Ubiquitin networks in cell death regulation and non-apoptotic signalling

Lead Research Organisation: Institute of Cancer Research
Department Name: Division of Breast Cancer Research

Abstract

The posttranslational modification with ubiquitin (Ub), a process referred to as ubiquitylation, controls important aspects of cell death and survival. Ub can be attached to pro- and anti-apoptotic proteins as a single moiety or in the form of polymeric chains in which successive ubiquitin molecules are connected through specific isopeptide bonds. Reminiscent of a code, the various ubiquitin modifications adopt distinct conformations and lead to different outcomes in cells. To prevent ubiquitylation from being constitutively on, modifications are reversed by de-ubiquitylating enzymes (DUB) that cleave off the Ub adduct. While the consequence of Ub-attachment is intensely studied, little is known with regards to the effects of deconjugating enzymes that remove the Ub-adduct. The aim of the proposed programme is to unravel how the ubiquitin-signal is conjugated and edited to modulate cellular processes that are required during normal development and tissue homeostasis. We will use a series of interlocking genetic and biochemical approaches to identify and characterise DUBs that regulate caspase-mediated cell death and non-apoptotic signalling. In a pilot study we identified several DUBs that, when removed, suppress cell death in vivo. Here we propose to investigate the physiological role and molecular mechanism of five of these DUBs. Particularly, we will assess whether they function as integral and evolutionarily conserved components of the tissue repair process. Importantly, a growing body of evidence indicate that caspase-mediated signalling generates the release of signals that communicate with the cellular environment to coordinate compensatory proliferation, tissue regeneration and wound healing. Hence, we will also assess whether loss of the identified DUBs also affects caspase activation required for adaptation to tissue stress. Taken together, we propose to investigate the dynamics and specificity of Ub networks and study how Ub conjugation and deconjugation impact on signaling outcomes. Unravelling how DUBs regulate cell death and non-apoptotic signalling is critically important as these processes play fundamental physiological roles in animal development and tissue homeostasis.

Technical Summary

The conjugation and de-conjugation of Ubiquitin (Ub) to target proteins influence diverse biological processes that can contribute to tumour formation when deregulated. Protein levels and activity of many pro- and anti-apoptotic molecules are controlled by E3-mediated conjugation of Ub. While the consequence of Ub-attachment is intensely studied, little is known about the deconjugating enzymes that remove the Ub-adduct. Here, we propose to investigate the dynamics and specificity of Ub networks and study how Ub conjugation and deconjugation impact on cell survival. We will use a series of interlocking genetic and biochemical approaches to identify and characterize deubiquitylating enzymes (DUBs) that regulate cell death. In a pilot study we identified several DUBs that, when knocked-down, suppress cell death in vivo. Here we propose to investigate the physiological role and molecular mechanism of four of these DUBs. Particularly, we will assess whether they function as integral and evolutionarily conserved components of the apoptosis machinery. Since cell death signalling components also execute nonapoptotic functions, we will assess whether loss of these DUBs also affects caspase activation required for cell migration and cell-fate decisions. Our preliminary data on one of these DUBs indicate that it is required for caspase activation following genotoxic stress, most likely because it removes Ub chains from the apoptosome. Intriguingly, we also found that genotoxic stress results in phosphorylation of this DUB, which appears to change its cleavage preference towards distinct Ub chain types. Moreover, we will also identify DUBs that regulate the Ripoptosome, a novel mammalian cell death-inducing platform that is negatively regulated in an Ub-dependent manner. Unravelling how DUBs regulate cell death and non-apoptotic signalling is critically important as these processes play fundamental roles in animal development and tissue homeostasis.

