Reconstitution of Human Necrosome Interactions in Saccharomyces cerevisiae

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.

Human Gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signaling in budding yeast

“…To allow comparison of both human proteins, in the case of MLKL, we artificially produced the phosphomimetic mutant MLKL(PM), as well as a truncated MLKL(1-182) version, which should structurally compare to the constitutively active NTD of GSDMD released by Caspase-1 cleavage. Although it has been shown that human RIPK3 and MLKL co-expression in yeast enhances the intrinsic toxicity of the former in this model [40], in our hands a human phosphomimetic MLKL T357E/S358D [MLKL(PM)] behaved like the wild type. This agrees with studies arguing that mouse MLKL(PM) gains activity while the human protein mutated in the equivalent phosphorylation sites may not.…”

“…MLKL is activated through phosphorylation by RIPK3 in mammalian cells [41, 42]. A recent report showed that human MLKL is not phosphorylated when heterologously expressed in yeast unless it is co-expressed with RIPK3 [40]. To by-pass the phosphorylation step, we cloned both the wild-type MLKL gene and a phosphomimetic T357E/S358D version, referred hereafter to as MLKL(PM), in the same expression vector used for GSDMD (Fig.…”

“…In the absence of homologous pathways, yeast supplies a cellular environment to study the fundamental properties of these pore-forming effectors. Furthermore, our previous work demonstrated that GSDMD can be processed by Caspase-1 in the yeast cell [39], and work by Ji et al [40],…”

Reconstitution of human pyroptotic cell death in Saccharomyces cerevisiae

“…To comprehensively determine the activities of the executors of pyroptosis in yeast, we measured the maximal growth rates, changes in membrane integrity and clonogenicity of yeast transformants overexpressing full-length and N-terminal domains of GSDMD and GSDME. Murine MLKL, which has been demonstrated to cause yeast lethality when overexpressed 35 , acted as a positive control. While neither the wild type GSDMD nor GSDME compromised yeast viability, the N-terminal truncation mutants of both gasdermins (hereafter abbreviated GSDMD 1–275 and GSDME 1–270 ) dramatically suppressed yeast growth rates (Fig.…”

“…Previously, we modeled necroptotic cell death mediated by RIPK1, RIPK3 and MLKL in yeast, demonstrating that reconstituted mammalian lytic cell death in yeast provides a tool for investigating intra- and intermolecular mechanisms of pore-forming proteins, and establishing a platform for high-throughput screening for functional domains or potential inhibitors 35 .…”