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

A V Jacobsen,M F Van Delft,E J Petrie,J M Hildebrand,M C Tanzer,I S Lucet,K N Lowes,J Silke,J M Murphy,S A Conos,Z Liu,J-G Zhang,G Lessene,D C S Huang

doi:10.1038/cddis.2015.386

Abstract

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

Highlights

Reduced capacity to undergo necroptosis has been correlated to increased aggressiveness of cancers;[9,10] and therapeutic initiation of necroptosis is currently being investigated as a cancer therapy.[11,12] there is emerging evidence that the necroptotic signalling pathway has a general role in the modulation of inflammation.[13,14,15,16,17] As such, unravelling the molecular events governing necroptosis, and potential avenues for therapeutic intervention, is of enormous interest
Necroptosis was induced in wild-type murine dermal fibroblasts (MDFs) using conventional necroptotic stimuli: tumour necrosis factor (TNF; T), to activate TNF receptor 1; the Smac mimetic, Compound A (S), to inhibit cIAP-mediated ubiquitylation of RIPK1 and permit its participation in cell death signalling; and the pan-caspase inhibitor, Q-VD-OPh (Q), to inhibit caspase-8 and permit necroptosis (Figure 1a)
The same library of 438 compounds was tested in Mlkl− / − MDFs reconstituted with the Q343A mutant Mixed Lineage Kinase domain-Like (MLKL), which we previously showed to induce cell death in the absence of necroptotic stimuli, independently of RIPK3 activity (Figure 1b).[25]

Summary

Introduction

Reduced capacity to undergo necroptosis has been correlated to increased aggressiveness of cancers;[9,10] and therapeutic initiation of necroptosis is currently being investigated as a cancer therapy.[11,12] there is emerging evidence that the necroptotic signalling pathway has a general role in the modulation of inflammation.[13,14,15,16,17] As such, unravelling the molecular events governing necroptosis, and potential avenues for therapeutic intervention, is of enormous interest. Necroptosis is initiated through activation of death receptors, such as Tumour Necrosis Factor Receptor 1 (TNFR1), or through microbial activation of pattern recognition receptors, such as Toll-like receptors or intracellular viral DNA sensors.[3,18,19,20] Receptor ligation initiates a signalling cascade, whereby Receptor Interacting Protein Kinase (RIPK)-3 oligomerises and is phosphorylated, a process known to be regulated by association with other effectors, such as the protein kinase RIPK1, TIR-domain-containing adapter-inducing. IFN-β (TRIF), or DNA-dependent activator of IFN regulatory factors (DAI), via their RIP Homotypic Interaction Motifs (RHIMs).[2,21,22] Once activated, RIPK3 phosphorylates the pseudokinase domain of Mixed Lineage Kinase domain-Like (MLKL), the most downstream known obligate effector of the necroptotic signalling pathway, to induce its activation.[23,24].

Methods

Results

Conclusion