Planned Impact

Impact and potential beneficiaries of this research:

-Scientific community:
While naturally occurring cell death was already observed 170 years ago, it was long considered a passive phenomenon and viewed as an inevitable end point of biological systems. This view began to change with studies of developmentally timed cell death in the silkworm and tadpole. These early studies showed that cell death can be delayed with inhibitors of protein or RNA synthesis and that neuronal cell survival requires extracellular survival factors termed neurotrophins. A breakthrough in elucidating the mechanism by which cells undergo programmed cell death came from genetic studies in the nematode C. elegans. It is now recognized that proper regulation of cell death plays a fundamental role in animal development and tissue homeostasis. Abnormal regulation of this process is associated with a wide variety of human diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Nevertheless, some aspects of cell death regulation remain obscure and limit our understanding of how tissue homeostasis is achieved and how organisms adapt to tissue malfunction. The successful conclusion of the proposed project is expected to provide a better understanding of the molecular mechanisms that control Ub-dependent regulation of signal transduction pathways that influence the ability of multi-cellular organisms to adapt to perturbations. As such our research addresses fundamental issues relevant to normal animal physiology and aging. Further, As ubiquitin-mediated regulation of cell death and tissue repair is involved in almost every aspect of life, the proposed project will also impact the health-care community. Of particular interest is the notion that cell death regulatory proteins also fulfill nonlethal functions in differentiation and tissue remodeling. It is now clear that the cell death machinery is involved in releasing signals to communicate with their cellular environment, to promote cell division, tissue regeneration, and wound healing. Unraveling the molecular details governing this process could lead to a better understanding of aging, tissue regeneration and cancer.

- Cancer patients:
One of the key problems in cancer is the adaptive nature of tumours. It is now recognised that the ubiquitin system modulates key signal transduction pathways that have direct implications in the 'evolvability' of tumours. Hence, cancer treatments would be significantly more successful if it was possible to target ubiquitin-dependent signalling events. Our research addresses a fundamental aspect of ubiquitin-dependent tissue repair. Ultimately, this work will provide new insights into the mechanisms through which ubiquitin-dependent signalling contributes to cancer, and how we can translate this information for patient benefit.

- Academia/Pharma based drug development teams:
Due to the growing interest in ubiquitin-dependent signalling events, and the notion that the conjugation and de-conjugation of ubiquitin to target proteins influence diverse biological processes that can contribute to aging as well as tumour formation when deregulated, it is likely that the results of this project will be of interest not only for academic but also private drug development groups. The identification of the molecular mechanisms through which adaptation to tissue stress and repair are co-ordinated in a Ub-dependent manner will reveal new signalling nodes and signal transduction pathways that could be targeted by treatment with inhibitors that block deubiquitylating enzymes.

Publications

10 25 50

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Riebeling T (2021) Primidone blocks RIPK1-driven cell death and inflammation. in Cell death and differentiation

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Moerke C (2019) The anticonvulsive Phenhydan® suppresses extrinsic cell death. in Cell death and differentiation

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Meier P (2016) Tissue Repair: How to Inflame Your Neighbours. in Current biology : CB

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Meier P (2015) Ubiquitin-Mediated Regulation of Cell Death, Inflammation, and Defense of Homeostasis. in Current topics in developmental biology

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Ciuffa R (2022) Novel biochemical, structural, and systems insights into inflammatory signaling revealed by contextual interaction proteomics. in Proceedings of the National Academy of Sciences of the United States of America

