Translating the ubiquitin code in mitotic cells
Lead Research Organisation:
University of Cambridge
Abstract
Regulated gene expression ensures that cells make the correct selection of genetically encoded protein components required for their function. However, cells also require a method to get rid of proteins once they are no longer required, or if they are faulty. Cellular 'digestion' of unwanted proteins is called proteolysis, and occurs very rapidly inside subcellular machines called proteasomes. Proteins are directed into proteasomes by specific tagging with multiple copies of a small ubiquitous protein known as 'ubiquitin', which forms chains that can be recognized by receptors on the lid of the proteasome. Ubiquitin tags also mediate other functions unrelated to proteolysis, and our growing knowledge of how different types of ubiquitin chain direct different outcomes has given rise to the concept of a 'ubiquitin code'.
A large fraction of the human genome encodes components of the Ubiquitin-Proteasome System (UPS), because almost all cellular processes require ubiquitin-mediated control. For example in regulation of cell division (mitosis) precise destruction of several key components, at exactly the right time and place, drives the whole process. The complexity of the UPS means that ubiquitin chains come in many varieties, and some are better than others at directing targeted proteins to the proteasome. In other words, the proteolysis of different proteins happens at different rates, depending on what type of ubiquitin chain they carry.
We still don't know very much about this part of the ubiquitin code, and how it is translated into proteolysis.
This proposal focuses on proteolysis of two key regulators of mitosis, called the Aurora kinases (A and B). Both are targeted by the same UPS pathway, but they are destroyed at very different rates at the end of mitosis. The resulting difference in timing of their eventual disappearance is critical to the correct sequence of events at mitotic exit. Our preliminary studies indicate that the different rates of proteolysis arise from differences in the ubiquitin code assembled on Aurora A versus Aurora B. We propose a detailed study of the ubiquitin code applied to Aurora kinases, to understand the part of the ubiquitin code that directs rapid destruction at the proteasome. We will use our new knowledge to design reagents that can be used to artificially manipulate cellular levels of Aurora kinases, or of other cellular targets to which they bind, by harnessing the UPS. A new generation of targeted therapies currently in development (called Protein Targeting Chimeras, or PROTACS) will in the future deliver the ability to target cellular proteins that are faulty, or expressed in the wrong time and place, as is often the case in disease. Understanding the ubiquitin code will assist the future design of these tools.
A large fraction of the human genome encodes components of the Ubiquitin-Proteasome System (UPS), because almost all cellular processes require ubiquitin-mediated control. For example in regulation of cell division (mitosis) precise destruction of several key components, at exactly the right time and place, drives the whole process. The complexity of the UPS means that ubiquitin chains come in many varieties, and some are better than others at directing targeted proteins to the proteasome. In other words, the proteolysis of different proteins happens at different rates, depending on what type of ubiquitin chain they carry.
We still don't know very much about this part of the ubiquitin code, and how it is translated into proteolysis.
This proposal focuses on proteolysis of two key regulators of mitosis, called the Aurora kinases (A and B). Both are targeted by the same UPS pathway, but they are destroyed at very different rates at the end of mitosis. The resulting difference in timing of their eventual disappearance is critical to the correct sequence of events at mitotic exit. Our preliminary studies indicate that the different rates of proteolysis arise from differences in the ubiquitin code assembled on Aurora A versus Aurora B. We propose a detailed study of the ubiquitin code applied to Aurora kinases, to understand the part of the ubiquitin code that directs rapid destruction at the proteasome. We will use our new knowledge to design reagents that can be used to artificially manipulate cellular levels of Aurora kinases, or of other cellular targets to which they bind, by harnessing the UPS. A new generation of targeted therapies currently in development (called Protein Targeting Chimeras, or PROTACS) will in the future deliver the ability to target cellular proteins that are faulty, or expressed in the wrong time and place, as is often the case in disease. Understanding the ubiquitin code will assist the future design of these tools.
Technical Summary
Progress in deciphering the ubiquitin code has relied on in vitro approaches using artificial ubiquitin chains. How are ubiquitin chains assembled and processed in vivo? In recent years we have developed tools to purify ubiquitinated proteins from cells and interrogate the composition of ubiquitin chains they carry, using linkage-specific antibodies and deubiquitinases. We discovered that two substrates, AURKA and AURKB, strongly conserved and both targeted by the Anaphase-Promoting Complex (APC/C) ubiquitin ligase, carry different configurations of ubiquitin chains. This difference translates into markedly different rates of proteolysis of the two substrates. AURKA and AURKB therefore present an important model for studying parameters of ubiquitin chain assembly and processing.
We propose to identify substrate-specific and signaling parameters mediating differential ubiquitin chain assembly on AURKA and AURKB, and the switch in ubiquitin chain specificity required for AURKA destruction. The study will focus on divergent N-terminal IDRs that are proposed to contain a number of functional Short Linear Interacting Motifs (SLiMs). We will use cell-based degradation assays and ubiquitination assays to correlate measurements of substrate proteolysis with substrate-specific ubiquitin linkage assembly, and in vitro proteasome binding and proteolysis assays using endogenously ubiquitinated substrates purified from mitotic cells. We will examine the role of SLiMs, and of cellular signaling pathways proposed to regulate them. Integration of bioinformatic and biophysical approaches with functional assays will advance the SLiM model of the proteome, describing molecular detail that underpins the notion of AURKA as a signalling hub.
Finally, we propose to design constrained peptide tools based on AURKA SLiMs and to test their ability to modulate AURK stability and function. These can be used to design chimeric peptides (PROTACS) to target other proteins in the cell.
We propose to identify substrate-specific and signaling parameters mediating differential ubiquitin chain assembly on AURKA and AURKB, and the switch in ubiquitin chain specificity required for AURKA destruction. The study will focus on divergent N-terminal IDRs that are proposed to contain a number of functional Short Linear Interacting Motifs (SLiMs). We will use cell-based degradation assays and ubiquitination assays to correlate measurements of substrate proteolysis with substrate-specific ubiquitin linkage assembly, and in vitro proteasome binding and proteolysis assays using endogenously ubiquitinated substrates purified from mitotic cells. We will examine the role of SLiMs, and of cellular signaling pathways proposed to regulate them. Integration of bioinformatic and biophysical approaches with functional assays will advance the SLiM model of the proteome, describing molecular detail that underpins the notion of AURKA as a signalling hub.
Finally, we propose to design constrained peptide tools based on AURKA SLiMs and to test their ability to modulate AURK stability and function. These can be used to design chimeric peptides (PROTACS) to target other proteins in the cell.
Planned Impact
Our research will have beneficiaries within academia and within the commercial sector (biotechnology, pharmaceutical) sectors and will benefit the general public on various timescales.
Academia will benefit through our acquisition and dissemination of new knowledge, through training of young scientists and future scientists in a world-class environment and through the new tools and methodology that we will generate, that will be shared with fellow academics upon request.
The commercial sector will benefit through new knowledge of cellular mechanisms relevant to disease and to the design of new therapeutic tools. New methodologies that we develop and promote in this research (purification of ubiquitin conjugates, characterization of degrons, measurements of protein stability, peptide design) are highly exploitable for drug discovery in ubiquitin-mediated pathways.
The general public will benefit from the potential healthcare benefits of the theoretical knowledge we generate and the biological tools we develop that may have therapeutic application in the future.
Academia will benefit through our acquisition and dissemination of new knowledge, through training of young scientists and future scientists in a world-class environment and through the new tools and methodology that we will generate, that will be shared with fellow academics upon request.
The commercial sector will benefit through new knowledge of cellular mechanisms relevant to disease and to the design of new therapeutic tools. New methodologies that we develop and promote in this research (purification of ubiquitin conjugates, characterization of degrons, measurements of protein stability, peptide design) are highly exploitable for drug discovery in ubiquitin-mediated pathways.
The general public will benefit from the potential healthcare benefits of the theoretical knowledge we generate and the biological tools we develop that may have therapeutic application in the future.
Publications

