Novel proteases with deubiquitylation activity: examination of function in T lymphocytes

Lead Research Organisation: University of Oxford
Department Name: Clinical Medicine

Abstract

Work over the last 25 years has established the importance of regulating protein ubiquitylation in a wide range of cellular functions including cell cycle control, transcriptional regulation, and cell signaling. These processes are disturbed in many human diseases such as cancer, neurodegeneration and immunological disorders. So far, most analyses have focused on mechanisms controlling ubiquitin ligation. However, the recognition of extensive classes of deubiquitylating enzymes (DUBs) strongly suggests a regulatory (as opposed to purely recycling) role for DUBs. Thus it is anticipated that the planned analysis of one class of these enzymes would shed light on important and medically relevant aspects of cellular physiology. A novel class of these enzymes containing an ovarian tumor domain (OTU), which encodes for a putative ubiquitin-specific cysteine protease, has been recently identified. This domain is conserved throughout evolution. Obubain 1 (OTUB1), a member of this protein family, does not contribute to the deubiquitylation of the bulk of cytosolic proteins. Preliminary evidence suggests that OTUB1 plays a specific role in the regulation of antigen responsiveness of T lymphocytes. Insights into this process could have important implications for our understanding of immune-modulation, and may identify OTU containing proteins as novel therapeutic targets.

Technical Summary

Work over the last 25 years has established the importance of regulating protein ubiquitylation in a wide range of cellular functions including cell cycle control, transcriptional regulation, and diverse aspects of cell signaling. These processes are disturbed in many human diseases such as cancer, neurodegeneration and immunological disorders involving abnormal accumulations of proteins in cells. So far, most analyses have focused on mechanisms controlling ubiquitin ligation. However, the recognition of extensive classes of deubiquitylating enzymes (DUBs) strongly suggests a regulatory (as opposed to purely recycling) role for DUBs. Thus it is anticipated that the planned analysis of one class of these enzymes would shed light on important and medically relevant aspects of cellular physiology. A novel class of these enzymes containing an ovarian tumor domain (OTU), which encodes for a putative ubiquitin-specific cysteine protease, has been recently identified. This domain is conserved throughout evolution. Obubain 1 (OTU1), a member of this protein family, does not contribute to the deubiquitylation of the bulk of cytosolic proteins. Preliminary evidence suggests that OTU1 plays a specific role in the regulation of antigen responsiveness of T lymphocytes. Further evaluation of this phenomenon will not only shed light on the role of ubiquitin-linked mechanisms in signaling in general, but also on how these proteins may regulate the T cell signaling cascade in particular. Insights into this process could have important implications for our understanding of immune-modulation, and may identify OTU containing proteins as novel therapeutic targets.

Publications

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Adams CJ (2008) ATM and Chk2 kinase target the p53 cofactor Strap. in EMBO reports

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Alexopoulou Z (2016) Deubiquitinase Usp8 regulates a-synuclein clearance and modifies its toxicity in Lewy body disease. in Proceedings of the National Academy of Sciences of the United States of America

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Batchelar ET (2008) Thioester hydrolysis and C-C bond formation by carboxymethylproline synthase from the crotonase superfamily. in Angewandte Chemie (International ed. in English)

 
Description ARC funding, Proteomic investigation in ankylosing spondylitis (co-PI with Dr. Paul Bowness, University of Oxford)
Amount £192,000 (GBP)
Organisation Versus Arthritis 
Start 03/2008 
End 03/2011
 
Description Accelerator Award
Amount £4,000,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2021
 
Description CRT/CRUK - DUB Alliance
Amount £208,899 (GBP)
Organisation Cancer Research Technology (CRT) 
Sector Private
Country United Kingdom
Start 11/2013 
End 10/2015
 
Description FP 7 PROLIFICA
Amount £35,000 (GBP)
Funding ID 265994 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 12/2012 
End 03/2013
 
Description ISSF Wellcome Trust
Amount £50,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2012 
End 07/2014
 
Description Innovative Target Exploration Network (ITEN) - Pfizer
Amount £550,000 (GBP)
Organisation Pfizer Global R & D 
Sector Private
Country United States
Start 04/2018 
End 03/2021
 
Description Oxford University John Fell Fund (Proteomics Bioinformatics)
Amount £44,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 03/2008 
End 03/2009
 
Description Substrate Peptidomimetic Inhibitors (SPIs) of the COP9 signalosome
Amount £350,000 (GBP)
Funding ID EP/N034295/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 12/2016 
End 11/2020
 
Description T cell based vaccine for HIV
Amount £1,200,000 (GBP)
Funding ID G1001757 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2011 
End 07/2016
 
Title DUBs expression plasmids 
Description We have created a series of mammalian and bacterial expression plasmids for the deubiquitinating enyzmes OTUB1 and OTUB2 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact Experimental results obtained with these expression plasmids were published in the following research article: 18954305 Since then, a number of other scientific groups have requested these reagents 
 
Title Proteomics MS data analysis pipeline 
Description In order to provide a better way to analyse mass spectrometry (MS) data in our laboratory, we have developed a novel easy-to-use proteomics analysis pipeline that is accessible over the web. 
Type Of Material Improvements to research infrastructure 
Year Produced 2009 
Provided To Others? Yes  
Impact Many research groups including our own are using this MS analysis pipeline to perform extensive data mining of large proteomic data sets. Many biological and biomedical research projects will highly benefit from this research tool in the future. A technology note describing this new tool is currently under review. 
 