 
Description The aim of this project is to unravel how cell death and survival decision are taken. We have used Drosophila as an entry point due to its reduced complexity and rapid in vivo validation. We have screened deubiquitylating enzymes and ubiquitin-related proteins to identify how cell death and inflammation are regulated. It is now clear that cell death and inflammatory processes are intimately linked. Common to both pathways is the conjugation of the small protein modifier Ubiquitin to target proteins. The conjugation of ubiquitin to target proteins modulates their activity and stability. We have discovered several Ub-related proteins that modulate either cell death or inflammation. We have analyzed two Drosophila melanogaster deubiquitylating enzymes (DUBs) of the OTU-family. The OTU family of DUBs is particularly interesting because many of these DUBs display specificity for one or several types of ubiquitin chain linkages. Our analysis revealed that two of the potential OTU DUBs from Drosophila melanogaster , CG3251 and Otu (shared homologues of human OTUD4) carry a serine (S, Ser) instead of the central cysteine in the catalytic triad. While a high number of serine proteases exist, ubiquitin-specific serine proteases have not been described. Analysis of the OTUD4 family proteins in other species revealed that loss of the catalytic cysteine seems to have occurred frequently in independent events. Taken together, our data suggest that the two Drosophila melanogaster OTUD4 homologues exert a DUB-independent function. We also investigated the function of Par-1, a Ub-associated protein which appears to be critically involved in the regulation of inflammation in both Drosophila and mammals. As these signal transduction pathways are used to control cell competition, our data provide a further link between cell death and inflammation. We are currently investigating how Par-1 modulates NFkB and MAPK signalling.

As part of this grant we also investigated how cell death is regulated during cell competition. Cell competition is a phenomenon where by less fit cells are eliminated by neighbouring fitter cells. This process is critically important during normal development and ageing to ensure tissue function. But it is highjacked in Cancer. Therefore a better understanding of the processes at play during cell competition will provide us with important new insight how to kill super-competitive cancer cells.
Exploitation Route It is now clear that cell death and inflammation are intimately linked, and that many age related disease pathologies are due to mal-adaptation of cell death and inflammation. Hence a better understanding of the complex relationship between cell death and inflammation will undoubtedly help to design better treatment strategies to tackle chronic auto-inflammatory diseases or defects in tissue homeostasis. Particularly, our findings on Cell competition will have important ramification as it indicates a new way of killing cancer cells. This is an exciting new approach that we are pursuing further.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title CRISPR mediated deletion of specific DNA regions 
Description we developed improved techniques to apply the CRISPR/CAS9 technique to delete specific genes from the genome 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? No  
Impact This will improve the way through which we analyse the function of specific genes. 
 
Title Collection of RHIM mutant proteins 
Description We have created a plasmid bank for all RHIM containing genes. Each of these genes are represented as WT or RHIM mutant, which will allow scientists to evaluate the physical interactions between these RHIM containing proteins. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact not available yet. 
 
Title MYOSIN-7A research reagent-2 
Description developed a proximity ligation assay that allows the detection of Myosin7A - caspase-8 complexes in vivo. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This methodology will enable researchers to evaluate Myosin7A-mediated regulation of caspase-8 using endogenous settings. 
 
Title Mouse model to study the role of MLKL ubiquitylation in health and disease 
Description We have used the CRISPR/Cas9 method to create a knockin mutation in MLKL that evades Ubiquitin-meidated regulation of MLKL. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2021 
Provided To Others? Yes  
Impact This mouse model has provided us with additional knowledge how lytic forms of cell death can be regulated. It is leading the way to develop new anti-cancer strategies. 
 
Title Organoid tumour models 
Description We have created various tumour organoid lines that we can use to study immunogenic cell death in C57BL/6 mice. 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? No  
Impact not available yet. But this reagent will allow us to create patient cohorts for the development of novel treatment protocols. 
 
Title PAR-1 research reagents 
Description we have generated various fly strains that express wild-type and mutant Par-1 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? No  
Impact not yet available 
 
Title RIPK1-PROTAC 
Description We have generated a PROTAC compound that targets RIPK1 for ubiquitin-mediated degradation. This reagents allows us to study the physiological and pathophysiological roles of RIPK1 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact This reagents will change our view of the role of RIPK1 in health and disease. 
 
Title TRIF-deficient KO cell lines 
Description To study the role of TRIF in health and disease we have created TRIF mutant knockout lines in L929, E0771, HT29 and HaCaT cells. 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? No  
Impact not available yet. 
 
