Oligomerization-driven MLKL ubiquitylation antagonizes necroptosis.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

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

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Maternal Circadian Disruption from Shift Work and the Impact on the Concentration of Melatonin in Breast Milk.

Background and Objective: Melatonin in breast milk exhibits a 24-hour circadian rhythm, present in nighttime breast milk but nearly undetectable in daytime breast milk. Shift work can disrupt the circadian timing of individuals, evident in changes in melatonin in saliva and urine samples. However, it is unknown whether these changes are also reflected in breast milk from a shift working mother. The aim of this study was to investigate whether maternal circadian rhythm disturbance from shift work impacts the melatonin concentration in breast milk. Materials and Methods: Breast milk and saliva samples were collected from 11 shift working mothers at four timepoints across five consecutive days. This included during their day shift or nonworkdays to act as a control, night shift, subsequent night shifts and postnight shift. Where possible, pre- and postfeed collections were also undertaken. Samples were grouped into four-time intervals: 12-6:30 am, 7-11:30 am, 12-6:30 pm, 7-11:30 pm, and melatonin levels (picogram per milliliter) in the breast milk and saliva samples were analyzed. Results: There was a significant decrease in breast milk melatonin (p = 0.026) at the 12-6:30 am time interval on subsequent night shifts, compared with control days. However, there was no overall time and shift type interaction effect (p = 0.70). In addition, no observed difference in melatonin levels was found in saliva samples, or when comparing pre- and postfeed breast milk. Breast milk melatonin however was found to be significantly higher compared with saliva (p > 0.001), at all but one time interval. Conclusion: The findings suggest that there is a potential effect of maternal circadian rhythm disruption from shift work on breast milk melatonin levels. This is an important step in exploring the role of maternal circadian timing and the effect on breast milk composition. Expansion of this research and exploration of other circadian rhythm misalignment sleep disorders on breast milk is highly recommended.

Chemo-sensitisation of HeLa cells to etoposide by a benzoxazine in the absence of DNA-PK inhibition.

The benzoaxines have been developed from structurally similar chromones as specific inhibitors of the PI3K family to sensitize cancer cells to the effects of chemotherapeutic agents; most have been shown to do this through specific inhibition of DNA-PK and DNA repair mechanisms. In this study we examined the benzoxazine, 2-((3-methoxybut-3en-2-yl)amino)-8methyl-4H-benzo[1,3]oxazin-4one (LTUSI54). This compound had no DNA-PK or PI3K inhibitory activity but still sensitized HeLa cells to the effects of Etoposide. LTUSI54 works synergistically with Etoposide to inhibit growth of HeLa cells and sub G1 analysis indicates that this is not due to an increase in apoptosis. LTUSI54 neither enhances DSB formation due to Etoposide nor does it delay the repair of such damage. Cell cycle analysis shows a clear G2 block with Etoposide alone while, in combination with LTUSI54 there is an additional S phase arrest. Phospho-kinase analysis indicated that LTUSI54 engages key regulators of cell cycle progression, specifically p38α, p53 and ERK 1/2. From our results we hypothesize that LTUSI54 is promoting the cell cycle arrest through activation of p38α pathways, independent of p53 mechanisms. This results in a decrease in p53 phosphorylation and hence, restricted apoptosis. Changes in cell number appear to be the result of p38α pathways disrupting cell cycle progression, at the S and G2 checkpoints. Further investigation into the finer mechanisms by which LTUSI54 effects cell cycle progression would be of great interest in assessing this compound as a chemosensitising agent.

Rapid Versus Slow Cooling Pasteurization of Donor Breast Milk: Does the Cooling Rate Effect Melatonin Reduction?

