Publications by authors named "Peter E Czabotar"

75 Publications

Yeast- and antibody-based tools for studying tryptophan C-mannosylation.

Nat Chem Biol 2021 Feb 4. Epub 2021 Feb 4.

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

Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions of this modification are poorly understood, with progress in the field hampered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a diverse array of proteins with and without tryptophan C-mannosylation and interrogate the modification's influence on protein stability and function. This system also enabled mutagenesis studies to identify residues of the glycosyltransferase and its protein substrates that are crucial for catalysis. The collection of modified proteins accrued during this work facilitated the generation and thorough characterization of monoclonal antibodies against tryptophan C-mannosylation. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation. This study revealed many new modification sites on proteins throughout the secretory pathway with both conventional and non-canonical consensus sequences.
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http://dx.doi.org/10.1038/s41589-020-00727-wDOI Listing
February 2021

The regulation of necroptosis by post-translational modifications.

Cell Death Differ 2021 Jan 18. Epub 2021 Jan 18.

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

Necroptosis is a caspase-independent, lytic form of programmed cell death whose errant activation has been widely implicated in many pathologies. The pathway relies on the assembly of the apical protein kinases, RIPK1 and RIPK3, into a high molecular weight cytoplasmic complex, termed the necrosome, downstream of death receptor or pathogen detector ligation. The necrosome serves as a platform for RIPK3-mediated phosphorylation of the terminal effector, the MLKL pseudokinase, which induces its oligomerization, translocation to, and perturbation of, the plasma membrane to cause cell death. Over the past 10 years, knowledge of the post-translational modifications that govern RIPK1, RIPK3 and MLKL conformation, activity, interactions, stability and localization has rapidly expanded. Here, we review current knowledge of the functions of phosphorylation, ubiquitylation, GlcNAcylation, proteolytic cleavage, and disulfide bonding in regulating necroptotic signaling. Post-translational modifications serve a broad array of functions in modulating RIPK1 engagement in, or exclusion from, cell death signaling, whereas the bulk of identified RIPK3 and MLKL modifications promote their necroptotic functions. An enhanced understanding of the modifying enzymes that tune RIPK1, RIPK3, and MLKL necroptotic functions will prove valuable in efforts to therapeutically modulate necroptosis.
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http://dx.doi.org/10.1038/s41418-020-00722-7DOI Listing
January 2021

RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells.

Elife 2020 12 2;9. Epub 2020 Dec 2.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8 T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.
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http://dx.doi.org/10.7554/eLife.63452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710356PMC
December 2020

Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association.

Structure 2021 Feb 22;29(2):114-124.e3. Epub 2020 Sep 22.

Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany. Electronic address:

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.
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http://dx.doi.org/10.1016/j.str.2020.09.002DOI Listing
February 2021

BAK core dimers bind lipids and can be bridged by them.

Nat Struct Mol Biol 2020 11 14;27(11):1024-1031. Epub 2020 Sep 14.

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

BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.
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http://dx.doi.org/10.1038/s41594-020-0494-5DOI Listing
November 2020

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

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

EMBO J 2020 09 26;39(18):e106275. Epub 2020 Aug 26.

The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
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http://dx.doi.org/10.15252/embj.2020106275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461020PMC
September 2020

Relating SMCHD1 structure to its function in epigenetic silencing.

Biochem Soc Trans 2020 08;48(4):1751-1763

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

The structural maintenance of chromosomes hinge domain containing protein 1 (SMCHD1) is a large multidomain protein involved in epigenetic gene silencing. Variations in the SMCHD1 gene are associated with two debilitating human disorders, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). Failure of SMCHD1 to silence the D4Z4 macro-repeat array causes FSHD, yet the consequences on gene silencing of SMCHD1 variations associated with BAMS are currently unknown. Despite the interest due to these roles, our understanding of the SMCHD1 protein is in its infancy. Most knowledge of SMCHD1 function is based on its similarity to the structural maintenance of chromosomes (SMC) proteins, such as cohesin and condensin. SMC proteins and SMCHD1 share similar domain organisation and affect chromatin conformation. However, there are important differences between the domain architectures of SMC proteins and SMCHD1, which distinguish SMCHD1 as a non-canonical member of the family. In the last year, the crystal structures of the two key domains crucial to SMCHD1 function, the ATPase and hinge domains, have emerged. These structures reveal new insights into how SMCHD1 may bind and regulate chromatin structure, and address how amino acid variations in SMCHD1 may contribute to BAMS and FSHD. Here, we contrast SMCHD1 with canonical SMC proteins, and relate the ATPase and hinge domain structures to their roles in SMCHD1-mediated epigenetic silencing and disease.
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http://dx.doi.org/10.1042/BST20200242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458401PMC
August 2020

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

Nat Commun 2020 06 19;11(1):3150. Epub 2020 Jun 19.