Title Ubiquitin restriction analysis 
Description We have improve the UbiCrest technique that allows the characterisation of Ubiquitin-dependent signalling events 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact This will allow to dissect the signalling events that control inflammation and cancer development 
 
Title ZBP1 mutant cell lines 
Description we have created reconstituted ZBP1 cell lines in which endogenous ZBP1 was replaced with various mutant versions. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact not available yet 
 
Title ZBP1-deficient KO cell lines 
Description To study the role of ZBP1 in nucleic acid sensing we generated ZBP1-KO CRISPR lines in L929, E0771, HT29 and HaCaT cell lines. 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? No  
Impact not available yet 
 
Description Caspase-mediated regulation of innate immunity in Drosophila 
Organisation University of Turku
Country Finland 
Sector Academic/University 
PI Contribution We identified that the effector caspase drICE cleaves the Inhibitor of Apoptosis (IAP) protein DIAP2 and that this cleavage is critically important to suppress inflammation under steady state conditions.
Collaborator Contribution Our collaborator in Turku validated our findings using Drosophila as a model system
Impact This work was published in 2023
Start Year 2017
 
Description Developmental control of cell death 
Organisation University of Massachusetts
Department Department of Cancer Biology
Country United States 
Sector Academic/University 
PI Contribution Established the developmental impact mediated by components of the apoptosome
Collaborator Contribution Established the developmental impact mediated by components of the apoptosome
Impact Characterise the developmental role of the apoptosome, and its regulation
Start Year 2006
 
Description Identification of specific Ubiquitin chains on components of the TNF receptor signalling complex 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution we have provide novel findings to address the question what types of signalling chains are conjugated to specific components of the TNF receptor signalling complex.
Collaborator Contribution The collaborator has provided us with recombinant enzymes that allowed us to investigate the Ubiquitin linkage types that are conjugated to components of the TNF receptor signalling complex.
Impact the study that reports the findings from this collaboration has been accepted for publication at Molecular Cell. This study has led to a better understanding of TNF signalling
Start Year 2015
 
Description MLKL-mediated Necroptosis 
Organisation Auburn University
Country United States 
Sector Academic/University 
PI Contribution This collaboration focusses on the regulation of necroptosis, a controlled form of lytic cell death. We have identified a regulatory mechanism through which the key effector MLKL triggers necroptosis. In particular we have generated a MLKL knock-in mouse model in which we changed a particular residue that is important for MLKL regulation. We have done all the preliminary characterisation of this model with respect to its sensitivity to cel death induction.
Collaborator Contribution Jason Upton, a world leader in virus induced necroptosis, at Auburn University tested the in vivo relevance of the MLKL mutation. He assessed whether the mutant animal, and cells thereof, is more susceptible to viral infection.
Impact This collaboration is multi-disciplinary. Discipline involved are: cell death immunity inflammation virology
Start Year 2019
 
Description MYOSIN-7A mediated regulation of cell survival 
Organisation University of California, Los Angeles (UCLA)
Department Jules Stein Eye Institute UCLA
Country United States 
Sector Academic/University 
PI Contribution using MYOSIN-7A reagents we made new discoveries implicating MYOsin-7A in the regulation of cell death, migration and cell invasion
Collaborator Contribution shared MYOSIN-7A reagents
Impact Identified the regulatory mechanism of MYOSIN-7A-mediated regulation of cell survival
Start Year 2008
 
Description RAC-mediated regulation of immune activation 
Organisation National Institute of Health and Medical Research (INSERM)
Department INSERM U1065 (Mediterranean Centre for Molecular Medicine - C3M)
Country France 
Sector Charity/Non Profit 
PI Contribution We will test the involvement of RAC in regulating immune activation.
Collaborator Contribution Laurent Boyer is providing us with constructs and fly lines to investigate the role of RAC in immune activation and cell competition.
Impact not yet available
Start Year 2016
 