Background and Objective: There is a question as to whether melatonin levels in breast milk are impacted by the cooling rate postpasteurization. Past research that has used in the Australian donor bank's breast milk Holder Pasteurization technique has reported varying findings regarding melatonin levels postpasteurization. Where breast milk was cooled slowly, a significant reduction in breast milk melatonin levels was observed. Conversely, where a rapid cooling method was used, there was no significant reduction in melatonin levels. The aim of this study was to investigate whether the cooling process between the different pasteurization techniques impact on melatonin levels in breast milk postpasteurization. Materials and Methods: Twenty-seven nighttime breast milk samples were collected, with each sample divided into three; one remained unpasteurized, one was pasteurized and rapidly cooled to 4°C, and the other was pasteurized and cooled slowly to 4°C. Results: Melatonin levels were significantly reduced in both the rapidly cooled and slow cooled breast milk samples when compared to their unpasteurized counterpart (p < 0.001). There was no significant difference in melatonin levels between the two cooling methods (p = 0.91). Conclusion: This study showed that both the rapid and cooling pasteurization processes had a similar reduction in melatonin levels in breast milk. However, even after pasteurization melatonin was still present. Therefore, it is recommended that donor banks still take into consideration circadian timing hormones such as melatonin and the time of day breast milk is expressed.

Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization.

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.

Co-expression of recombinant RIPK3:MLKL complexes using the baculovirus-insect cell system.

Pseudokinase domains are found throughout the kingdoms of life and serve myriad roles in cell signaling. These domains, which resemble protein kinases but are catalytically-deficient, have been described principally as protein interaction domains. Broadly, pseudokinases have been reported to function as: allosteric regulators of conventional enzymes; scaffolds to nucleate assembly and/or localization of signaling complexes; molecular switches; or competitors of signaling complex assembly. From detailed structural and biochemical studies of individual pseudokinases, a picture of how they mediate protein interactions is beginning to emerge. Many such studies have relied on recombinant protein production in insect cells, where endogenous chaperones and modifying enzymes favor bona fide folding of pseudokinases. Here, we describe methods for co-expression of pseudokinases and their interactors in insect cells, as exemplified by the MLKL pseudokinase, which is the terminal effector in the necroptosis cell death pathway, and its upstream regulator kinase RIPK3. These methods are broadly applicable to co-expression of other pseudokinases with their interaction partners from bacmids using the baculovirus-insect cell expression system.

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. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.

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. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

A family harboring an MLKL loss of function variant implicates impaired necroptosis in diabetes.

Maturity-onset diabetes of the young, MODY, is an autosomal dominant disease with incomplete penetrance. In a family with multiple generations of diabetes and several early onset diabetic siblings, we found the previously reported P33T PDX1 damaging mutation. Interestingly, this substitution was also present in a healthy sibling. In contrast, a second very rare heterozygous damaging mutation in the necroptosis terminal effector, MLKL, was found exclusively in the diabetic family members. Aberrant cell death by necroptosis is a cause of inflammatory diseases and has been widely implicated in human pathologies, but has not yet been attributed functions in diabetes. Here, we report that the MLKL substitution observed in diabetic patients, G316D, results in diminished phosphorylation by its upstream activator, the RIPK3 kinase, and no capacity to reconstitute necroptosis in two distinct MLKL-/- human cell lines. This MLKL mutation may act as a modifier to the P33T PDX1 mutation, and points to a potential role of impairment of necroptosis in diabetes. Our findings highlight the importance of family studies in unraveling MODY's incomplete penetrance, and provide further support for the involvement of dysregulated necroptosis in human disease.

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. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

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. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

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. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues.

The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.

Potent Inhibition of Necroptosis by Simultaneously Targeting Multiple Effectors of the Pathway.

Necroptosis is an inflammatory form of programmed cell death that has been implicated in various human diseases. Compound 2 is a more potent analogue of the published compound 1 and inhibits necroptosis in human and murine cells at nanomolar concentrations. Several target engagement strategies were employed, including cellular thermal shift assays (CETSA) and diazirine-mediated photoaffinity labeling via a bifunctional photoaffinity probe derived from compound 2. These target engagement studies demonstrate that compound 2 binds to all three necroptotic effector proteins (mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine protein kinase 1 (RIPK1) and receptor-interacting serine/threonine protein kinase 3 (RIPK3)) at different levels in vitro and in cells. Compound 2 also shows efficacy in vivo in a murine model of systemic inflammatory response syndrome (SIRS).

MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis.

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.

Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3.

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 kinase-domain-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.

Dissecting RAF Inhibitor Resistance by Structure-based Modeling Reveals Ways to Overcome Oncogenic RAS Signaling.

Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.