Title Ubiquitin active site probes 
Description Flexible ubiquitin probes Oxford inventors have designed ubiquitin- and ubiquitin-like active site probes which can be modified, using click-chemistry, to add functional groups such as fluorescent tags. These probes offer numerous advantages for profiling DUBs (deubiquitinating enzymes) in cell extracts, as well as intact cells. This strategy has the advantage of requiring minimal protein modification, affording an easy and generally applicable synthesis route. The click-chemistry feature also has the key advantage of enabling a variety of functional groups to be added, allowing further expansion and diversification of the ubiquitin-based probes. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact This resulted in a number of joint research publications, and is the basis for the collaboration with the DUB alliance (financed by pharma), with the aim to find DUB based inhibitors that can be developed into novel anti-cancer drugs. 
URL http://www.isis-innovation.com/licensing/8918.html
 
Description Cell biology of deubiquitinating enzymes 
Organisation Utrecht University
Country Netherlands 
Sector Academic/University 
PI Contribution The role of my group is to perform the biochemical experiments that describe the enyzmatic properties of the DUBs under study. Furthermore, my group is performing the proteomics and mass spectrometry related work to study the specificity of ubiquitination, the type of ubiquitin chains and mapping the sites of ubiquitination of DUB substrates.
Collaborator Contribution The collaboration with Dr. Madelon Maurice's group at the University of Utrecht (NL) is focussed on the cell biology of deubiquitinating enzymes (DUBs). We have previously described proteome changes upon the regulation of USP7/HAUSP expression, a DUB involved in cancer biology. Current experimental efforts are directed towards determining a role for the cylindromatosis suppressor gene CYLD and wnt signalling.
Impact Publication: 17927229 We also have a manuscript describing a role of the deubiquitinating enyzme CYLD in wnt signalling that is currently under review.
 
Description DUB Alliance - CRT/Forma/Celgene 
Organisation Cancer Research Technology (CRT)
Country United Kingdom 
Sector Private 
PI Contribution Development of DUB inhibitors for cancer treatment Alliance between my lab and Cancer Research Technologies (CRT), Forma Therapeutics and Celgene
Collaborator Contribution Development of small molecule DUB inhibitors, Ubiquitin research tools
Impact Novel DUB inhibitors (anti-cancer drug candidates) Multidisciplinary approach between HTS, biochemistry, proteomics & cell biology
Start Year 2013
 
Description DUB Alliance - CRT/Forma/Celgene 
Organisation Celgene
Country United States 
Sector Private 
PI Contribution Development of DUB inhibitors for cancer treatment Alliance between my lab and Cancer Research Technologies (CRT), Forma Therapeutics and Celgene
Collaborator Contribution Development of small molecule DUB inhibitors, Ubiquitin research tools
Impact Novel DUB inhibitors (anti-cancer drug candidates) Multidisciplinary approach between HTS, biochemistry, proteomics & cell biology
Start Year 2013
 
Description DUB Alliance - CRT/Forma/Celgene 
Organisation FORMA Therapeutics
Country United States 
Sector Private 
PI Contribution Development of DUB inhibitors for cancer treatment Alliance between my lab and Cancer Research Technologies (CRT), Forma Therapeutics and Celgene
Collaborator Contribution Development of small molecule DUB inhibitors, Ubiquitin research tools
Impact Novel DUB inhibitors (anti-cancer drug candidates) Multidisciplinary approach between HTS, biochemistry, proteomics & cell biology
Start Year 2013
 
Description DUBs in DNA Repair 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Application of mass spectrometry and proteomics to study DUBs involved in DNA double break strand repair mechanisms (DDR).
Collaborator Contribution DDR biochemistry and biology.
Impact Interesting novel mechanisms of DUBs function in the context of DDR. This will provide novel entry points for new drug development to treat cancer.
Start Year 2014
 
Description ITEN Pfizer 
Organisation Pfizer Global R & D
Country United States 
Sector Private 
PI Contribution Pfizer Establishes New Partnering Model for Early-Stage Academic Research
Collaborator Contribution The University of Cambridge and the University of Oxford are the first to participate in the ITEN model in the United Kingdom, and the University of Texas Southwestern (UTSW) is the first to participate in the United States. Pfizer is seeking to selectively include other institutions to be part of the ITEN model.
Impact Collaborative project on deubiquitylating enzymes early target discovery
Start Year 2017
 
