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1.
Cell Death Differ ; 23(11): 1827-1838, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27419363

ABSTRACT

Caspase-1 cleaves and activates the pro-inflammatory cytokine interleukin-1 beta (IL-1ß), yet the mechanism of IL-1ß release and its dependence on cell death remains controversial. To address this issue, we generated a novel inflammasome independent system in which we directly activate caspase-1 by dimerization. In this system, caspase-1 dimerization induced the cleavage and secretion of IL-1ß, which did not require processing of caspase-1 into its p20 and p10 subunits. Moreover, direct caspase-1 dimerization allowed caspase-1 activation of IL-1ß to be separated from cell death. Specifically, we demonstrate at the single cell level that IL-1ß can be released from live, metabolically active, cells following caspase-1 activation. In addition, we show that dimerized or endogenous caspase-8 can also directly cleave IL-1ß into its biologically active form, in the absence of canonical inflammasome components. Therefore, cell death is not obligatory for the robust secretion of bioactive IL-1ß.


Subject(s)
Caspase 1/metabolism , Interleukin-1beta/metabolism , Animals , Caspase 8/metabolism , Cell Death , Cell Survival , DNA Gyrase/metabolism , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Inflammasomes/metabolism , Mice , Protein Multimerization , Recombinant Fusion Proteins/metabolism
2.
Cell Death Dis ; 7: e2051, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26775703

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.


Subject(s)
HSP90 Heat-Shock Proteins/genetics , Protein Kinases/genetics , Animals , Apoptosis , Cell Death , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Necrosis , Phosphorylation , Protein Kinases/metabolism , Translocation, Genetic
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