death. Ubiquitination of the LUBAC catalytic component HOIP itself is found to control apoptosis in mammalian cells and TNF-dependent development and systemic inflammation in mice. Multiple lysine residues of RBR-type E3 ligase HOIP are modified with linear and mixed-linkage polyubiquitin chains. Ubiquitination of human HOIP at K784 within the IBR domain controls the apoptotic pathway in fibroblasts. K784R mutation in HOIP does not affect E3 ligase activity or ability to generate Met1-linked ubiquitin chains. The equivalent K778R mutation in murine HOIP does not affect mouse development. Synthetic embryonic lethality in SHARPIN-deficient HOIPK778R/K778R mice is rescued by TNFR1 knockout. Introduction The linear ubiquitin chain assembly complex (LUBAC) is a critical regulator of inflammation in humans and mice (Walczak, 2011; Ikeda, 2015; Sasaki & Iwai, 2015; Peltzer & Walczak, 2019). LUBAC influences the inflammatory response by regulating the tumor necrosis factor (TNF)-signaling pathway. Upon TNF binding, the TNF receptor (TNFR) forms TNFR complex I (TNFR1), consisting of TNF receptor type 1-associated DEATH domain (TRADD), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis protein (cIAP) 1/2 and LUBAC. TNFR complex I promotes cell survival via downstream signaling cascades such as NF-κB through the key kinase complex IκB kinase (IKK) consisting of IKKα/β and NF-κB essential modifier (NEMO). Post-translational modifications, including ubiquitination, regulate multiple events in this signaling cascade. Linear/Met1-, Lys11-, and Lys63-ubiquitin linkage types regulate the recruitment of specific signaling complexes (Peltzer et al, 2016; Witt & Vucic, 2017), whereas Lys48-linked ubiquitin chains trigger degradation by the ubiquitin-proteasome system. As part of TNFR complex I, LUBAC generates linear/Met1-ubiquitin chains on NEMO (Haas et al, 2009; Tokunaga et al, 2009) and RIPK1 (Gerlach et al, 2011) to promote NF-κB signaling. NF-κB activation leads to induction of anti-apoptosis genes such as cellular FLICE-like inhibitory protein (cFLIP), thus known as an anti-apoptosis pathway (Lamkanfi et al, 2007; Peltzer & Walczak, 2019). On the other hand, when the NF-κB pathway is disturbed, TNF can promote apoptosis via formation of the TNFR complex II, which consists of RIPK1, TRADD, FAS-associated death domain (FADD) and caspase 8 (Justus & Ting, 2015; Witt & Vucic, 2017). TNFR complex II formation also appears to be regulated by LUBAC (Asaoka & Ikeda, 2015; Sasaki & Iwai, 2015; Peltzer & Walczak, 2019), but the mechanisms are unclear. LUBAC consists of the E3 ligase HOIP/RNF31 and two subunits HOIL-1L/RBCK1 and SHARPIN/SIPL1 (Gerlach et al, 2011; Ikeda et al, 2011; Tokunaga et al, 2011; Rittinger & Ikeda, 2017). Genetic loss of HOIP or HOIL-1L triggers embryonic lethality in mice due to upregulation of apoptosis, uncovering their essential roles in mouse embryonic development and cell death regulation (Emmerich et al, 2013; Peltzer et al, 2014; Meier et al, 2015; Hrdinka & Gyrd-Hansen, 2017). In contrast, SHARPIN-deficient mice (Sharpincpdm/cpdm) suffer from systemic inflammation accompanied with chronic proliferative dermatitis at the age of 6–8 weeks (Seymour et al, 2007; Kumari et al, 2014). Skin tissues derived from Sharpincpdm/cpdm mice show immune cell infiltrations and upregulation of keratinocyte apoptosis (Seymour et al, 2007). The inflammatory phenotypes of these genetically modified mice are at least partially rescued by TNFR1 knockout, suggesting that LUBAC attenuates apoptosis downstream of the TNF signaling cascade (Kumari et al, 2014; Rickard et al, 2014). HOIL-1L and HOIP mutations are observed in patients with autoimmune diseases, implicating LUBAC in the regulation of immune responses in humans (Boisson et al, 2012; Boisson et al, 2015). At the molecular level, HOIP is a RING-IBR-RING (RBR) type of E3 ligase that specifically generates linear/Met1-linked ubiquitin chains with SHARPIN and HOIL-1L. Linear ubiquitin chains are atypical chains linked via the C-terminal Gly of one ubiquitin moiety to the N-terminal Met1 of another ubiquitin moiety. The catalytic center of HOIP is in the second RING domain (Stieglitz et al, 2012), and the linear Ub chain determining domain (LDD) provides its unique ability to generate linear ubiquitin chains (Smit et al, 2012). Thus far, HOIP is the only ligase known to generate linear ubiquitin chains (Kirisako et al, 2006; Dove & Klevit, 2017). In vitro, HOIP requires HOIL-1L or SHARPIN to generate linear ubiquitin chains (Kirisako et al, 2006; Gerlach et al, 2011; Ikeda et al, 2011; Tokunaga et al, 2011). However, the HOIP RBR-LDD fragment is active in the absence of HOIL-1L and SHARPIN, suggesting a self-inhibitory mechanism (Smit & Sixma, 2014; Walden & Rittinger, 2018). LUBAC generates linear/Met1 ubiquitin chains at lysine residues on substrates, which depends on HOIL-1L (Smit et al, 2013). In cells, HOIP exists mostly in complex with SHARPIN or HOIL-1L (Kirisako et al, 2006; Tokunaga et al, 2011); thus, the LUBAC complex is expected to be active. Yet, the LUBAC-dependent downstream cascades are dependent on stimuli like TNF. The mechanisms that regulate LUBAC activity are unclear. In particular, it is not known how inflammatory stimuli modulate the interactions between LUBAC and its substrates. Two deubiquitinases (DUBs), called "OTU DUB with LINear linkage specificity" (OTULIN) and CYLD, hydrolyze linear ubiquitin chains and regulate inflammatory signaling cascades. Both OTULIN and CYLD can form a complex with HOIP (Fiil et al, 2013; Keusekotten et al, 2013; Elliott et al, 2014; Schaeffer et al, 2014; Takiuchi et al, 2014; Elliott et al, 2016; Hrdinka et al, 2016; Kupka et al, 2016; Wagner et al, 2016). However, loss-of-function of OTULIN and CYLD in mice does not result in the expected phenotypes compared with LUBAC-deficient mice (Reiley et al, 2006; Zhang et al, 2006; Damgaard et al, 2016); knock-in mice expressing OTULIN C129A (a dominant negative mutant) are embryonic lethal with increased apoptosis signals, partially overlapping with the phenotypes of HOIP and HOIL-1L knockout mice, and the main phenotype known for CYLD deficient mice is in tumor development (Peltzer et al, 2014; Heger et al, 2018; Peltzer et al, 2018). Recently, it was shown that the OTULIN mutant (Cys129Ala) increases ubiquitination signal of all three LUBAC components in cells (Heger et al, 2018). Hyper-ubiquitinated LUBAC in the OTULIN mutant Cys129Ala expressing cells is not recruited to TNFR complex I, leading to suppression of this branch of the TNF-induced signaling cascade (Heger et al, 2018). These observations collectively suggest that LUBAC activity and linear ubiquitination of LUBAC components are tightly regulated. Yet, the post-translational mechanisms controlling LUBAC and its inflammatory outcomes are poorly understood. Results Human HOIP is polyubiquitinated in cells To investigate how HOIP is regulated by ubiquitination, we transiently expressed Myc-HOIP in HEK293T cells. Linear ubiquitin chains were below the detection limit in HEK293T extracts (Appendix Fig S1A), which may reflect their deubiquitination. To stabilize linear ubiquitin chains, we co-expressed catalytically inactive OTULIN (C129A), which acts as dominant-negative (Fiil et al, 2013; Keusekotten et al, 2013; Heger et al, 2018). In addition, to enrich for proteins modified with linear ubiquitin chains, we performed pulldown assays using a known enrichment matrix called GST-linear-TUBE, which consists of GST fused to three tandem repeats of the linear ubiquitin binding-domain UBAN immobilized on a glutathione sepharose resin (Fig 1A; Asaoka et al, 2016). We detected modified HOIP in the pulldown by immunoblotting (Appendix Fig S1A, lane 8, and Fig 1B, lane 1) suggesting that HOIP is ubiquitinated at least partially by linear ubiquitin chains, as reported previously with OtulinC129A/C129A knock-in mouse embryonic fibroblasts (MEFs) (Heger et al, 2018). Figure 1. Human HOIP is ubiquitinated at K784 and regulates NF-κB A scheme of procedures for the UbiCRest-based assays employed to analyze HOIP modification with ubiquitin chains. Total cell extracts from HEK293T cells transiently expressing Myc-HOIP and Myc-OTULIN C129A, a catalytic inactive mutant, subjected to GST-linear-TUBE pulldown followed by UbiCRest using recombinant deubiquitinases (vOTU, OTULIN and USP21). UbiCRest assays to evaluate ubiquitin chain types on HOIP examined by immunoblotting. Immunoblotting of