Description Molecular mechanisms of oxygen sensing 
Organisation University of Oxford
Department Nuffield Department of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Proteomics and mass spectrometry based analysis of protein modifications controlled by oxygen sensing
Collaborator Contribution Financed two research positions (RA and bioinformatician) Joint publications: PMID: 18936059 PMID: 17573339 PMID: 17339318 PMID: 20583823 PMID: 19574390 PMID: 18972478
Impact Financed two research positions (RA and bioinformatician) Joint publications: PMID: 18936059 PMID: 17573339 PMID: 17339318 PMID: 20583823 PMID: 19574390 PMID: 18972478
 
Description Role of the ubiquitin proteasome system in regulation of transcription 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Scientific collaboration using mass spectrometry and proteomics approaches to assess the role of the ubiquitin proteasome system in the regulation of transcription
Collaborator Contribution Scientific collaboration on the role of the ubiquitin proteasome system in regulation of transcription Publication PMID: 20516216
Impact PMID: 20516216
Start Year 2006
 
Description The ubiquitin-proteasome system in aging 
Organisation Karolinska Institute
Department Department of Neuroscience
Country Sweden 
Sector Academic/University 
PI Contribution Biochemical characterisation of proteasome complexes isolated from young and old rat muscle tissue, proteomics analysis
Collaborator Contribution Swedish postdoctoral fellowship joint publications: PMID: 20940294 PMID: 17503500
Impact Swedish postdoctoral fellowship Joint publications: PMID: 20940294 PMID: 17503500
Start Year 2006
 
Description Ubiquitin processing enzymes in DNA repair 
Organisation University of Oxford
Department Cancer Epidemiology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution My group has contributed with biochemical experiments to isolate ubiquitin processing enzymes that contribute to control DNA repair mechanisms
Collaborator Contribution Scientific collaboration, biochemistry expertise, molecular biology
Impact Publication 19713937
Start Year 2007
 
Title Flexible ubiquitin probes 
Description Rapid, direct detection of deubiquinating enzymes in cell lysates and whole cells with high-throughput and multiplexing possibilities. http://patentscope.wipo.int/search/en/detail.jsf?docId=WO2013144656&recNum=3&docAn=GB2013050848&queryString=PA/%22isis%20innovation%22%20&maxRec=821 Website: http://www.isis-innovation.com/licensing/8918.html 
IP Reference WO2013144656 
Protection Patent application published
Year Protection Granted 2012
Licensed Commercial In Confidence
Impact These ubiquitin-based probes will find utility as research tools for cellular and molecular biology and proteomics and may be particularly useful for screening compounds in order to identify potential inhibitors of deubiquitinating enzymes as novel anti-cancer and anti-viral agents.
 
Title DUB inhibitors for cancer research through the DUB Alliance 
Description DUB inhibitors for cancer research through the DUB Alliance (CRT-Cancer Research Technologies in collaboration with five academic labs, Forma Therapeutics and Celgene 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2016
Development Status Under active development/distribution
Impact DUB inhibitors have not yet been used in man, but the DUB Alliance will be one of the first trying to conduct this for the development of novel anticancer therapeutics. 
 
Company Name UBIq 
Description Development of tools to study the ubiquitin proteasome system 
Year Established 2009 
Impact Novel reagents are being developed and offered to the research community, which long-term potential to be used for the discovery of clinical biomarkers.
 
Description Publication for a wider audience not only restricted to academia 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact General description of novel technological advances in analysing biological samples

The journal in which this article was published is widely read amongst professional in the pharma industry Publication: The Column http://www.nxtbook.com/nxtbooks/advanstaruk/thecolumn1007/index.php
Year(s) Of Engagement Activity 2007
 
Description Race for Life 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Team building exercise
Websites:
http://www.ccmp.ox.ac.uk/public-engagement
http://www.raceforlifesponsorme.org/kessler-group/eurl.axd/289728e11b02e2478608452b80d5ec5e

Raising awareness for sport as a way to increase health
Year(s) Of Engagement Activity 2012
 
Description Science day Junior School 
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 Regional
Primary Audience Schools
Results and Impact A "Science Day" was organized by members of the Kessler group for the Haddenham Community Junior School, Buckinghamshire, year 5 pupils (25th March 2010).

Website:
http://www.ccmp.ox.ac.uk/public-engagement

Enthusiastic response from pupils and teachers proposed to repeat this event in the future.
Year(s) Of Engagement Activity 2010
 
Description Work experience for student 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Organized a day of work experience for sixth form student

Student decided to study medical sciences at the University of Cambridge...
Year(s) Of Engagement Activity 2008