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

iScience. 2022 Jun 17;25(7):104632. doi: 10.1016/j.isci.2022.104632. eCollection 2022 Jul 15.

Authors

Daniel Frank^{1

2}, Sarah E Garnish^{1

2}, Jarrod J Sandow^{1

2}, Ashley Weir^{1

2}, Lin Liu^{1

2}, Elise Clayer^{1

2

3}, Lizeth Meza^{1

2}, Maryam Rashidi^{1

2}, Simon A Cobbold^{1

2}, Simon R Scutts^{1

2}, Marcel Doerflinger^{1

2}, Holly Anderton^{1

2}, Kate E Lawlor^{4

5}, Najoua Lalaoui^{1

2}, Andrew J Kueh^{1

2}, Vik Ven Eng^{4

5}, Rebecca L Ambrose^{4

5}, Marco J Herold^{1

2}, Andre L Samson^{1

2}, Rebecca Feltham^{1

2}, James M Murphy^{1

2}, Gregor Ebert^{1

2

6}, Jaclyn S Pearson^{4

5

7}, James E Vince^{1

2}

Affiliations

¹ The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, VIC 3052, Australia.
² Department of Medical Biology, The University of Melbourne, Parkville, VIC 3050, Australia.
³ Zentrum für Allergie und Umwelt München (ZAUM), Zentrum München, 80333 Munich, Bayern, Germany.
⁴ Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC 3168, Australia.
⁵ Department of Molecular and Translational Science, Monash University, Clayton, VIC 3168, Australia.
⁶ Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, 80802 Munich, Bayern, Germany.
⁷ Department of Microbiology, Monash University, Clayton, VIC 3168, Australia.

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.

Keywords: Cell biology; Molecular biology.