Abstract
Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer. Receptor-interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like protein (MLKL) are required for necroptosis activation. Specifically, RIPK3-dependent MLKL phosphorylation promotes the assembly of disulfide bond-dependent MLKL polymers that drive the execution of necroptosis. However, how MLKL disulfide bond formation is regulated is not clear. In this study we discovered that the MLKL-modifying compound necrosulfonamide cross-links cysteine 86 of human MLKL to cysteine 32 of the thiol oxidoreductase thioredoxin-1 (Trx1). Recombinant Trx1 preferentially binds to monomeric MLKL and blocks MLKL disulfide bond formation and polymerization in vitro Inhibition of MLKL polymer formation requires the reducing activity of Trx1. Importantly, shRNA-mediated knockdown of Trx1 promotes MLKL polymerization and sensitizes cells to necroptosis. Furthermore, pharmacological inhibition of Trx1 with compound PX-12 induces necroptosis in multiple cancer cell lines. Altogether, these findings demonstrate that Trx1 is a critical regulator of necroptosis that suppresses cell death by maintaining MLKL in a reduced inactive state. Our results further suggest new directions for targeted cancer therapy in which thioredoxin inhibitors like PX-12 could potentially be used to specifically target cancers expressing high levels of MLKL or MLKL short isoforms.
Highlights
Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer
Receptor-interacting protein kinase 3 (RIPK3)-dependent mixed lineage kinase domainlike protein (MLKL) activation leads to the formation of lethal MLKL polymers [36]
Current models of necroptosis indicate that MLKL polymers are responsible for disrupting the integrity of the plasma membrane
Summary
Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer. Most of our understanding of the necroptotic machinery has emerged through studies of the TNF-␣-signaling pathway These works led to the discovery of serine/threonine kinases RIPK1/3 and MLKL as critical mediators of necroptosis (18 – 24). Together, these proteins form the core of the necrosome complex that is central for transducing necroptotic signals. MLKL mutant with multiple cysteine residues changed to serine residues could not form intermolecular disulfide bonds and is greatly compromised in its ability to induce necroptosis [36] These results suggest that MLKL activity is tightly regulated by its redox state. Whether similar oxidative events drive MLKL activation in human cells remains a topic of much debate
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