Description RIPK1-mediated regulation of immunogenic cell death 
Organisation Auburn University
Country United States 
Sector Academic/University 
PI Contribution Here we are testing the role of RIPK1 in regulating immunogenic cell death. We have developed a pharmacological PROTAC compound that targets RIPK1 for degradation. This enables us to evaluate the role of RIPK1 in controlling intracellular signalling events that control the immunogenicity of cell death events.
Collaborator Contribution To study the role of RIPK1 in regulating TNFR1, TLR3 and ZBP1-mediated necroptosis (a lytic form of cell death), we have collaborated with Manolis Pasparakis who is providing us with bone marrow from various knockout animals. We are also collaborating with Jason Upson who is an expert in ZBP1-mediated necroptosis. Jason is in the possession of various viruses that can be used to trigger ZBP1-induced necroptosis. With the help of our RIPK1-PROTAC it will be possible to evaluate the role of RIPK1 in this process.
Impact These experiments will provide us with a deep understanding of the signalling processes that regulate the immunogenicity of cell death.
Start Year 2021
 
Description RIPK1-mediated regulation of immunogenic cell death 
Organisation University of Cologne
Department CECAD Research Center
Country Germany 
Sector Academic/University 
PI Contribution Here we are testing the role of RIPK1 in regulating immunogenic cell death. We have developed a pharmacological PROTAC compound that targets RIPK1 for degradation. This enables us to evaluate the role of RIPK1 in controlling intracellular signalling events that control the immunogenicity of cell death events.
Collaborator Contribution To study the role of RIPK1 in regulating TNFR1, TLR3 and ZBP1-mediated necroptosis (a lytic form of cell death), we have collaborated with Manolis Pasparakis who is providing us with bone marrow from various knockout animals. We are also collaborating with Jason Upson who is an expert in ZBP1-mediated necroptosis. Jason is in the possession of various viruses that can be used to trigger ZBP1-induced necroptosis. With the help of our RIPK1-PROTAC it will be possible to evaluate the role of RIPK1 in this process.
Impact These experiments will provide us with a deep understanding of the signalling processes that regulate the immunogenicity of cell death.
Start Year 2021
 
Description Regulation of TNF signalling 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Here we worked with the Aebersold lab to characterise the interactome of the TNF-receptor-I signalling complex. We trained members of the Aebersold lab to purify TNF-receptor signalling complex-I and -II from cells. Moreover, we helped with advise and prioritisation of leads.
Collaborator Contribution The Aebersold lab identified the composition, stoichiometry, temporal organization and cellular requirements for the formation of the TNF-receptor signaling complex (TNF-RSC). Overall, they showed that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function.
Impact The above mentioned approach resulted in the identification of 2 new components of the TNF-RSC, namely WRNIP1 and UBASH3b. Moreover, it produced large amount of interaction data, highlighting the complexity of the TNF signalling. This project was only possible due to its multi-disciplinary nature. Disciplines: Mass spectrometry Cellular Biology
Start Year 2021
 
Description Regulation of caspase-8 mediated signalling 
Organisation Medical Research Council (MRC)
Department MRC Toxicology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab analysed the biochemical consequence of the Caspase-8 mutation Casp8F122G/L123G (Casp8mDED2). This mutation abrogates Caspase-8 homodimerisation but not its heterodimerisation with cFLIP. Hence, this Casp8mDED2 mutant enables to study the physiological function of caspase-8:cFLIP heterodimers. We evaluated cells of mutant mice and identified the functional consequence of this mutation.
Collaborator Contribution The Pasparakis lab generated Casp8F122G/L123G (Casp8mDED2) mutant animals and crossed them to various disease models.
Impact This study resulted in a detailed understanding of how caspase-8 is activated and how active caspase-8 regulates cellular processes.
Start Year 2020
 
