HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

Jacobsen, Annette V.; Lowes, Kym N.; Tanzer, Maria C.; Lucet, Isabelle S; Hildebrand, Joanne M; Petrie, Emma J.; Delft, Mark F. van; Liu, Zikou; Conos, Stephanie A.; Jg, Zhang; Huang, David C.S.; Silke, John; Lessène, Guillaume; Murphy, James M.

doi:10.1038/cddis.2015.386

Cited by 137 publications

(131 citation statements)

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“…Figure 3) [35]. These observations are in line with other studies which have shown that crucial regulators of necroptosis and autophagy are HSP90 client proteins whose degradation upon HSP90 inhibition interferes with the coordinate execution of these processes [45–47]. …”

Section: Resultssupporting

confidence: 85%

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer

et al. 2016

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The chaperone heat shock protein 90 (HSP90) crucially supports the maturation, folding, and stability of a variety of client proteins which are of pivotal importance for the survival and proliferation of cancer cells. Consequently, targeting of HSP90 has emerged as an attractive strategy of anti-cancer therapy, and it appears to be particularly effective in the context of molecular sensitization towards radiotherapy as has been proven in preclinical models of different cancer entities. However, so far the clinical translation has largely been hampered by suboptimal pharmacological properties and serious hepatotoxicity of first- and second-generation HSP90 inhibitors. Here, we report on NW457, a novel radicicol-derived member of the pochoxime family with reduced hepatotoxicity, how it inhibits the DNA damage response and how it synergizes with ionizing irradiation to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer in vitro and in vivo.

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Section: Resultssupporting

confidence: 85%

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer

et al. 2016

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“…A recent report suggests that Hsp90 can modulate the stability of the RIPK3/MLKL complex and the oligomerization of MLKL and that an Hsp90 inhibitor can protect against Necroptosis[31]. To confirm that cpd 1 is not acting through Hsp90 inhibition, we compared the thermal stability of the Hsp90α catalytic domain bound to a positive control compound 17-AAG vs. cpd 1 .…”

Section: Resultsmentioning

confidence: 99%

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis

Marcotte

Paramasivam

et al. 2016

PLoS ONE

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MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an “out” position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.

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“…12 Taken together, these studies led us to hypothesize that cell type-specific factors, such as proteins that modulate MLKL activation and 4HB domain exposure, oligomerization, membrane translocation and permeabilization are necessary for MLKL 4HB domain-induced cell death (summarized in Figure 6a). The identities of these proteins (termed X1-X4 in Figure 6a) are of enormous interest to the field and we have recently identified one such factor, the co-chaperone system Cdc37-HSP90, as an important mediator of MLKL activation, 23 which can be considered as putative X1 proteins. In addition, we have shown that phosphorylation of the MLKL activation loop coincides with MLKL oligomerization and membrane translocation but that these events precede membrane permeabilization by several hours, 15 again strongly implying that this is a regulated, rate-limiting stage in MLKL killing and imply the existence of X4 factors.…”

Section: Discussionmentioning

confidence: 99%

Evolutionary divergence of the necroptosis effector MLKL

et al. 2016

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The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species. Necroptosis is a form of programmed cell death that can be induced following ligation of death ligand and Toll-like receptors (TLRs). Most experimental work has focused on necroptosis induced by tumour necrosis factor (TNF). The key effectors in the pathway are the protein kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, 1-4 and the mixed-lineage kinase domain-like (MLKL) pseudokinase. [5][6][7][8] RIPK3 phosphorylates the pseudokinase domain of MLKL, the most terminal known essential component of the pathway, 5,6 which is believed to induce a conformational change and unleash the N-terminal four-helix bundle (4HB) domain of MLKL: an executioner domain. 5,9,10 Several models have been proposed for how this 4HB domain might induce cell death, including activation of downstream effectors, such as ion channels, 11,12 direct permeabilization of membranes and/or formation of a transmembrane pore, 13,14 all of which remain the subject of debate. The consensus from these and other studies is that in order to kill, MLKL must translocate to membranes and assemble into high molecular weight signalling complexes, which are likely to be MLKL oligomers, although the stoichiometry of these MLKL oligomers remains an open question. [10][11][12][13][14] Nonetheless, phosphorylation appears to be a key cue for MLKL activation 15 and, as the most terminal known post-translational modification in the pathway, could potentially be utilized as a biomarker in pathologies arising from necroptotic cell death. 14,16 A model whereby RIPK3-mediated phosphorylation of the MLKL pseudokinase domain activation loop (S345 in mouse;...

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HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

Cited by 137 publications

References 73 publications

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis

Evolutionary divergence of the necroptosis effector MLKL

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