Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

Meng, Yanxiang; Ka, Davies; Fitzgibbon, Cheree; Young, Samuel N.; Garnish, Sarah E; Horne, Christopher R; Luo, Chu; Garnier, Jean‐Marc; Liang, Lung-Yu; Cowan, A.D.; Samson, André L.; Lessène, Guillaume; Sandow, Jarrod J.; Czabotar, P.E.; Murphy, James M.

doi:10.1038/s41467-021-27032-x

Cited by 57 publications

(73 citation statements)

References 78 publications

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“…Current models for how MLKL kills downstream of RIPK3 phosphorylation posit that phosphorylation causes a conformational change in MLKL that exposes its NTD [ 26 , 35 , 36 , 65 ]. Expression of the mouse MLKL NTD is sufficient to kill mouse cells, suggesting that it contains the necessary oligomerisation and membrane permeabilisation activities required to kill cells that are normally held in check by the pseudokinase domain [ 25 – 27 , 72 ].…”

Section: Discussionmentioning

confidence: 99%

“…This induces the autophosphorylation and activation of both kinases, allowing RIPK3 to recruit the pseudokinase, mixed lineage kinase domain-like (MLKL), the most downstream known obligate effector of the necroptosis pathway [ 23 – 25 ]. RIPK3 then phosphorylates the activation loop within MLKL’s pseudokinase domain, initiating a chain of events involving a conformational transition, oligomerisation and subsequent translocation of MLKL to cellular membranes, where membrane disruption mediated by MLKL’s N-terminal four-helix bundle (4HB) domain leads to death of the cell [ 25 – 36 ]. Several pathway interactors and modulators of necroptotic signalling have been identified [ 37 – 46 ], however the precise mechanism and regulation of MLKL-mediated plasma membrane disruption is less clear, with endosomal sorting complex proteins, flotillins, phosphatidylinositol phosphates, and ion channels all proposed to have important roles [ 27 – 29 , 47 – 52 ].…”

Section: Introductionmentioning

confidence: 99%

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The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

et al. 2022

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Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

et al. 2022

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“…SAXS data were collected at the Australian Synchrotron SAXS/WAXS beamline using an inline co-flow size-exclusion chromatography setup to minimize sample dilution and maximize signal-to-noise (Kirby et al, 2016; Ryan et al, 2018). Samples were analysed largely in-line with established methods (Meng et al, 2021; Oliver et al, 2021). Specifically, protein was injected (80 µL) onto an inline Superdex 200 5/150 Increase GL column (Cytiva), equilibrated with size exclusion buffer (50 mM Tris-HCl, pH 8, 300 mM NaCl, 5% glycerol) at 12 °C, at a flow rate of 0.2 mL min −1 .…”

Section: Methodsmentioning

confidence: 99%

Nanobodies identify an activated state of the TRIB2 pseudokinase

Jamieson

Pudjihartono

Horne

et al. 2022

Preprint

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Tribbles proteins (TRIB1–3) are a pseudokinase-only branch of the human kinome, which recruit substrates to the COP1 ubiquitin-ligase for ubiquitination. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukaemia oncogene, by way of recruiting the C/EBPa transcription factor for degradation by COP1. Here we report selection and characterisation of nanobodies against the TRIB2 pseudokinase domain from a synthetic yeast surface-display library. We identified nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of Nb4.103 in complex with TRIB2 identified a mode of binding to the N-terminal lobe of the pseudokinase, in a manner that enables specific recognition of TRIB2 over TRIB1 and TRIB3. In the nanobody-stabilised state, TRIB2 adopts an activated conformation that is remarkably similar to the C/EBPa-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilise a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors, and nanobody tools for future investigation of TRIB2 function.

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“…translocates to the membrane and induces plasma membrane rupture, leading to the uncontrolled release of cellular content, which is thought to drive a strong inflammatory signal [21][22][23][24][25][26].…”

Section: Tnf Signalling and Cell Deathmentioning

confidence: 99%

A proteomic perspective on TNF-mediated signalling and cell death

Tanzer

2022

Biochemical Society Transactions

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The tumour necrosis factor (TNF) is the most potent inducer of cell death amongst cytokines. It is crucial for processes including homeostasis, the development of the immune system and fighting infections. However, high levels of TNF due to genetic disorders or persistent infections can contribute to autoinflammatory and autoimmune diseases or life-threatening conditions like sepsis. These diseases generally display increased levels of cell death, which, downstream of the TNF receptor, can either be caspase-dependent (apoptosis) or caspase-independent (necroptosis). Significant efforts have been invested in unravelling and manipulating signalling mechanisms regulating these two different types of cell death. Here I discuss how modern proteomic approaches like phosphoproteomics and secretomics provide a novel perspective on this central cytokine and its effect on inflammation and cell survival.

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Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

Cited by 57 publications

References 78 publications

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

Nanobodies identify an activated state of the TRIB2 pseudokinase

A proteomic perspective on TNF-mediated signalling and cell death

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