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

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
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http://dx.doi.org/10.1038/s41467-020-16819-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305203PMC
June 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

Crystal structure of the hinge domain of Smchd1 reveals its dimerization mode and nucleic acid-binding residues.

Sci Signal 2020 06 16;13(636). Epub 2020 Jun 16.

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

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.
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http://dx.doi.org/10.1126/scisignal.aaz5599DOI Listing
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

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Nat Chem Biol 2019 11 7;15(11):1057-1066. Epub 2019 Oct 7.

Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.
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http://dx.doi.org/10.1038/s41589-019-0365-8DOI Listing
November 2019

BAX, BAK, and BOK: A Coming of Age for the BCL-2 Family Effector Proteins.

Cold Spring Harb Perspect Biol 2020 04 1;12(4). Epub 2020 Apr 1.

Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Parkville, Victoria 3052, Australia.

The BCL-2 family of proteins control a key checkpoint in apoptosis, that of mitochondrial outer membrane permeabilization or, simply, mitochondrial poration. The family consists of three subgroups: BH3-only initiators that respond to apoptotic stimuli; antiapoptotic guardians that protect against cell death; and the membrane permeabilizing effectors BAX, BAK, and BOK. On activation, effector proteins are converted from inert monomers into membrane permeabilizing oligomers. For many years, this process has been poorly understood at the molecular level, but a number of recent advances have provided important insights. We review the regulation of these effectors, their activation, subsequent conformational changes, and the ensuing oligomerization events that enable mitochondrial poration, which initiates apoptosis through release of key signaling factors such as cytochrome We highlight the mysteries that remain in understanding these important proteins in an endeavor to provide a comprehensive picture of where the field currently sits and where it is moving toward.
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http://dx.doi.org/10.1101/cshperspect.a036319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111251PMC
April 2020

Characterization of a novel venetoclax resistance mutation (BCL2 Phe104Ile) observed in follicular lymphoma.

Br J Haematol 2019 09 24;186(6):e188-e191. Epub 2019 Jun 24.

Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Australia.

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http://dx.doi.org/10.1111/bjh.16069DOI Listing
September 2019

Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations.

Nat Commun 2019 06 3;10(1):2385. Epub 2019 Jun 3.

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

Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.
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http://dx.doi.org/10.1038/s41467-019-10363-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547681PMC
June 2019

Neutralising antibodies block the function of Rh5/Ripr/CyRPA complex during invasion of Plasmodium falciparum into human erythrocytes.

Cell Microbiol 2019 07 24;21(7):e13030. Epub 2019 Apr 24.

Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine.
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http://dx.doi.org/10.1111/cmi.13030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594224PMC
July 2019

Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy.

EMBO J 2019 01 20;38(2). Epub 2018 Dec 20.

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

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co-ordinate the removal of damaged mitochondria. We report that following mitochondrial damage-induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3-only proteins and its homo-dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX-mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK-dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1-dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro-inflammatory effect.
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http://dx.doi.org/10.15252/embj.201899916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331729PMC
January 2019

Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.

Nature 2019 01 12;565(7737):118-121. Epub 2018 Dec 12.

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

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.
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http://dx.doi.org/10.1038/s41586-018-0779-6DOI Listing
January 2019

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia.

Cancer Discov 2019 03 4;9(3):342-353. Epub 2018 Dec 4.

Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Victoria, Australia.

