Publications by authors named "Cheree Fitzgibbon"

11 Publications

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

Nat Commun 2021 06 7;12(1):3364. Epub 2021 Jun 7.

The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK.

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.
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http://dx.doi.org/10.1038/s41467-021-23474-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184782PMC
June 2021

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

Nat Commun 2021 04 13;12(1):2211. Epub 2021 Apr 13.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41467-021-22400-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044208PMC
April 2021

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

Cell Death Dis 2021 04 1;12(4):345. Epub 2021 Apr 1.

Research Department, Sidra Medicine, Doha, 26999, Qatar.

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.
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http://dx.doi.org/10.1038/s41419-021-03636-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016849PMC
April 2021

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

Cell Death Differ 2021 Jul 15;28(7):2126-2144. Epub 2021 Feb 15.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41418-021-00742-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257593PMC
July 2021

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

ACS Chem Biol 2020 10 22;15(10):2702-2713. Epub 2020 Sep 22.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.

Necroptosis is an inflammatory form of programmed cell death that has been implicated in various human diseases. Compound is a more potent analogue of the published compound 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 . These target engagement studies demonstrate that compound 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 and in cells. Compound also shows efficacy in a murine model of systemic inflammatory response syndrome (SIRS).
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http://dx.doi.org/10.1021/acschembio.0c00482DOI Listing
October 2020

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

Nat Commun 2020 06 19;11(1):3060. Epub 2020 Jun 19.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.

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.
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http://dx.doi.org/10.1038/s41467-020-16823-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305131PMC
June 2020

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

Nat Commun 2020 06 19;11(1):3151. Epub 2020 Jun 19.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41467-020-16887-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305196PMC
June 2020

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

Proc Natl Acad Sci U S A 2020 04 31;117(15):8468-8475. Epub 2020 Mar 31.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;

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.
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http://dx.doi.org/10.1073/pnas.1919960117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165463PMC
April 2020

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

Cell Rep 2019 09;28(13):3309-3319.e5

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.08.055DOI Listing
September 2019

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

Cell Syst 2018 08 11;7(2):161-179.e14. Epub 2018 Jul 11.

Systems Biology Ireland, University College Dublin, Dublin, Ireland; Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin, Ireland; School of Medicine and Medical Science, University College Dublin, Dublin, Ireland; Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cels.2018.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149545PMC
August 2018

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

Invest New Drugs 2013 Dec 22;31(6):1466-75. Epub 2013 Sep 22.

La Trobe Institute of Molecular Science, La Trobe University, Bendigo, Australia.

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.
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http://dx.doi.org/10.1007/s10637-013-0031-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3825418PMC
December 2013
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