Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death

Daniel Frank; Sarah E. Garnish; Jarrod J. Sandow; Ashley Weir; Lin Liu; Elise Clayer; Lizeth Meza; Maryam Rashidi; Simon A. Cobbold; Simon R. Scutts; Marcel Doerflinger; Holly Anderton; Kate E. Lawlor; Najoua Lalaoui; Andrew J. Kueh; Vik Ven Eng; Rebecca L. Ambrose; Marco J. Herold; Andre L. Samson; Rebecca Feltham; James M. Murphy; Gregor Ebert; Jaclyn S. Pearson; James E. Vince

doi:10.1016/j.isci.2022.104632

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death

Daniel Frank
, Sarah E. Garnish
, Jarrod J. Sandow
, Ashley Weir
, Lin Liu
, Elise Clayer
, Lizeth Meza
, Maryam Rashidi
, Simon A. Cobbold
, Simon R. Scutts
, Marcel Doerflinger
, Holly Anderton
, Kate E. Lawlor
, Najoua Lalaoui
, Andrew J. Kueh
, Vik Ven Eng
, Rebecca L. Ambrose
, Marco J. Herold
, Andre L. Samson
, Rebecca Feltham

James M. Murphy, Gregor Ebert, Jaclyn S. Pearson, James E. Vince^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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. Ripk3^K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3^K469R/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.

Original language	English
Article number	104632
Pages (from-to)	1-28
Number of pages	28
Journal	iScience
Volume	25
Issue number	7
Early online date	17 Jun 2022
DOIs	https://doi.org/10.1016/j.isci.2022.104632
Publication status	Published - 15 Jul 2022

Keywords

Cell biology
Molecular biology

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Copyright © 2022 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Frank, D., Garnish, S. E., Sandow, J. J., Weir, A., Liu, L., Clayer, E., Meza, L., Rashidi, M., Cobbold, S. A., Scutts, S. R., Doerflinger, M., Anderton, H., Lawlor, K. E., Lalaoui, N., Kueh, A. J., Eng, V. V., Ambrose, R. L., Herold, M. J., Samson, A. L., ... Vince, J. E. (2022). Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death. iScience, 25(7), 1-28. Article 104632. https://doi.org/10.1016/j.isci.2022.104632

@article{e04e84a9f56c43979c84789f2a66c579,

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: 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 Research 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 ).",

year = "2022",

month = jul,

day = "15",

doi = "10.1016/j.isci.2022.104632",

language = "English",

volume = "25",

pages = "1--28",

journal = "iScience",

issn = "2589-0042",

publisher = "Elsevier Inc.",

number = "7",

}

Frank, D, Garnish, SE, Sandow, JJ, Weir, A, Liu, L, Clayer, E, Meza, L, Rashidi, M, Cobbold, SA, Scutts, SR, Doerflinger, M, Anderton, H, Lawlor, KE, Lalaoui, N, Kueh, AJ, Eng, VV, Ambrose, RL, Herold, MJ, Samson, AL, Feltham, R, Murphy, JM, Ebert, G, Pearson, JS & Vince, JE 2022, 'Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death', iScience, vol. 25, no. 7, 104632, pp. 1-28. https://doi.org/10.1016/j.isci.2022.104632

TY - JOUR

T1 - Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death

AU - Frank, Daniel

AU - Garnish, Sarah E.

AU - Sandow, Jarrod J.

AU - Weir, Ashley

AU - Liu, Lin

AU - Clayer, Elise

AU - Meza, Lizeth

AU - Rashidi, Maryam

AU - Cobbold, Simon A.

AU - Scutts, Simon R.

AU - Doerflinger, Marcel

AU - Anderton, Holly

AU - Lawlor, Kate E.

AU - Lalaoui, Najoua

AU - Kueh, Andrew J.

AU - Eng, Vik Ven

AU - Ambrose, Rebecca L.

AU - Herold, Marco J.

AU - Samson, Andre L.

AU - Feltham, Rebecca

AU - Murphy, James M.

AU - Ebert, Gregor

AU - Pearson, Jaclyn S.

AU - Vince, James E.

N1 - Funding: 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 Research 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 ).

PY - 2022/7/15

Y1 - 2022/7/15

N2 - 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.

AB - 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.

KW - Cell biology

KW - Molecular biology

U2 - 10.1016/j.isci.2022.104632

DO - 10.1016/j.isci.2022.104632

M3 - Article

AN - SCOPUS:85133405654

SN - 2589-0042

VL - 25

SP - 1

EP - 28

JO - iScience

JF - iScience

IS - 7

M1 - 104632

ER -

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death

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