Specificity in the ubiquitin system

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

The cells in our body need to respond to signals of many kinds, and adapt appropriately to ever changing environments. This is achieved to some extent by direct modification of cellular proteins. Closer examination reveals that virtually every cellular protein is regulated by modifications, which regulate its the life-time, activity, interactions with other proteins, and its localisation within the cell.
Our programme studies one such modification, named ubiquitination. This process has in recent years emerged as a central mechanism to relay signals in response to signals, in particular to a wide variety of stress such as DNA damage, or infection. Importantly, a large number of human diseases, from cancer to infection and inflammation, emerge when the ubiquitin system is disturbed.
At this stage of research, much mechanistic detail of the process itself has to be uncovered. We are planning to gain insight into the molecular mechanism behind ubiquitination, by using biochemical and structural biology methods to understand this process at the atomic level. A number of the studied proteins are direct regulators of disease, and hence atomic structure information might aid in the development of novel drugs.

Technical Summary

Protein ubiquitination emerges as a key regulatory posttranslational modification, which is utilised both in degradative processes regulating the half-life of target proteins, as well as in signal transduction where it serves non-degradative functions.
During ubiquitination, the small protein ubiquitin is attached covalently to target proteins. Ubiquitin can be ubiquitinated itself, generating ubiquitin polymers. The versatility of the ubiquitin system is achieved by the ability of ubiquitin to form at least eight types of polymers, and proteomic analysis has shown that all different ubiquitin linkages are present in cells. To date, functional and structural data is available for only three types of polyubiquitin chains. These are the well-studied Lys48-linked polyubiquitin chain, that targets modified proteins for proteasomal degradation, as well as Lys63-linked polyubiquitin that are implicated in cell signalling processes. Independent functions for the remaining chain types are emerging.
We aim to generate and study the remaining ubiquitin chain types, and understand their roles in the cell. For this, we need to study ubiquitin chain assembly systems. This work has led us to determine the structure of the E3 ubiquitin ligase Parkin, explaining many mutations of individuals with early-onset Parkinson’s disease, shedding new light on the role of ubiquitin in neurodegenerative disorders.
Moreover, we have identified E2 and E3 enzymes to generate Lys6- and Lys11-linked polyubiquitin chains. Availability of novel chain linkages is important to understand the molecular basis for specificity in the ubiquitin system, especially specificity of deubiquitinases and ubiquitin binding domains.
Deubiquitinating enzymes hydrolyse ubiquitin chains, and are therefore key regulators of the ubiquitin system. Currently, limited information is available as how this important class of enzymes is regulated. We perform structural and functional studies on deubiquitinases of the Ovarian Tumour family, to understand how they affect cell signalling pathways at the molecular level.

Publications

10 25 50
 
Description DFG Postdoctoral Research Fellowship
Amount £50,000 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 07/2011 
End 06/2012
 
Description DUBs Alliance
Amount £392,961 (GBP)
Organisation Cancer Research Technology (CRT) 
Sector Private
Country United Kingdom
Start 10/2013 
End 06/2018
 
Description EMBO Long Term Fellowship - Kulathu
Amount £30,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 04/2012 
End 01/2013
 
Description EMBO Long Term Fellowship - Pruneda
Amount £52,000 (GBP)
Funding ID EMBO ALTF 589-2013 
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 08/2013 
End 07/2015
 
Description EMBO Young Investigator Programme
Amount £8,500 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 01/2011 
End 12/2013
 
Description ERC Consolidator Grant
Amount € 1,990,125 (EUR)
Funding ID ERC-2016-CoG-724804 - UbiArchitect 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 10/2017 
End 10/2018
 
Description ERC Starting Grant
Amount € 1,480,285 (EUR)
Funding ID ERC-20120-StG-309756 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 10/2012 
End 09/2017
 
Description Erasmus Plus - Bompadre
Amount € 3,000 (EUR)
Organisation erasmus + 
Sector Public
Country United Kingdom
Start 06/2015 
End 08/2015
 
Description Fellowship - R Damgaard
Amount kr 800,000 (DKK)
Organisation Lundbeck Foundation 
Sector Charity/Non Profit
Country Denmark
Start 06/2017 
End 05/2019
 
Description Fellowship - Shibata
Amount ¥4,500,000 (JPY)
Organisation Naito Foundation 
Sector Charity/Non Profit
Country Japan
Start 04/2017 
End 04/2018
 