samples using antibodies as indicated. Ponceau S staining used for monitoring GST-liner-TUBE input. Representative data shown from three independent experiments. Mass spectrometry spectra corresponding to a peptide containing HOIP-K784 with double Gly (114 + K). Domains of human HOIP and identified ubiquitination sites at K454, K458, K735, and K784. Multiple sequence alignment of different HOIP orthologues illustrating the position K735 and K784 according to the ClustalX color scheme. Sequences were retrieved from the NCBI protein database with the following accessions: Homo sapiens (NP_060469.4), Canis lupus (XP_005623312.1), Mus musculus (NP_919327.2), Monodelphis domestica (XP_007479924.1), Xenopus laevis (NP_001090429.1), Alligator mississippiensis (XP_006259801.1), Takifugu rubripes (XP_003968217.2), and Drosophila melanogaster (NP_723214.2). Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wild type (WT) or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody was used to monitor protein loading. Representative data shown from three independent experiments. Luciferase-based NF-κB gene reporter assays using Myc-HOIP wild type (WT), a catalytic inactive mutant C885A, ubiquitination-site mutants of K454R, K458R, K735R, K784R co-transfected with HOIL-1L-HA and Flag-SHARPIN. Luciferase signal was normalized to an internal Renilla control signal. Data information: In (G), data are presented as mean ± SD. **P ≤ 0.01, ****P ≤ 0.0001 (ANOVA). n = 4. Source data are available online for this figure. Source Data for Figure 1 [embj2019103303-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint To verify modification of HOIP, we used the ubiquitin chain restriction (UbiCRest) method (Hospenthal et al, 2015), a DUB-based analysis to differentiate the linkage types of ubiquitin chains (Fig 1A). The observed HOIP modification disappeared upon treatment with USP21, which hydrolyzes all linkage types of ubiquitin chains, verifying that the modification is ubiquitination (Fig 1B, lane 4). Treatment with either vOTU, which cleaves Lys-linked ubiquitin chains but not linear ubiquitin chains, or with OTULIN, which specifically cleaves linear ubiquitin chains, partially reduced the modification of HOIP (Fig 1B, lane 2 and 3). These data suggest that HOIP is polyubiquitinated with mixed linkage types of Lys and linear. OTULIN treatment diminished the levels of high-molecular-weight HOIP, suggesting that linear ubiquitin chains are added on the Lys-linked ubiquitin chains. Using mass spectrometry, we uncovered four ubiquitinated residues within human HOIP: lysine (K)454, K458, K735, K784 (Fig 1C and D, Appendix Fig S1B–D). K454 and K458 are not within any of the annotated HOIP domains and are not well-conserved (Fig 1D, Appendix Fig S1E). In contrast, K784 is within the "In Between Ring fingers" (IBR) domain, and K735 is located within the "Really Interesting New Gene" (RING) 1 domain (Fig 1D), and both residues are conserved in a wide range of species (Fig 1E). HOIP K784 regulates NF-κB activation without affecting LUBAC complex formation in cells To evaluate the functional role of these ubiquitination sites in HOIP, we generated HOIP K454R, K458R, K735R, and K784R mutants. Given that LUBAC is a regulator of NF-κB signaling, we examined these HOIP mutants using standard NF-κB reporter assays in which luciferase expression is under the control of NF-κB-response elements. Transfected cells expressed similar levels of wild-type (WT) HOIP, the ubiquitination-site mutants (K454R, K458R, K735R, K784R), or a negative control (catalytically inactive mutant, C885A) (Fig 1F, Appendix Fig S1F). As expected, we observed an increased luciferase signal in cells that co-express SHARPIN and HOIL-1L with HOIP WT, but not with HOIP C885A (Fig 1G). The luciferase signal was also significantly reduced in cells co-expressing HOIP K784R, whereas it was significantly increased, albeit mildly, in cells co-expressing HOIP K454R, K458R, or K735R (Fig 1G). We observed similar results in assays without HOIL-1L or SHARPIN (Appendix Fig S1G and H). We chose to pursue the HOIP K784 site, given that it is conserved and promotes NF-κB signaling. To investigate how linear ubiquitination at K784 affects LUBAC formation, we transiently