Protein shapeshifting in necroptotic cell death signaling.

Necroptosis is a mode of programmed cell death executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following its activation by the upstream receptor-interacting protein kinase-3 (RIPK3), subsequent to activation of death, Toll-like, and pathogen receptors. The pathway originates in innate immunity, although interest has surged in therapeutically targeting necroptosis owing to its dysregulation in inflammatory diseases. Here, we explore how protein conformation and higher order assembly of the pathway effectors - Z-DNA-binding protein-1 (ZBP1), RIPK1, RIPK3, and MLKL - can be modulated by post-translational modifications, such as phosphorylation, ubiquitylation, and lipidation, and intermolecular interactions to tune activities and modulate necroptotic signaling flux.

MLKL deficiency elevates testosterone production in male mice independently of necroptotic functions.

Mixed lineage kinase domain-like (MLKL) is a pseudokinase, best known for its role as the terminal effector of the necroptotic cell death pathway. MLKL-mediated necroptosis has long been linked to various age-related pathologies including neurodegeneration, atherosclerosis and male reproductive decline, however many of these attributions remain controversial. Here, we investigated the role of MLKL and necroptosis in the adult mouse testis: an organ divided into sperm-producing seminiferous tubules and the surrounding testosterone-producing interstitium.

Divergent roles of RIPK3 and MLKL in high-fat diet-induced obesity and MAFLD in mice.

Cell death frequently occurs in the pathogenesis of obesity and metabolic dysfunction-associated fatty liver disease (MAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in the essential necroptotic regulators, receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL), as well as mice lacking apoptotic caspase-8 in myeloid cells combined with RIPK3 loss, that RIPK3/caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and MAFLD upon high-fat diet feeding.

In situ visualization of endothelial cell-derived extracellular vesicle formation in steady state and malignant conditions.

Endothelial cells are integral components of all vasculature within complex organisms. As they line the blood vessel wall, endothelial cells are constantly exposed to a variety of molecular factors and shear force that can induce cellular damage and stress. However, how endothelial cells are removed or eliminate unwanted cellular contents, remains unclear.

Mapping where and when necroptotic cell death occurs in disease.

A series of recent studies have established where the necroptosis machinery — RIPK1, RIPK3, MLKL, ZBP1 and Caspase-8 — occur and are activated in mouse and human tissues, and how ZBP1 splicing might regulate necroptosis. These studies highlight the importance of mapping necroptosis spatially to understand how pathway dysregulation can lead to disease.

An immunohistochemical atlas of necroptotic pathway expression.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues.

Barriers and facilitators to implementing a Canadian shared-care ADHD program in pediatric settings in Shanghai: a consolidated framework for implementation research approach.

Objectives: The vast majority of children with Attention-Deficit Hyperactivity Disorder (ADHD) do not have access to proper diagnosis and treatment in China. The goal of this project is to identify the challenges and facilitators in implementing a Canadian ADHD Shared Care Pathways program in pediatric settings in Shanghai region.

Methods: Purposive semi-structured focus groups were conducted on a total of 13 healthcare practitioners from the Shanghai Xinuha, Ninghai and Chongming hospitals.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL in biological membranes and MLKL overriding pharmacological and endogenous inhibition of MLKL.

Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1.

MLKL deficiency protects against low-grade, sterile inflammation in aged mice.

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl and Ripk3 mice on a congenic C57BL/6 J genetic background.

Endothelial Caspase-8 prevents fatal necroptotic hemorrhage caused by commensal bacteria.

Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown.

Ubiquitylation of RIPK3 beyond-the-RHIM can limit RIPK3 activity and cell death.

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469.

Development of a carotid artery thrombolysis stroke model in mice.

Recanalization with restored cerebral perfusion is the primary goal of thrombolytic therapy in acute ischemic stroke. The identification of adjunctive therapies that can be safely used to enhance thrombolysis in stroke remains an elusive goal. We report here the development of a mouse in situ carotid artery thrombolysis (iCAT) stroke model involving graded cerebral ischemia to induce unihemispheric infarction after thrombotic occlusion of the common carotid artery (CCA).

The web of death: the expanding complexity of necroptotic signaling.

The past decade has seen the emergence of the necroptosis programmed cell death pathway as an important contributor to the pathophysiology of myriad diseases. The receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase executioner protein, mixed lineage kinase domain-like (MLKL), have grown to prominence as the core pathway components. Depending on cellular context, these proteins also serve as integrators of signals, such as post-translational modifications and protein or metabolite interactions, adding layers of complexity to pathway regulation.

Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling.

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling.

Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation).

Implementing a Canadian shared-care ADHD program in Beijing: Barriers and facilitators to consider prior to start-up.

Background: The shared care pathway for ADHD is a program developed in Canada with two main strategies: (a) implement a shared care pathway between general practitioners (GPs) and specialists, and (b) step up or down care so that the patient is treated at the most appropriate level of care, depending on the complexity or outcome of their illness. The current study aims to identify the challenges and facilitators of implementing this program in a Chinese mental health service setting.

Methods: Two focus groups were conducted using semi-structured interviews with a total of 7 health care providers in Beijing.

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL.

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined.

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL.

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death.

Ferroptosis mediates selective motor neuron death in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord; however, the primary cell death pathway(s) mediating motor neuron demise remain elusive. We recently established that necroptosis, an inflammatory form of regulated cell death, was dispensable for motor neuron death in a mouse model of ALS, implicating other forms of cell death. Here, we confirm these findings in ALS patients, showing a lack of expression of key necroptotic effector proteins in spinal cords.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis.

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase.

Meaningful relationships as a driving force in the experience of parents of a child living with polyposis conditions.

While much research has been conducted on the experiences of individuals with inflammatory bowel diseases, there remains a dearth of research conducted on those affected by polyposis conditions. As a result, little is known about the lived experiences of those with polyposis conditions, especially in the cases of parents of pediatric patients with these conditions. Using a hermeneutical phenomenological qualitative research approach, this study sought to explore the lived experiences of parents of children with polyposis conditions, with specific attention paid to the processes in which parents engage in order to adapt to their realities.

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain.

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

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death.