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 FelthamJames 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

Access to Document

10.1016/j.isci.2022.104632Licence: CC BY-NC-ND

Frank-2022-Ubiquitylation-of-RIPK3-25-7-104632-CCBYNCND
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/).
Final published version, 5.18 MBLicence: CC BY-NC-ND

Cite this

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

Abstract

Keywords

Access to Document

Fingerprint

Cite this