co-expressed HOIP WT or HOIP K784R with HOIL-1L and SHARPIN in HEK293T cells and analyzed their interactions by co-immunoprecipitation. HOIP WT and HOIP K784R each interacted with HOIL-1L and SHARPIN (Fig 1F), suggesting that HOIP K784R supports LUBAC complex formation in cells. These data suggest that HOIP K784R reduces NF-κB reporter activity without compromising LUBAC complex formation. HOIP K784R generates unanchored linear ubiquitin chains and ubiquitinates NEMO According to crystal structure analysis, HOIP K784 is on the surface of an alpha helix in the IBR domain, not in contact with the E2 (UbcH5) or the ubiquitin loaded on E2, and distant from the active site C885 (Lechtenberg et al, 2016). To determine how mutations in HOIP affect its activity, we purified recombinant HOIP proteins in various forms and performed in vitro ubiquitination assays. As expected, the HOIP C885A catalytic mutant did not generate unanchored linear ubiquitin chains nor did it ubiquitinate NEMO (Fig 2A, Appendix Fig S2). Furthermore, HOIP C885A was not polyubiquitinated (Fig 2A, Appendix Fig S2), suggesting that HOIP is modified dependently on its own catalytic activity in vitro. Both HOIP WT and HOIP K784R generated unanchored linear ubiquitin chains and ubiquitinated the LUBAC substrate NEMO, when co-incubated with SHARPIN and HOIL-1L (Fig 2A). In the absence of SHARPIN, the ubiquitination signal was reduced in reactions with HOIP K784R compared to HOIP WT (Appendix Fig S2). These data suggest that the HOIP K784R mutant, in a complex with both HOIL-1L and SHARPIN, can ubiquitinate substrates in vitro, though with altered kinetics compared to HOIP WT. Figure 2. The human HOIP K784R mutant as a part of the LUBAC generates linear ubiquitin chains and ubiquitinates its substrate NEMO in vitro and in cells In vitro ubiquitination assays for the indicated times using the recombinant proteins of ubiquitin (Ub), E1, E2 (UbcH7), HOIP (WT, K784R or C885A mutant), HOIL-1L, SHARPIN, and NEMO. Immunoblotting of Linear ubiquitin chains, NEMO, HOIP, HOIL-1L, and SHARPIN detected by using antibodies as indicated. Representative data from three independent experiments. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wild-type (WT), Myc-HOIP K784R, or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS–PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody was used to monitor loading. Representative data from three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embj2019103303-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint To test HOIP activity in cells, we transiently expressed HOIP WT, HOIP K784R, or HOIP C885A with HOIL-1L and SHARPIN in HEK293T cells. Cells expressing HOIP WT and HOIP K784R displayed similar levels of linear ubiquitin chains and polyubiquitinated NEMO (Fig 2B), whereas cells expressing HOIP C885A lacked both ubiquitination events. These results collectively indicate that HOIP K784R, as a part of LUBAC, can ubiquitinate NEMO in vitro and in cells. The observations of HOIP ubiquitination dependent on its catalytic site C885 (Fig 2A and B) and HOIP ubiquitination by mixed linkage types of chains in cells (Fig 1B) suggest that HOIP ubiquitination is at least partially self-ubiquitination in cells. HoipK778R/K778R mice do not have overt developmental defects Given that human HOIP K784R disrupted NF-κB signaling in the reporter assay without substantially affecting HOIP catalytic activity or LUBAC formation in HEK293T cells, we investigated the endogenous function of HOIP K784 in vivo. We used CRISPR-Cas9 to generate homozygous knock-in mice with a substitution at HOIP K778, the equivalent residue to K784 in mice (HoipK778R/K778R, Appendix Fig S3A and B). HoipK778R/K778R mice were born at the nearly expected ratio from crosses of Hoip+/K778R mice and displayed no obvious developmental phenotypes (Fig 3A and B). These observations are in contrast to HOIP loss-of-function mice, which are embryonic lethal (Emmerich et al, 2013; Peltzer et al, 2014; Hrdinka & Gyrd-Hansen, 2017). Figure 3. No obvious developmental defect in HoipK778R/K778R knock-in mice is observed while TNF-responses are suppressed in HoipK778R/K778R cells A. Numbers of weaned mice of the indicated genotypes from Hoip+/K778R crosses. B. A gross appearance image of Hoip+/+ and HoipK778R/K778R male mice at 6 weeks old. C, D. Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20 ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-tubulin antibody shown for monitoring loading amount. Representative data from three independent experiments. E. Induction of TNF-dependent NF-κB target genes, ICAM, VCAM and IκB-α in Hoip+/+ or HoipK778R/K778R immortalized MEFs determined by qRT–PCR. RNA extraction and cDNA synthesis from MEFs treated with hTNF (20 ng/ml) for the indicated time subjected to examine transcripts of ICAM, VCAM, and IκB-α. Normalization to β-actin. Representative data from three independent experiments, n = 3. F. TNF-dependent induction of cleavage of PARP and caspase 3 in primary Hoip+/+ or HoipK778R/K778R MEFs determined by immunoblotting. Total cell extracts of MEFs treated with hTNF (100 ng/ml) and CHX (1 µg/ml) for the indicated times subjected to SDS–PAGE followed by immunoblotting using antibodies as indicated. Anti-α-tubulin antibody was used to monitor loading amount. Representative data of two independent experiments. G. TNF-dependent induction of caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100 ng/ml) and CHX (1 µg/ml) for 4 h subjected to the caspase 3 activity assays. H. TNF-induced caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100 ng/ml) with or without cycloheximide (CHX) (1 µg/ml) or z-VAD (20 µM). Representative data from three independent experiments, n = 4. I. TNF receptor complex II formation in Hoip+/+ or HoipK778R/K778R immortalized MEFs. Total cell extracts of MEFs treated with hTNF (100 ng/ml), CHX (1 µg/ml) and z-VAD (25 µM) for the indicated time immunoprecipitated using an anti-FADD antibody. Recruitment of RIPK1, HOIP, and SHARPIN monitored by immunoblotting. The anti-Vinculin antibody blot shown for monitoring loading amount. The black arrows indicating HOIP and SHARPIN in the IP samples and unmodified RIPK in the input samples, respectively. Representative data of two independent experiments. Data information: In (E, G and H), data are presented as mean ± SD. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (ANOVA, n = 4 in G). CHX (Cycloheximide), hTNF (human Tumor Necrosis Factor), z-VAD (Z-Val-Ala-Asp fluoromethyl ketone). Source data are available online for this figure. Source Data for Figure 3 [embj2019103303-sup-0008-SDataFig3.pdf] Download figure Download PowerPoint TNF-induced NF-κB activation is suppressed in HoipK778R/K778R MEFs To analyze if HOIP K778 is involved in the regulation of TNF-dependent NF-κB signaling, we derived MEF lines from Hoip+/+ and HoipK778R/K778R mice and stimulated them with TNF (Fig 3C and D, Appendix Fig S3C). We found that TNF-induced phosphorylation of IκB-α is prolonged and degradation of IκB-α was very mildly reduced in HoipK778R/K778R MEFs compared with Hoip+/+ MEFs (Fig 3C). In these cells, phosphorylation of IKK was also slightly decreased (Fig 3D). Furthermore, the TNF-induced transcription of some NF-κB target genes, such as ICAM, VCAM, and IκB-α, was significantly reduced in HoipK778R/K778R MEFs compared to Hoip+/+ MEFs (Fig 3E), whereas TNF-induced gene induction of A20 was unaffected (Appendix Fig S3C). To elucidate the step of TNF-dependent signaling that is affected in HoipK778R/K778R MEFs, we examined the formation of TNFR complex I. Upon TNF-treatment, RIPK1, HOIP, SHARPIN, and NEMO co-immunoprecipitated with TNF in both Hoip+/+ and HoipK778R/K778R MEFs (Appendix Fig S3D), indicating recruitment to TNFR complex I. Recruitment of RIPK1, HOIP, and SHARPIN was similar in HoipK778R/K778R MEFs compared to WT MEFs. Collectively, these data indicate that HOIP K778R mildly but significantly suppresses the TNF-induced NF-κB signaling cascade in MEFs. Apoptotic markers are increased in HoipK778R/778KR MEFs LUBAC plays a role in the anti-apoptotic branch of the TNF pathway (Walczak, 2011; Asaoka & Ikeda, 2015; Sasaki & Iwai, 2015). Therefore, we assessed the ability of HoipK778R/K778R MEFs to resist TNF-dependent apoptosis. To this end, we examined TNF-mediated induction of the active form of caspase 3 (cleaved caspase 3), which is a so-called apoptosis executi
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