Chemical tools for studying conventional and non-conventional modes of ubiquitylation
Lead Research Organisation:
University of Dundee
Department Name: UNLISTED
Abstract
Our health and well-being are dependent on the correct functioning of the cells in our body. It is therefore imperative that we have a thorough understanding of the biochemical processes that take place within them. It is these processes that become faulty in certain diseases therefore being armed with the knowledge of their intricacies will place in a position where we can rationally develop new therapies that alleviate their symptoms of or even cure them.
A cellular regulatory mechanism that affects all processes is known as protein ubiquitylation. Protein ubiquitylation involves the attachment of a small protein called ubiquitin to other proteins. This step is carried out by enzymes known as E3. Challenges with fully understanding protein ubiquitylation arise from a lack of research technologies that enable the activities of even a single E3 in a cell to be measured. Additionally, we now have strong knowledge about which proteins are tagged with ubiquitin but our ability to identify the E3 responsible is extremely difficult and new tools are urgently needed.
We have recently developed some exciting and powerful technology that allows not only a single E3s activity in a cell to be measured, but the activity of tens of E3s to be measured simultaneously. This technology will revolutionise our ability to understand the roles of E3s and protein ubiquitylation in both normal and diseased cells.
A cellular regulatory mechanism that affects all processes is known as protein ubiquitylation. Protein ubiquitylation involves the attachment of a small protein called ubiquitin to other proteins. This step is carried out by enzymes known as E3. Challenges with fully understanding protein ubiquitylation arise from a lack of research technologies that enable the activities of even a single E3 in a cell to be measured. Additionally, we now have strong knowledge about which proteins are tagged with ubiquitin but our ability to identify the E3 responsible is extremely difficult and new tools are urgently needed.
We have recently developed some exciting and powerful technology that allows not only a single E3s activity in a cell to be measured, but the activity of tens of E3s to be measured simultaneously. This technology will revolutionise our ability to understand the roles of E3s and protein ubiquitylation in both normal and diseased cells.
Technical Summary
E3 ligases (E3s) mediate the transfer of ubiquitin to specific substrates and play important roles in both health and disease. The cellular regulation and function of the vast majority of the >600 E3s remain poorly understood. The activity of E3s can be regulated by posttranslational mechanisms, including phosphorylation, and aberrant E3 activity is emerging as a hallmark of certain diseases. Development of new tools that allow the unbiased assessment of the activity state of E3s in normal and diseased cells and tissues would provide field-changing insight into the cellular roles of E3s, and may uncover novel therapeutic targets. We have developed new chemical biological tools which for the first time allow the activity of endogenous HECT and RBR E3s to be determined directly. We have validated these tools against the E3 termed Parkin that is mutated in Parkinson’s disease. Our work demonstrated that these tools can be used to garner valuable insights into the regulatory aspects of a disease-relevant E3 and that they have value as a biomarker platform for assessing disease susceptibility. We have also discovered a novel class of E3 that that was only possible through the application of our tools. E3s were previously believed to be composed of RING, HECT and RBR subtypes. We have shown that there is fourth type of E3 which we term RCR. The apparent sole member of the RCR class is a neuron-associated E3 known as MYCBP2. MYCBP2 is a positive regulator of an axon degeneration pathway and hence may be a valuable therapeutic target for addressing a range of neurological disorders. Remarkably, MYCBP2 has a unique mechanism and transfers the ubiquitin molecule onto non-lysine residues within protein substrates. Future work in our lab will involve the functional studies on MYCBP2 and non-lysine ubiquitylation in axon degeneration. We will also be applying our tools to try and understand which other poorly characterised E3s might regulate disease-relevant signalling pathways.
