ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1.

Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues.

Caspase cleavage of RIPK3 after Asp is dispensable for mouse embryogenesis.

The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp is dependent on downstream caspase-3.

Activity of caspase-8 determines plasticity between cell death pathways.

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR1, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL. Mice that lack caspase-8 display MLKL-dependent embryonic lethality, as do mice that express catalytically inactive CASP8(C362A). Casp8Mlkl mice die during the perinatal period, whereas Casp8Mlkl mice are viable, which indicates that inactive caspase-8 also has a pro-death scaffolding function.

Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.

The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis. In humans, caspase-8 deficiency is associated with immunodeficiency or very early onset inflammatory bowel disease.

OTULIN limits cell death and inflammation by deubiquitinating LUBAC.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease and embryonic lethality during mouse development. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1).

CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction.

RIPK1 inhibits ZBP1-driven necroptosis during development.

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL.

Structural Insights into WD-Repeat 48 Activation of Ubiquitin-Specific Protease 46.

Protein ubiquitination patterns are an important component of cellular signaling. The WD-repeat protein WDR48 (USP1-associated factor UAF-1) stimulates activity of ubiquitin-specific proteases USP1, USP12, and USP46. To understand how WDR48 exerts its effect on the USP scaffold, we determined structures of the ternary WDR48:USP46:ubiquitin complex.

Ubiquitin chain elongation requires E3-dependent tracking of the emerging conjugate.

Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood.

Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis.

Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 trigger pro-inflammatory cell death termed "necroptosis." Studies with RIPK3-deficient mice or the RIPK1 inhibitor necrostatin-1 suggest that necroptosis exacerbates pathology in many disease models. We engineered mice expressing catalytically inactive RIPK3 D161N or RIPK1 D138N to determine the need for the active kinase in the whole animal.

Ubiquitin-dependent regulation of COPII coat size and function.

Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive.

Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation.

Antibodies that specifically recognize polyubiquitin chains containing ubiquitins linked at a particular lysine residue are powerful tools for interrogating endogenous protein modifications. Here, we describe protocols for revealing K11-, K48-, and K63-linked polyubiquitin chains by western blotting, immunoprecipitation, or immunostaining.

K11-linked ubiquitin chains as novel regulators of cell division.

Modification of proteins with ubiquitin chains is an essential regulatory event in cell cycle control. Differences in the connectivity of ubiquitin chains are believed to result in distinct functional consequences for the modified proteins. Among eight possible homogenous chain types, canonical Lys48-linked ubiquitin chains have long been recognized to drive the proteasomal degradation of cell cycle regulators, and Lys48 is the only essential lysine residue of ubiquitin in yeast.

The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2.

Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential noncovalent interaction that occurs in addition to the thioester bond at the E2 active site.

K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin.

Identification of a physiological E2 module for the human anaphase-promoting complex.

Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C.

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells.

Heat shock inhibits caspase-1 activity while also preventing its inflammasome-mediated activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase-1. We report that heat-shocking LT-sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase-1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen-activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen-activated protein kinase cleavage and degradation of an unknown substrate by the proteasome.

Killing of macrophages by anthrax lethal toxin: involvement of the N-end rule pathway.

Macrophages from certain inbred mouse strains are rapidly killed (< 90 min) by anthrax lethal toxin (LT). LT cleaves cytoplasmic MEK proteins at 20 min and induces caspase-1 activation in sensitive macrophages at 50-60 min, but the mechanism of LT-induced death is unknown. Proteasome inhibitors block LT-mediated caspase-1 activation and can protect against cell death, indicating that the degradation of at least one cellular protein is required for LT-mediated cell death.

Anthrax lethal toxin-induced inflammasome formation and caspase-1 activation are late events dependent on ion fluxes and the proteasome.

Anthrax lethal toxin (LT) is cytotoxic to macrophages from certain inbred mouse strains. The gene controlling macrophage susceptibility to LT is Nalp1b. Nalp1b forms part of the inflammasome, a multiprotein complex involved in caspase-1 activation and release of interleukin (IL)-1beta and IL-18.

Oxidized ATP protection against anthrax lethal toxin.

Bacillus anthracis lethal toxin (LT) induces rapid lysis (<90 min) of murine macrophages from certain inbred strains. The mechanism for LT-induced cytolysis is currently unknown. We hypothesized that the ATP-activated macrophage P2X7 receptors implicated in nucleotide-mediated macrophage lysis could play a role in LT-mediated cytolysis and discovered that a potent P2X7 antagonist, oxidized ATP (o-ATP), protects macrophages against LT.