Description Regulation of caspase-8 mediated signalling 
Organisation University of Cologne
Department CECAD Research Center
Country Germany 
Sector Academic/University 
PI Contribution My lab analysed the biochemical consequence of the Caspase-8 mutation Casp8F122G/L123G (Casp8mDED2). This mutation abrogates Caspase-8 homodimerisation but not its heterodimerisation with cFLIP. Hence, this Casp8mDED2 mutant enables to study the physiological function of caspase-8:cFLIP heterodimers. We evaluated cells of mutant mice and identified the functional consequence of this mutation.
Collaborator Contribution The Pasparakis lab generated Casp8F122G/L123G (Casp8mDED2) mutant animals and crossed them to various disease models.
Impact This study resulted in a detailed understanding of how caspase-8 is activated and how active caspase-8 regulates cellular processes.
Start Year 2020
 
Description Regulation of cell competition 
Organisation Columbia University
Department Department of Genetics and Development
Country United States 
Sector Academic/University 
PI Contribution We have provide a novel molecular link in the regulation of cell competition and inflammatory signalling. In addition, we have provided reagents and research tools to our collaborator.
Collaborator Contribution Our collaborator investigated the involvement of our key protein in their assay system to gain a better understand of the regulatory mechanisms of cell competition.
Impact currently there are no outputs or outcomes of this collaboration
Start Year 2015
 
Description Regulation of immune activation 
Organisation Cornell University
Department Department of Entomology
Country United States 
Sector Academic/University 
PI Contribution In collaboration with Nicolas Buchon, a world leader in immune activation in Drosophila, we are investing the role of positive and negative regulators of IMD signalling. While we are functionally characterising identified targets, Nicoas Buchon investigates the role of these genes in modulating the transcriptional outputs of immune activation. In particular, Nicolas Buchon conducts RNAseq experiments for us.
Collaborator Contribution In collaboration with Nicolas Buchon, a world leader in immune activation in Drosophila, we are investing the role of positive and negative regulators of IMD signalling. While we are functionally characterising identified targets, Nicoas Buchon investigates the role of these genes in modulating the transcriptional outputs of immune activation. In particular, Nicolas Buchon conducts RNAseq experiments for us.
Impact This collaboration led to a better understanding of immune regulation in Drosophila
Start Year 2016
 
Description Regulation of innate immunity in Drosophila 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution To study how innate immune signalling is regulated we have set up a collaboration with Professor Ioannis P. Nezis at the school of life science. Using mass spectrometry, we discovered that selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis. We identified the interaction between TAK1 and TAB2/SH3PX1.
Collaborator Contribution Our collaborator functionally validated this interaction and identified that selective autophagy regulates TAK1 signalling.
Impact The paper reporting this finding was published.
Start Year 2019
 
Description Regulation of necroptosis 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Country Australia 
Sector Academic/University 
PI Contribution Here we are studying the molecular mechanism through which the necroptosis effector MLKL is activated and how its cytotoxic potential is regulated. The key aspect is to understand how we can induce necroptosis in cancer cells. Necroptosis is an immunogenic form of cell death that will greatly help to activate a patient's own immune system against cancer.
Collaborator Contribution James Murphy is providing structural support and advise in studying MLKL, a key effector of necroptosis. James previously crystallised MLKL (Immunity. 2013 Sep 19;39(3):443-53; Biochem J. 2013 Nov 13), which has greatly helped us in resolving how MLKL is regulated.
Impact not applicable yet.
Start Year 2019
 
Description Regulation of the pseudokinase MLKL 
Organisation Auburn University
Country United States 
Sector Academic/University 
PI Contribution We discovered that the pseudokinase MLKL is regulated via post-translational modification and this modification controls its cytotoxic potential, protecting us from viruses.
Collaborator Contribution Prof. Jason Upton provided us with vital reagents. In addition he conducted virus infection assays, which demonstrated that ubiquitylation of MLKL is essential for virus-induced necroptosis and restriction of viral titres.
Impact We identified that postr-translational modification of MLKL is required to protect us from viral infection. We found that ubiquitylation of MLKL restricts viral propagation. This data was published in Nat. Comms 2021.
Start Year 2020
 