Description Lister Prize
Amount £200,000 (GBP)
Funding ID n/a 
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description Lister Studentship
Amount £2,000 (GBP)
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2017 
End 09/2017
 
Description Marie Curie Initial Training Network
Amount £179,619 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 10/2011 
End 09/2015
 
Description Marie Curie Long Term Fellowship - Kulathu
Amount € 180,103 (EUR)
Funding ID FP7-People-2009-IEF-253389 
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 04/2010 
End 03/2012
 
Description Marie Sklodowska-Curie Long Term Fellowship Damgaard
Amount € 183,455 (EUR)
Funding ID H2020-MSCA-IF-2014-654019 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 04/2015 
End 03/2017
 
Description Michael J Fox Foundation
Amount $150,000 (USD)
Funding ID PO#47254 
Organisation FORMA Therapeutics 
Sector Private
Country United States
Start 05/2017 
End 10/2018
 
Description PhD Scholarship - J Usher
Amount £130,000 (GBP)
Organisation Higher Education Funding Council for England 
Department Gates Cambridge Scholarship
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description SNSF Studentship - Michel
Amount SFr. 55,000 (CHF)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 02/2017 
End 03/2018
 
Title DUB assay 
Description Fluorescence based assay for deubiquitinase enzymes. Patented UK application no. 1009941.4. Licensed by Boston Biochem. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact New assay to generate fluorescence output from DUB activity. Goes beyond current assays as diubiquitin is the substrate. useful for DUB drug discovery. 
 
Title K11-linked polyubiquitin 
Description This reagent is a polymer of ubiquitin molecules linked via an isopeptide bond to Lys11 (K11). This was generated by protein engineering of an K11-assembly enzyme. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact K11-linked ubiquitin chains are a novel signal in cells involved in the cell cycle. We have been the first to generate this material in large quantities. This will be useful to identify new cell cycle regulators which may be involved in cancer. 
 
Title K6 and K29-linked diubiquitin 
Description ubiquitin molecules linked via K6 (1) or K29 (2) into a diubiquitin. this was generated by chemical synthesis described in Virdee et al, Nat Chem Biol 2009. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact K6- and K29-linked polyubiquitin exist in cells but their function is unknown. With this material we have been able to identify a specific enzyme that cleaves the K29-linked ubiquitin chains specifically. These reagents have not been available before. 
 
Title K6-linked polyUb (enzymatic) 
Description We have developed an enzymatic assembly method for Lys6-linked polyubiquitin. This is described in Hospenthal et al, NSMB 2013. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact This enabled research on the unstudied posttranslational modification of Lys6-linked polyubiquitin. 
 
Title OTU DUBs 
Description We generated a suite of linkage-specific OTU DUBs that are sued in Ub chain restriction analysis. These enzymes have been evaluated by a leading Ubiquitin reagent supplier for a fee. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact many collaborations started 
 
Title ubiquitin chain restriction analysis (UbiCRest) 
Description We have discovered a class of human deubiquitinating enzymes that can be used analytically to study the type and topology of ubiquitin chains on a substrate. The discovered deubiquitinases have different properties and specificities. This will allow ubiquitin chain sequencing, which we believe may become an important future tool in biochemical studies. UPDATE: Boston Biochem has licensed this technology, and is now selling it. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact This method will allow quick and easy assessment of the Ub chain types present on a protein. this is currently a challenging but very important problem. 
URL http://www.bostonbiochem.com/ubicrest-technology-exclusively-licensed-boston-biochem-laboratory-prof...
 
Description Affimer collaboration 
Organisation Avacta Group
Country United Kingdom 
Sector Private 
PI Contribution We have access to new research reagents through a collaboration with Avacta LifeSciences, which has resulted in generating new insights into the ubiquitin system. The collaboration is restricted to ubiquitin and ubiquitin chain specific reagents, which we receive from Avcacta and characterise structurally, biochemically and in cell biologically. A first paper is planned on these new tools. Once we help improve reagents, MRC LMB will benefit through royalty payments.
Collaborator Contribution Avacta LifeSciences has a novel technology to evolve a small proteins scaffold to bind protein targets with very high affinity. They have generated reagents that we were seeking, and upon testing, we engaged in a fully-fleshed collaboration.
Impact A first paper is in preparation, for submission in summer 2016.
Start Year 2015
 
