Thioredoxin-1 Inhibition Promotes MLKL Activation: Biochemical Insights Into a Suppressor of the Necroptotic Pathway
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Necroptosis is an immunogenic caspase-independent cell death program. While mainly serving to defend against viral infection, dysregulated necroptotic signaling contributes to the pathology of a growing number of human diseases associated with inflammation, neurodegeneration, and ischemic injuries. Since its emergence, many key components of the mammalian necroptotic pathway have been discovered including RIP1/3 kinases and MLKL, which together, form the core of the necrosome complex. RIP3-dependent phosphorylation of MLKL promotes the formation of high molecular weight MLKL polymers that disrupt the integrity of the plasma membrane leading to cell death. However, the mechanistic details of MLKL polymerization and its precise function in executing necroptosis remain poorly understood. To gain insight into this process, the crosslinking property of the specific MLKL inhibitor Necrosulfonamide (NSA) was exploited as a strategy for identifying novel MLKL interacting proteins. Immunoprecipitation of NSA crosslinked MLKL complexes followed by mass spectrometric analysis revealed Thioredoxin-1, an essential cytosolic thiol oxidoreductase, to be a potential MLKL regulator. Based on its ability to reduce disulfide bonds on specific protein targets, it was hypothesized that Thioredoxin-1 actively maintains MLKL in a reduced monomeric state to suppress the occurrence of spontaneous necroptosis. Therefore, genetic and pharmacological approaches were taken to perturb Thioredoxin-1 activity as a means for testing whether it would induce necroptosis in human cells. The data presented here demonstrates that inhibiting Thioreoxin-1 activity using the compound PX-12 promoted RIP3-dependent MLKL S358 phosphorylation, polymer formation, and caspase-independent necrotic cell death. Additionally, PX12-induced cell death was rescued by co-treating cells with NSA highlighting the involvement of MLKL in this cell death pathway. These results were corroborated by shRNA-mediated knockdown of Thioreoxin-1 mRNA. Altogether, these findings point to Thioredoxin-1 as a critical regulator of MLKL activity and necroptosis in human cells.