The BCL2 inhibitor venetoclax induces high rates of durable remission in patients with previously treated chronic lymphocytic leukemia (CLL). However, despite continuous daily treatment, leukemia recurs in most patients. To investigate the mechanisms of secondary resistance, we analyzed paired pre-venetoclax and progression samples from 15 patients with CLL progression enrolled on venetoclax clinical trials. The novel Gly101Val mutation in BCL2 was identified at progression in 7 patients, but not at study entry. It was first detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affinity of BCL2 for venetoclax by ∼180-fold in surface plasmon resonance assays, thereby preventing the drug from displacing proapoptotic mediators from BCL2 in cells and conferring acquired resistance in cell lines and primary patient cells. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. SIGNIFICANCE: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1119DOI Listing
March 2019

The Structural Basis of Necroptotic Cell Death Signaling.

Trends Biochem Sci 2019 01 30;44(1):53-63. Epub 2018 Nov 30.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

The recent implication of the cell death pathway, necroptosis, in innate immunity and a range of human pathologies has led to intense interest in the underlying molecular mechanism. Unlike the better-understood apoptosis pathway, necroptosis is a caspase-independent pathway that leads to cell lysis and release of immunogens downstream of death receptor and Toll-like receptor (TLR) ligation. Here we review the role of recent structural studies of the core machinery of the pathway, the protein kinases receptor-interacting protein kinase (RIPK)1 and RIPK3, and the terminal effector, the pseudokinase mixed lineage kinase domain-like protein (MLKL), in shaping our mechanistic understanding of necroptotic signaling. Structural studies have played a key role in establishing models that describe MLKL's transition from a dormant monomer to a killer oligomer and revealing important interspecies differences.
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http://dx.doi.org/10.1016/j.tibs.2018.11.002DOI Listing
January 2019

Ensemble Properties of Bax Determine Its Function.

Structure 2018 10 16;26(10):1346-1359.e5. Epub 2018 Aug 16.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, VIC 3052, Australia. Electronic address:

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.
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http://dx.doi.org/10.1016/j.str.2018.07.006DOI Listing
October 2018

Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis.

Nat Commun 2018 06 21;9(1):2422. Epub 2018 Jun 21.

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

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.
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http://dx.doi.org/10.1038/s41467-018-04714-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013482PMC
June 2018

Enhanced antimalarial activity of plasmepsin V inhibitors by modification of the P position of PEXEL peptidomimetics.

Eur J Med Chem 2018 Jun 18;154:182-198. Epub 2018 May 18.

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, 3010, Australia. Electronic address:

Plasmepsin V is an aspartyl protease that plays a critical role in the export of proteins bearing the Plasmodium export element (PEXEL) motif (RxLxQ/E/D) to the infected host erythrocyte, and thus the survival of the malaria parasite. Previously, development of transition state PEXEL mimetic inhibitors of plasmepsin V have primarily focused on demonstrating the importance of the P Arg and P Leu in binding affinity and selectivity. Here, we investigate the importance of the P position by incorporating both natural and non-natural amino acids into this position and show disubstituted beta-carbon amino acids convey the greatest potency. Consequently, we show analogues with either cyclohexylglycine or phenylglycine in the P position are the most potent inhibitors of plasmepsin V that impair processing of the PEXEL motif in exported proteins resulting in death of P. falciparum asexual stage parasites.
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http://dx.doi.org/10.1016/j.ejmech.2018.05.022DOI Listing
June 2018

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)-LRR oligomerization interface.

J Allergy Clin Immunol 2018 12 17;142(6):1956-1967.e6. Epub 2018 May 17.

Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; Department of Medical Biology, University of Melbourne, Parkville, Australia; Immunology Laboratory, Guangzhou Institute of Paediatrics, Guangzhou, China. Electronic address:

Background: Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4).

Objective: Here we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease.

Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively.

Results: The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1-dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4-mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex.

Conclusion: This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.
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http://dx.doi.org/10.1016/j.jaci.2018.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281029PMC
December 2018

Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Cell 2018 05;173(5):1217-1230.e17

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

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, BokBaxBak animals exhibited more severe defects and died earlier than BaxBak mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing BokBaxBak triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult BokBaxBak mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.
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http://dx.doi.org/10.1016/j.cell.2018.04.036DOI Listing
May 2018

The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.

Cell Death Differ 2018 09 14;25(9):1567-1580. Epub 2018 Feb 14.