Description Collaboration on the role of linear ubiquitin chains in NFkB signalling 
Organisation Imperial College London
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided reagents in form of ubiquitin chains and expertise
Collaborator Contribution We collaborated to understand the role of linear ubiquitin chains in TNF signallingWe collaborated on the role of linear ubiquitin chain binding to NEMO
Impact The results were pubished in 2 high-profile papers: a) Rahighi et al, Cell 2009 (collaboration Frankfurt) b) Haas et al, Mol Cell (Collaboration Imperial)
Start Year 2008
 
Description Collaboration on the role of linear ubiquitin chains in NFkB signalling 
Organisation University of Frankfurt
Department Institute for Biochemistry II
Country Germany 
Sector Academic/University 
PI Contribution We provided reagents in form of ubiquitin chains and expertise
Collaborator Contribution We collaborated to understand the role of linear ubiquitin chains in TNF signallingWe collaborated on the role of linear ubiquitin chain binding to NEMO
Impact The results were pubished in 2 high-profile papers: a) Rahighi et al, Cell 2009 (collaboration Frankfurt) b) Haas et al, Mol Cell (Collaboration Imperial)
Start Year 2008
 
Description DUB Alliance 
Organisation Cancer Research Technology (CRT)
Country United Kingdom 
Sector Private 
PI Contribution This Alliance, led by CRT, has set as an aim to develop small molecule inhibitors for deubiquitinases. The Alliance will fund a scientist in my lab to contribute to the effort.
Collaborator Contribution target identification, lead discovery, SAR, crystallography, in vivo validation, phase 1 clinical trials
Impact no non-confidential research outputs as of yet.
Start Year 2013
 
Description DUB Alliance 
Organisation FORMA Therapeutics
Country United States 
Sector Private 
PI Contribution This Alliance, led by CRT, has set as an aim to develop small molecule inhibitors for deubiquitinases. The Alliance will fund a scientist in my lab to contribute to the effort.
Collaborator Contribution target identification, lead discovery, SAR, crystallography, in vivo validation, phase 1 clinical trials
Impact no non-confidential research outputs as of yet.
Start Year 2013
 
Description DUB Alliance 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution This Alliance, led by CRT, has set as an aim to develop small molecule inhibitors for deubiquitinases. The Alliance will fund a scientist in my lab to contribute to the effort.
Collaborator Contribution target identification, lead discovery, SAR, crystallography, in vivo validation, phase 1 clinical trials
Impact no non-confidential research outputs as of yet.
Start Year 2013
 
Description DUB Alliance 
Organisation University of Liverpool
Department School of Life Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution This Alliance, led by CRT, has set as an aim to develop small molecule inhibitors for deubiquitinases. The Alliance will fund a scientist in my lab to contribute to the effort.
Collaborator Contribution target identification, lead discovery, SAR, crystallography, in vivo validation, phase 1 clinical trials
Impact no non-confidential research outputs as of yet.
Start Year 2013
 
Description DUB Alliance 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution This Alliance, led by CRT, has set as an aim to develop small molecule inhibitors for deubiquitinases. The Alliance will fund a scientist in my lab to contribute to the effort.
Collaborator Contribution target identification, lead discovery, SAR, crystallography, in vivo validation, phase 1 clinical trials
Impact no non-confidential research outputs as of yet.
Start Year 2013
 
Description Gyrd-Hansen lab 
Organisation Ludwig Institute for Cancer Research
Department Oxford Branch
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We collaborated with the labs of Mads Gyrd-Hansen on the cellular role of OTULIN, a new deubiquitinase we discovered in humans. The Gyrd-Hansen lab recently relocated to Oxford, from Copenhagen.
Collaborator Contribution - technical expertise and analysis - know-how - generating large amounts of data for publications.
Impact Keusekotten et al, Cell 2013 Fiil et al, Mol Cell 2013 Elliott et al, submitted
Start Year 2012
 
Description Hofmann lab 
Organisation University of Cologne
Department Institute for Genetics
Country Germany 
Sector Academic/University 
PI Contribution We collaborate with the bioinformatics group of Prof Kay Hofmann, who has provided great insights and predictions on the DUBs in humans and bacterial species.
Collaborator Contribution bioinformatical predictions of deubiquitinases and other proteins int he ubiquitin system
Impact published papers: Ye et al, Mol BioSyst 2009 Kulathu et al, NSMB 2009 Keusekotten et al, Cell 2013
Start Year 2008
 