Description Regulation of the pseudokinase MLKL 
Organisation Skidmore College
Country United States 
Sector Academic/University 
PI Contribution We identified that the pseudokinase MLKL is ubiquitylated at K219 and that this ubiquitylation influences its ability to trigger lytic cell death (necroptosis).
Collaborator Contribution Aurelia Ball performed extensive all-atom molecular dynamics (MD) simulations of unmodified MLKL (WT), MLKL phosphorylated at S345 (S345phos), MLKL both phosphorylated at S345 and mono ubiquitylated at K219 (K219ub S345phos), starting from the crystal structure of mMLKL (PBD: 4BTF). Overall these simulations indicated a conformational change in the activation loop helix and 4HB domain upon S345 phosphorylation, leading to MLKL activation. In addition, the conjugation of Ub to K219 seemed to influence the 4HB domain flexibility of P-MLKL, which might be an important feature for MLKL polymerisation.
Impact This collaboration helped us to understand the activation mechanism of ubiquitylated MLKL, and resulted in a publication.
Start Year 2020
 
Description Regulation of the pseudokinase MLKL 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Country Australia 
Sector Academic/University 
PI Contribution In collaboration with Prof. James M Murphy we characterised the mechanism of MLKL activation during health and disease. We discovered that ubiquitylation of MLKL at residue K219 contributes to its activation, driving tissue damage and inflammatory responses. We generated point mutants of MLKL, created an knock-in animal and characterised the functional consequence of MLKL-K219R ex vivo and in vivo.
Collaborator Contribution Professor James M Murphy characterised the MLKL-K219R mutant using in vitro assays, such as the thermal shift assays, which established that this mutant folds as wild-type MLKL.
Impact This collaboration established that endogenous MLKL is ubiquitylated during necroptosis, and that ubiquitylation at K219 plays a critical role for the execution of MLKL-dependent cell death. Moreover, we demonstrated that K219 ubiquitylation is required for MLKLdependent pathogenesis in vivo, and contributes to MLKL-mediated viral clearance. Mechanistically, we provided evidence that ubiquitylation of MLKL facilitates the formation of higher order polymers required for membrane rupture and lytic cell death. Therefore, we identified that K219 ubiquitylation regulates the cytotoxic potential of MLKL.
Start Year 2020
 
Description Ubiquitin-mediated regulation of caspases 
Organisation University of Massachusetts
Department Molecular, Cell and Cancer Biology (MCCB)
Country United States 
Sector Academic/University 
PI Contribution we generated genetically engineered flies expressing tagged versions of Ubiquitin
Collaborator Contribution Our collaborators received our material and investigated how ubiquitylation of caspases regulates their activity.
Impact PMID: 28207763
Start Year 2015
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation Canadian Institutes of Health Research
Department Institute of Cancer Research
Country Canada 
Sector Public 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation Max Planck Society
Department Max Planck Institute for Terrestrial Microbiology
Country Germany 
Sector Academic/University 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description mass spectrometric identification of components of the TNF-receptor signalling complex 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution We have provided purified samples for mass spectrometric analysis.
Collaborator Contribution Our collaborators have analysed our samples using state-of-the art mass spectrometry. Moreover, using Saint Analysis, this has helped us to identify putative binding partners of components of the TNF signalling complex.
Impact The manuscript that the reports these mass spectrometric findings has been accepted for publication Cell Reports.
Start Year 2014
 
Description Awareness event at the House of Lords 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Talk and discussion groups brought together clinicians, fundamental biologists, researchers from pharmaceutical companies, policymakers and end users to discuss how breast cancer research, and novel treatment regiments, could be improved via a change in clinical practice for patient benefit.

