Abstract
The necrosome is a large-molecular-weight complex in which the terminal effector of the necroptotic pathway, Mixed Lineage Kinase Domain-Like protein (MLKL), is activated to induce necroptotic cell death. The precise mechanism of MLKL activation by the upstream kinase, Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) and the role of Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) in mediating MLKL activation remain incompletely understood. Here, we reconstituted human necrosome interactions in yeast by inducible expression of these necrosome effectors. Functional interactions were reflected by the detection of phosphorylated MLKL, plasma membrane permeabilization, and reduced proliferative potential. Following overexpression of human necrosome effectors in yeast, MLKL aggregated in the periphery of the cell, permeabilized the plasma membrane and compromised clonogenic potential. RIPK1 had little impact on RIPK3/MLKL-mediated yeast lethality; however, it exacerbated the toxicity provoked by co-expression of MLKL with a RIPK3 variant bearing a mutated RHIM-domain. Small molecule necroptotic inhibitors necrostatin-1 and TC13172, and viral inhibitors M45 (residues 1–90) and BAV_Rmil, abated the yeast toxicity triggered by the reconstituted necrosome. This yeast model provides a convenient tool to study necrosome protein interactions and to screen for and characterize potential necroptotic inhibitors.
Highlights
Necroptosis is an inflammatory form of programmed cell death characterized by necrotic morphological features, which has been observed in multiple human diseases [1,2].Necroptosis can be triggered by death receptors and pattern recognition receptors, such as Tumor Necrosis Factor receptor 1 (TNFR1), Toll-Like Receptor 3/4 (TLR3/4) and ZDNA-Binding Protein 1 (ZBP1), leading to the formation of a large-molecular-weight complex, the necrosome [3,4]
We investigated the impact of ectopic expression of necroptotic proteins in Saccharomyces cerevisiae, first investigating the activity of each individual component prior to exploring their interactions in the yeast context
Consistent with a recent report [40], human Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) had no significant impact on yeast growth, nor did human Mixed-Lineage Kinase Domain-Like Protein (MLKL), mouse RIPK1 or mouse Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) (Figure 1A)
Summary
Necroptosis is an inflammatory form of programmed cell death characterized by necrotic morphological features, which has been observed in multiple human diseases [1,2].Necroptosis can be triggered by death receptors and pattern recognition receptors, such as Tumor Necrosis Factor receptor 1 (TNFR1), Toll-Like Receptor 3/4 (TLR3/4) and ZDNA-Binding Protein 1 (ZBP1), leading to the formation of a large-molecular-weight complex, the necrosome [3,4]. Active RIPK3 phosphorylates and activates Mixed-Lineage Kinase Domain-Like Protein (MLKL) at T357/S358 in its Pseudokinase Domain (PsKD) to trigger oligomerization and membrane localization of MLKL and subsequent membrane rupture [6,7,8]. PsKD domain connected by a two-helix linker [9]. The 4HB domain contains a positively charged interface that forms electrostatic interactions with phospholipids of the plasma membrane [12]. This exposure occurs following activation of the PsKD, which triggers MLKL to oligomerize and orients the positively charged interface of the 4HB domain such that it can engage with the plasma membrane [11,13].
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