Description Klenerman / Jackson labs 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have an ongoing collaboration applying single molecule methods (at the Chemistry Dept) to the ubiquitin system (our area and contribution).
Collaborator Contribution The Klenerman and Jackson labs have excellent single molecule expertise and apparatus which we use to gain insight into our biological systems.
Impact published paper: Ye et al, Nature 2012
Start Year 2011
 
Description bacterial effectors 
Organisation Duke University
Country United States 
Sector Academic/University 
PI Contribution We work on bacterial effector mechanisms and structure
Collaborator Contribution the partners provide cell biological insights (U Liv) and microbiology insights (Duke)
Impact paper in preparation
Start Year 2016
 
Description bacterial effectors 
Organisation University of Liverpool
Department Department of Geography and Planning
Country United Kingdom 
Sector Academic/University 
PI Contribution We work on bacterial effector mechanisms and structure
Collaborator Contribution the partners provide cell biological insights (U Liv) and microbiology insights (Duke)
Impact paper in preparation
Start Year 2016
 
Description ubiquitin chemical biology 
Organisation Netherlands Cancer Institute (NKI)
Department Chemical Biology Laboratory
Country Netherlands 
Sector Public 
PI Contribution We collaborate with the team of Dr Huib Ovaa at NKI Amsterdam to analyse linkage specificity of deubiquitinases. using the tools provided by the Ovaa group, we were able to discover linkage-specificity in a family of human deubiquitinases. Furthermore, some different tools helped us to determine crystal structures of previously inaccessible protein complexes
Collaborator Contribution Huib Ovaa lab assembles Ub chains as substrates for our assays, and ubiquitin suicide probes as chemical modifiers for deubiquitinases.
Impact several papers published in 2013. Mevissen et al, Cell 2013 Ekkebus et al, JACS 2013 1 paper published: Licchesi et al, NSMB 2012.
Start Year 2010
 
Description Dr P. Marco-Casanova: Cambridge Science Festival / MRC LMB Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact These activities were about teaching kids to use microscopes and doing preparations of their own cheek cells to look at them under the microscope. Dr Paola Marco-Casanova participated in the events that were organised by Dr Rodrigo-Brenni from MRC LMB.


- generate awareness of cell biology and science in young pupils
- represent MRC scientists to the general public
Year(s) Of Engagement Activity 2013
 
Description Interview on our research on ubiquitin in disease. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We were interviewed by a scientific writer writing for the American Society of Biochemistry and Molecular Biology (ASBMB) to contribute to a piece about our recent research on OTULIN and linear ubiquitin chains in human disease. In 2016, we published a Cell paper in which we discovered a new human disease. While this was not covered by MRC press offices, it is nice that the American colleagues picked this up and gave it the deserved platform.
Year(s) Of Engagement Activity 2016
URL http://www.asbmb.org/asbmbtoday/201701/Feature/ChainOfEvents/
 
Description LMB Open Day June 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Members of the research group organised all-age activities under the theme "Ubiquitin everywhere". Activities included games, demonstrations and talking to the public about the role of ubiquitin. Children and adults engaged with the science and feedback was very positive.
Year(s) Of Engagement Activity 2017
 
Description Outreach events in Belfast - Rune Damgaard 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Rune Damgaard visited Belfast to present his work in basic biological research to a school audience, to trigger interest of pupils in a career in science.
Year(s) Of Engagement Activity 2017
 
Description Talk to Trade & Investment Officers from British Embassies 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was chosen to present LMB to Trade & Investment Officers from British Embassies and discussed with them our activities - not only research but also eg drug discovery. Afterwards, I had plenty of interest from officers from the US and Germany. Officers realised the power of our location in Cambridge as ideal for research and technology.

We represented MRC and LMB and altered them of our work.
Year(s) Of Engagement Activity 2014
 
Description Workshop - Understanding Protein-Ubiquitin Interactions 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Results

Impacts
Year(s) Of Engagement Activity 2014
 
Description visit to Parliament - Rune Damgaard / Marie Curie Fellowship event 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Rune Damgaard participated in his role as an Marie Curie Fellow in an event at the British Parliament in London, to speak to MPs and policymakers about the benefit of European Funding, freedom of movement and mobility, for his own career and UK science.
Rune was one of two selected fellows from Marie Curie Fellowship, and one of 20 invited experts.
Year(s) Of Engagement Activity 2017