title: HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.
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.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: A V
authors.initials: AV
authors.lastname: Jacobsen
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: K N
authors.initials: KN
authors.lastname: Lowes
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: M C
authors.initials: MC
authors.lastname: Tanzer
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: I S
authors.initials: IS
authors.lastname: Lucet
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Hildebrand
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: E J
authors.initials: EJ
authors.lastname: Petrie
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: M F
authors.initials: MF
authors.lastname: van Delft
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: S A
authors.initials: SA
authors.lastname: Conos
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: J-G
authors.initials: JG
authors.lastname: Zhang
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: D C S
authors.initials: DC
authors.lastname: Huang
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: J
authors.identifier: http://orcid.org/0000-0002-7611-5774
authors.initials: J
authors.lastname: Silke
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.|Department of Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Lessene
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.|Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Murphy
mesh_terms: D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009336:Necrosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D014178:Translocation, Genetic
chemical_list: D018841:HSP90 Heat-Shock Proteins; C570694:MLKL protein, mouse; D011494:Protein Kinases