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.
 
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 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 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 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 
 
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 Fibrotic collagen accumulation and immune activation in Drosophila adipocytes 
Organisation University of Science and Technology of China USTC
Department School of Life Sciences
Country China 
Sector Academic/University 
PI Contribution We discovered a putative role of Par-1 in regulating fibrotic collagen accumulation and immune activation in Drosophila adipocytes.
Collaborator Contribution Using reagents that we generated our collaborator will address the role of Par-1 in a fly model of fibrosis. Our collaborator will test whether depletion of Par-1 results in Collagen deposition (Vkg-GFP accumulation) in an NFkB-dependent manner. This will establish whether modulation of of Par-1 causes collagen deposition due to immune activation.
Impact this is ongoing and no outcomes are yet available.
Start Year 2015
 
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 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 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 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 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
 
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 2006,2007,2008,2009,2010