@article{c6f489597a38404eaed19b01209f5f70,
title = "Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death",
abstract = "Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.",
keywords = "Cell biology, Molecular biology",
author = "Daniel Frank and Garnish, {Sarah E.} and Sandow, {Jarrod J.} and Ashley Weir and Lin Liu and Elise Clayer and Lizeth Meza and Maryam Rashidi and Cobbold, {Simon A.} and Scutts, {Simon R.} and Marcel Doerflinger and Holly Anderton and Lawlor, {Kate E.} and Najoua Lalaoui and Kueh, {Andrew J.} and Eng, {Vik Ven} and Ambrose, {Rebecca L.} and Herold, {Marco J.} and Samson, {Andre L.} and Rebecca Feltham and Murphy, {James M.} and Gregor Ebert and Pearson, {Jaclyn S.} and Vince, {James E.}",
note = "Funding Information: We thank Kim Newton and Vishva Dixit (Genentech) for providing us the p-RIPK3 T231/S232 antibody (#GEN-135-35-9) and Ripk3 −/− mouse strain; David L. Vaux for intellectual input; Annette Jacobsen, Zikou Liu and Emma Petrie for kindly providing the MDF cell lines; David Komander for providing the plasmid for producing GST-TUBE. The generation of the Ripk3 K469R/K469R mice used in this study was supported by Phenomics Australia and the Australian Government through the National Collaborative Research Infrastructure Strategy ( NCRIS ) program. MJH is a National Health and Medical Research Council of Australia ( NHMRC ) Senior Research Fellow ( 1156095 ) and supported by the Leukemia and Lymphoma Society SCOR grant 7015-18 . K.E.L was supported by NHMRC project grants ( 1145788 , 1162765 ) and ideas grant ( 1181089 ), and is an Australian Reserch Council ( ARC ) Future Fellow ( FT19100266 ); JEV was supported by NHMRC project grants ( 1145788 and 1101405 ), an ideas grant ( 1183070 ), a fellowship ( 1141466 ) and an investigator grant ( 2008692 ); ALS was supported by NHMRC ideas grant ( 2002965 ); JMM was supported by an NHMRC grant ( 1172929 ). This work was also supported by operational infrastructure grants through the Australian Government Independent Research Institute Infrastructure Support Scheme ( 9000719 ) and the Victorian State Government Operational Infrastructure Support, Australia. The schematic in the Graphical Abstract and Figure S6 F was created with the aid of BioRender. Funding Information: We thank Kim Newton and Vishva Dixit (Genentech) for providing us the p-RIPK3T231/S232 antibody (#GEN-135-35-9) and Ripk3−/− mouse strain; David L. Vaux for intellectual input; Annette Jacobsen, Zikou Liu and Emma Petrie for kindly providing the MDF cell lines; David Komander for providing the plasmid for producing GST-TUBE. The generation of the Ripk3K469R/K469R mice used in this study was supported by Phenomics Australia and the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. MJH is a National Health and Medical Research Council of Australia (NHMRC) Senior Research Fellow (1156095) and supported by the Leukemia and Lymphoma Society SCOR grant 7015-18. K.E.L was supported by NHMRC project grants (1145788, 1162765) and ideas grant (1181089), and is an Australian Reserch Council (ARC) Future Fellow (FT19100266); JEV was supported by NHMRC project grants (1145788 and 1101405), an ideas grant (1183070), a fellowship (1141466) and an investigator grant (2008692); ALS was supported by NHMRC ideas grant (2002965); JMM was supported by an NHMRC grant (1172929). This work was also supported by operational infrastructure grants through the Australian Government Independent Research Institute Infrastructure Support Scheme (9000719) and the Victorian State Government Operational Infrastructure Support, Australia. The schematic in the Graphical Abstract and Figure S6F was created with the aid of BioRender. The project was conceived by DF and JEV; the experiments were designed by DF, SEG, AJK, MJH, JEV; the experiments were performed and analyzed by DF, SEG, JJS, AW, LL, ALS, MR, HA, KEL, NL, RLA, VVE, GE, JSP; expert advice and reagents were provided by EC, LM, SAC, SRS, MD, HA, AJK, MJH, RF, KEL, NL, JMM; the manuscript written by DF and JEV. All authors assisted with manuscript editing. J.M.M. and A.L.S. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper self-identifies as living with a disability. Publisher Copyright: {\textcopyright} 2022 The Authors",
year = "2022",
month = jul,
day = "15",
doi = "10.1016/j.isci.2022.104632",
language = "English",
volume = "25",
journal = "iScience",
issn = "2589-0042",
publisher = "Elsevier",
number = "7",
}