Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3

Abstract

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinasedomain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction.

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

“…protein interactions | cell death | RIPK3 | programmed necrosis | protein engineering N ecroptosis is a caspase-independent cell death pathway that has been implicated in host defense to counter pathogens (1)(2)(3)(4)(5)(6)(7)(8), and its dysregulation, in the pathology of inflammatory diseases (9)(10)(11)(12)(13). Necroptotic signaling can be initiated by death receptor ligands, including tumor necrosis factor (TNF), engaging their cognate receptors.…”

“…At the heart of this complex, the RIPK1 and RIPK3 kinases form a heterooligomeric complex in which RIPK3 is activated by autophosphorylation. Although the details of the next steps in necroptosis signaling are incompletely understood, necrosomal RIPK3 is thought to recruit and phosphorylate MLKL to promote its oligomerization, membrane translocation, and lytic permeabilization of the plasma membrane (8,(14)(15)(16)(17)(18)(19)(20)(21).…”

A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

“…As several other necroptotic proteins are lysine-rich, we considered whether the choice of fixative was a critical variable for robust immunodetection of MLKL, RIPK3 and RIPK1 in human and mouse cells. Accordingly, we compared the performance of 22 antibodies for immunofluorescent staining of human HT29 cells and mouse dermal fibroblasts (MDFs) -two cellular models that are well-characterized to undergo necroptosis when treated with TNF, Smacmimetic and IDN-6556 (herein referred to as TSI) 5,28,29,44,47 . To quantitatively gauge the performance of each antibody, their immunosignals were characterized in four ways: 1) A ratio of the immunofluorescent signals between a positive and negative control.…”

“…CC-BY-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.26.356063 doi: bioRxiv preprint 8 antibody, clone 1H2 (source: in-house) 5 , detected RIPK3 via immunofluorescence in methanol-fixed, but not paraformaldehyde-fixed cells. However, most of the immunofluorescent signals produced by 1H2 were non-specific (Fig.…”

“…Cell death by necroptosis is thought to have originated as an ancestral host defence mechanism, which is reflected in the breadth of pathogen-encoded proteins that inhibit the pathway 1,2,3,4,5,6 . In addition to reported innate immunity functions 7,8,9 , the dysregulation of necroptosis has been implicated in a range of pathologies, including ischemic-reperfusion injuries, such as in the kidney 10,11 and heart 12 , and inflammatory diseases 13,14,15,16 , including inflammatory bowel disease 17 .…”