Apart from a positive feedback, there were no recordable impacts.
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019,2020
 
Description Cell Death Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact The activity was to educate medical student and Vets about the different forms of cell deaths.
Year(s) Of Engagement Activity 2020,2021
 
Description Chair of Site Visit review committee of the Nantes Cancer Research Centre 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was acting chair of the Science Review Panel for the Nantes Cancer Centre.
Year(s) Of Engagement Activity 2021
 
Description Fundraising event at the Breast Cancer Now Research Centre 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A series of fund raising events took place throughout these years, each event saw 20 -30 people taking part. Ongoing research was communicated orally, which led to lively exchanges of questions and answers

Fundraisers liked the event, which prompted many repeat venues in which the current project was explained in great details
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016,2017,2018,2019,2020,2023
 
Description Lecture on Cell Death 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was invited to give lectures on cell death. Below is a list of invited lectures of the last 5 years:
2023 University of Freiburg, Germany
2023 Life Science 2, University of Zürich, Switzerland
2023 University of Innsbruck, Austria
2023 University College London, UK
2022 Novartis Institutes for Biomedical Research, Basel, Switzerland
2022 Gordon Research Conference on Cell Death and Inflammation, Les Diablerets, Switzerland
2022 EMBO Conference on Ubiquitin and Ubiquitin-like proteins, Cavtat, Croatia
2022 EMBO workshop on phagocytosis, Ghent, Belgium
2022 EMBO workshop on Cell Death and Inflammation, Crete, Greece
2021 2nd Fusion Conference on Ubiquitin signalling in Health and Disease, virtual
2021 18th International TNF conference, Les Diablerets, Switzerland
2021 Virtually Dead Meeting, virtual
2020 Symposium 2020 - For2036, Obergurgl, Austria
2019 EMBO Workshop on Cell Death and Immunity, Crete, Greece
2019 Cold Spring Harbor Meeting on Cell Death, New York, USA
2019 Beatson International Cancer Conference, Glasgow, UK
2019 17th International TNF conference, Monterey, California, USA
2019 Cancer, Inflammation and Immunity Conference, Cambridge, UK
2019 EMBO Workshop on Pathogens and Signalling, Oxford, UK
2018 Swiss Apoptosis Meeting, Bern, Switzerland
2018 Gordon Research Conference on Cell Death, Maine, USA
2018 CDD Cancer and Cell Death conference, Cambridge, UK
2018 Technical University, Copenhagen, Denmark
2018 Fusion Conference on Ubiquitin signalling in health and disease, Bahamas
2018 Keystone Symposium on Cell Death, Inflammation and Adaptation to Tissue Stress, Breckenridge, Colorado, USA
2017 The Banbury Centre Cold Spring Harbor Laboratory Meeting on Regulated Necrosis - Pathways and Mechanisms, Banbury, USA
2017 Keystone Symposium on Cell Death and Immunity, Dublin, Ireland
2017 16th International TNF Conference, Singapore
2017 University of Milan, Italy
2017 University of Bath, UK
2017 EMBO workshop on Cell Death and Inflammation, Obergurgl, Austria
2017 Fusion Conference on Cell Death, Stress and Metabolism, Cancun, Mexico
2016 NCRI Cancer Conference, Liverpool, UK
2016 Gordon Research Conference on Cell Death, Spain
2016 'At the Cross-road' symposium, University of Cologne, Germany
2016 Cell Death and Inflammation, Crete, Greece
2016 European Cell Death Workshop, Rome, Italy
2016 NCRI cancer conference, Liverpool, UK
2016 Champalimaud Centre for the unknown, Lisbon, Portugal
2016 University of Cologne, Germany
2016 Cancer Course, University of Birmingham, UK
2016 University of Manchester, UK
2016 Max F. Perutz Laboratories (MFPL) Vienna, Austria
2016 Gulbenkian Institute, Lisbon, Portugal
2016 Gordon Research Conference on Ubiquitin, NF-kB and MAPK signalling, Whistler, Canada
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019,2020,2021,2022,2023