People |
ORCID iD |
Satpal Virdee (Principal Investigator) |
Publications
Barnsby-Greer L
(2023)
An Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates
Barnsby-Greer L
(2024)
UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4.
in Nature structural & molecular biology
Bustos F
(2022)
Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome.
in Life science alliance
De Cesare V
(2021)
Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases.
in Proceedings of the National Academy of Sciences of the United States of America
Kwasna D
(2018)
Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability
in Molecular Cell
Mabbitt PD
(2020)
Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.
in Nature chemical biology
Mathur S
(2020)
Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases.
in Cell chemical biology
Pao KC
(2018)
Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity.
in Nature
Spear LA
(2022)
Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering.
in Journal of molecular biology
Squair DR
(2022)
A new dawn beyond lysine ubiquitination.
in Nature chemical biology
Virdee S
(2022)
An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration.
in Neural regeneration research
Virdee S
(2022)
Beyond Lysine Ubiquitination
in The FASEB Journal
Related Projects
Project Reference | Relationship | Related To | Start | End | Award Value |
---|---|---|---|---|---|
MC_UU_00018/1 | 01/04/2018 | 31/03/2024 | £4,394,000 | ||
MC_UU_00018/2 | Transfer | MC_UU_00018/1 | 01/04/2018 | 31/03/2024 | £2,542,000 |
MC_UU_00018/3 | Transfer | MC_UU_00018/2 | 01/04/2018 | 31/03/2024 | £3,121,000 |
MC_UU_00018/4 | Transfer | MC_UU_00018/3 | 01/04/2018 | 31/03/2024 | £2,751,000 |
MC_UU_00018/5 | Transfer | MC_UU_00018/4 | 01/04/2018 | 31/03/2024 | £3,744,000 |
MC_UU_00018/6 | Transfer | MC_UU_00018/5 | 01/04/2018 | 31/03/2024 | £2,520,000 |
MC_UU_00018/7 | Transfer | MC_UU_00018/6 | 01/04/2018 | 31/03/2024 | £2,557,000 |
MC_UU_00018/8 | Transfer | MC_UU_00018/7 | 01/04/2018 | 31/03/2024 | £2,128,000 |
Title | Model substrates for deubiquitinating enzymes with non-lysine activity |
Description | We have developed model substrates that are ubiquitinated on threonine and serine residues. These are valuable tools for discovering and studying deubiquitinating enzymes (DUBs) with non-lysine activity. We have also made fluorescent variants allowing quantitative assessment of DUB activity. The substrates are prepared by exploiting the threonine and serine ubiquitination activity of MYCBP2 we discovered. A patent has been granted relating to this procedure. These tools slowed us to discover a class of DUBs with no-lysine activity. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Mission Therapeutics, a biotech developing DUB inhibitors, have expressed interest in these reagents. |
Description | Collaboration with Tim Clausen (Institute of Molecular Pathology, Vienna) |
Organisation | Research Institute of Molecular Pathology (IMP) |
Country | Austria |
Sector | Academic/University |
PI Contribution | We are collaborating on the study of the Moyamoya Disease associated ubiquitin E3 ligase RNF213. The Clausen lab are carrying out structural work and we are providing chemical probe technology to assist them. We are carrying our cellular and biochemical assays of E3 activity. |
Collaborator Contribution | Partner has been very successful in obtaining the structure of a chemically stabilised ternary E3 ligase complex we prepared. |
Impact | No outcomes yet. Multidisciplinary: structural biology, chemistry, cell biology, biochemistry, proteomics |
Start Year | 2020 |
Description | Collaboration with bioinformatics lab in Cologne |
Organisation | University of Cologne |
Department | Institute for Genetics |
Country | Germany |
Sector | Academic/University |
PI Contribution | The project is our own and the collaboration is merely to provide a minor but expert bioinformatics complement. |
Collaborator Contribution | Bioinformatic analyses, contribution of figures for publication. |
Impact | None yet |
Start Year | 2017 |
Description | Research collaboration with team at the MRC Centre for Reproductive Health |
Organisation | University of Edinburgh |
Department | MRC Centre for Reproductive Health |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We are working with a team at the MRC Centre for Reproductive Health. Using our unique chemical biology technology we have helped the team prepare a homogenously dimethylated protein RNA binding protein. |
Collaborator Contribution | The collaborators have the expertise and assays required for studying the effects of dimethylation on the protein under investigation. |
Impact | Preliminary data showing that we can make dimethylated PABP1 contributed to the Gray and Brook labs being successfully awarded a BBSRC grant in March 2017. The project is multidisciplinary: biochemical biophysical chemical biology structural biology |
Start Year | 2016 |
Title | LIGASE SCREENING ASSAY |