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

The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.
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http://dx.doi.org/10.1038/s41418-018-0061-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143630PMC
September 2018

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Authors:
Lorenzo Galluzzi Ilio Vitale Stuart A Aaronson John M Abrams Dieter Adam Patrizia Agostinis Emad S Alnemri Lucia Altucci Ivano Amelio David W Andrews Margherita Annicchiarico-Petruzzelli Alexey V Antonov Eli Arama Eric H Baehrecke Nickolai A Barlev Nicolas G Bazan Francesca Bernassola Mathieu J M Bertrand Katiuscia Bianchi Mikhail V Blagosklonny Klas Blomgren Christoph Borner Patricia Boya Catherine Brenner Michelangelo Campanella Eleonora Candi Didac Carmona-Gutierrez Francesco Cecconi Francis K-M Chan Navdeep S Chandel Emily H Cheng Jerry E Chipuk John A Cidlowski Aaron Ciechanover Gerald M Cohen Marcus Conrad Juan R Cubillos-Ruiz Peter E Czabotar Vincenzo D'Angiolella Ted M Dawson Valina L Dawson Vincenzo De Laurenzi Ruggero De Maria Klaus-Michael Debatin Ralph J DeBerardinis Mohanish Deshmukh Nicola Di Daniele Francesco Di Virgilio Vishva M Dixit Scott J Dixon Colin S Duckett Brian D Dynlacht Wafik S El-Deiry John W Elrod Gian Maria Fimia Simone Fulda Ana J García-Sáez Abhishek D Garg Carmen Garrido Evripidis Gavathiotis Pierre Golstein Eyal Gottlieb Douglas R Green Lloyd A Greene Hinrich Gronemeyer Atan Gross Gyorgy Hajnoczky J Marie Hardwick Isaac S Harris Michael O Hengartner Claudio Hetz Hidenori Ichijo Marja Jäättelä Bertrand Joseph Philipp J Jost Philippe P Juin William J Kaiser Michael Karin Thomas Kaufmann Oliver Kepp Adi Kimchi Richard N Kitsis Daniel J Klionsky Richard A Knight Sharad Kumar Sam W Lee John J Lemasters Beth Levine Andreas Linkermann Stuart A Lipton Richard A Lockshin Carlos López-Otín Scott W Lowe Tom Luedde Enrico Lugli Marion MacFarlane Frank Madeo Michal Malewicz Walter Malorni Gwenola Manic Jean-Christophe Marine Seamus J Martin Jean-Claude Martinou Jan Paul Medema Patrick Mehlen Pascal Meier Sonia Melino Edward A Miao Jeffery D Molkentin Ute M Moll Cristina Muñoz-Pinedo Shigekazu Nagata Gabriel Nuñez Andrew Oberst Moshe Oren Michael Overholtzer Michele Pagano Theocharis Panaretakis Manolis Pasparakis Josef M Penninger David M Pereira Shazib Pervaiz Marcus E Peter Mauro Piacentini Paolo Pinton Jochen H M Prehn Hamsa Puthalakath Gabriel A Rabinovich Markus Rehm Rosario Rizzuto Cecilia M P Rodrigues David C Rubinsztein Thomas Rudel Kevin M Ryan Emre Sayan Luca Scorrano Feng Shao Yufang Shi John Silke Hans-Uwe Simon Antonella Sistigu Brent R Stockwell Andreas Strasser Gyorgy Szabadkai Stephen W G Tait Daolin Tang Nektarios Tavernarakis Andrew Thorburn Yoshihide Tsujimoto Boris Turk Tom Vanden Berghe Peter Vandenabeele Matthew G Vander Heiden Andreas Villunger Herbert W Virgin Karen H Vousden Domagoj Vucic Erwin F Wagner Henning Walczak David Wallach Ying Wang James A Wells Will Wood Junying Yuan Zahra Zakeri Boris Zhivotovsky Laurence Zitvogel Gerry Melino Guido Kroemer

Cell Death Differ 2018 03 23;25(3):486-541. Epub 2018 Jan 23.

Paris Descartes/Paris V University, Paris, France.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
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http://dx.doi.org/10.1038/s41418-017-0012-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864239PMC
March 2018

Conversion of Bim-BH3 from Activator to Inhibitor of Bak through Structure-Based Design.

Mol Cell 2017 Nov;68(4):659-672.e9

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, VIC 3052, Australia. Electronic address:

Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.
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http://dx.doi.org/10.1016/j.molcel.2017.11.001DOI Listing
November 2017