Description | The present invention relates to a method for identifying a MYCBP2 modulator. Suitable modulators are identified by modulation of MYCBP2 ubiquitin E3 ligase activity via covalent modification of either of two catalytic cysteines (C4520 and C4572) or by impeding the motion of a newly presented dynamic, so-called, mediator loop region where C4520 resides. The present invention also relates to the use of hydroxy group- containing small molecules and peptides as proxy substrates for measuring MYCBP2 ligase activity and their use in the method of identifying modulators. |
IP Reference | WO2019175609 |
Protection | Patent application published |
Year Protection Granted | 2019 |
Licensed | No |
Impact | The filling of this patent has facilitated venture capital investment into a spin out company. Company has been incorporated and investors are progressing through diligence before final commitment. |
Company Name | OUTRUN THERAPEUTICS LIMITED |
Description | `I have founded a biotech company that will develop next generation therapeutics that target the ubiquitin system. Two high profile venture capital investors have invested a combined £4,000,000 |
Year Established | 2019 |
Impact | Reagents produced by the MRC Reagents and Services facility have been provided to the company thereby generating notable revenue. |
Description | Charity Cycle |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | June 2021: Mathieu Soetens (postdoc in my lab) cycled 666 miles from the lab to the Northernmost point of the UK, Hermaness point in Unst (Shetlands), emphasising bird watching, local travelling, low carbon tourism and raising awareness of PArkinsons research being carried out in Dundee. Three conferences were given to local naturalism societies: Angus and Dundee birding club, Dundee Naturalist society, and one to the supporters on Zoom. Total £3124 donated https://www.youtube.com/watch?v=Y_3dtO0aVRc |
Year(s) Of Engagement Activity | 2021 |
Description | Charity cycle ride 2 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | June 2022: Mathieu Soetens (postdoc in my lab) cycled 365 miles from the lab to(wards) UK's westernmost point, St Kilda (Outer Hebrides), emphasising bird watching, local travelling, and low carbon tourism. Five conferences were given to local naturalism societies: Angus and Dundee birding club, Dundee Naturalist society, Fife birding club, RSPB local group, and Caloosa bird club (Florida USA). Total £1333 donated for Parkinsons research at Dundee. https://www.youtube.com/watch?v=PVDlksSfwRY |
Year(s) Of Engagement Activity | 2022 |
Description | Discovery Days presentation |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Discovery Days presentation sharing the importance of my labs research with the general public. This was a university initiative to concide with my promotion to chair of chemical biology. |
Year(s) Of Engagement Activity | 2021 |
Description | Front page headline of The Herald "Scientists in breakthrough over Parkinson's and MS" |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Front page print headline article for The Herald 4th August 2020 and https://www.heraldscotland.com/news/18625807.enzyme-breakthrough-dundee-scientists-pave-way-treatments-parkinsons-ms-mnd/ |
Year(s) Of Engagement Activity | 2020 |
Description | Immersive Cell Sci-Art Workshop & Resources Development |
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 | Immersive Cell Sci-Art Workshop & Resources Development: online co-production workshop with External Artist & Baldragon Academy Science & Art Dept Heads & teachers (finalise & schedule workshops & live Q&A sessions) |
Year(s) Of Engagement Activity | 2021 |
Description | Lab member participation in Soapbox Science event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Lucy Barnsby in my lab participated in a Soapbox Science event which is a novel public outreach platform for promoting women and non-binary scientists and the science they do. This involved engaging members of the public and raising awareness locally. |
Year(s) Of Engagement Activity | 2022 |
Description | UoD Press Release: - Researchers uncover new neurodegeneration drug target |
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 | Public/other audiences |
Results and Impact | Press release to concide with our work unrevelling the ubiquitin transfer mechanism of the neuronal E3 ligase MYCBP2, and, our findings that inactivation of its distinct enzyme activity with point mutation confered axon protein in an animal model. |
Year(s) Of Engagement Activity | 2020 |
Description | UoD Press Release: Discovery raises possibility of treating neurological disorders |
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 | Media (as a channel to the public) |
Results and Impact | This was a University of Dundee press release to highlight our discovery of a novel ubiquitin enzyme module in the protein MYCBP2. MYCBP2 mutations are associated with a neurological disorder but its loss also confers potent axon protein, so is a potential therapeutic target. |
Year(s) Of Engagement Activity | 2018 |