Abdelbaki A
(2020)
AURKA destruction is decoupled from its activity at mitotic exit but is essential to suppress interphase activity.
in Journal of cell science

Abdelbaki A
(2023)
Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1.
in Life science alliance

Asteriti IA
(2023)
AurkA nuclear localization is promoted by TPX2 and counteracted by protein degradation.
in Life science alliance

Cacioppo R
(2023)
Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A
in eLife


Cacioppo R
(2023)
Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A.
in eLife

Esposito M
(2020)
USP13 controls the stability of Aurora B impacting progression through the cell cycle.
in Oncogene

Naso FD
(2021)
Nuclear localisation of Aurora-A: its regulation and significance for Aurora-A functions in cancer.
in Oncogene

Naso FD
(2020)
Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.
in Cells

Okoye CN
(2022)
Counting Degrons: Lessons From Multivalent Substrates for Targeted Protein Degradation.
in Frontiers in physiology
Description | We have furthered characterization of motifs in AURKA that are responsible for its regulated destruction by the Anaphase-Promoting Complex (APC/C), manuscript under review. We have identified a deubiquitinase enzyme that regulates AURKB localization and activity during mitosis, one manuscript published, another in preparation. We have discovered why AURKA is destroyed at mitotic exit, manuscript published. We have characterized a novel small molecule PROTAC tool against AURKA, manuscript published. We have secured a funded PhD studentship from AstraZeneca in order to develop this PROTAC work further. We have designed and tested targeted protein degraders for AURKA based on repeat protein scaffolds. |
Exploitation Route | We have published three manuscripts and preparing another four articles. I have been invited to speak at an industry meeting where our findings on targeted protein degradation can be disseminated to researchers in pharma, to contribute to drug discovery efforts. I have been invited to guest edit a special edition on PROTACs for the European Journal of Pharmaceutical Sciences, as another opportunity to disseminate outcomes of this funding to a broad audience of academic and industrial scientists. Our specific findings on differential activity of a PROTAC against distinct subcellular pools of the target can be taken forwards in designing drugs to target such pools. |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Our finding that different subcellular pools of a protein target display differential sensitivity to targeted protein degradation (mediated by a small molecule PROTAC) may be key to the success of future therapeutics. This finding has been communicated to the research field through publication and research talks (2nd TPD Europe meeting, ELRIG) and discussed with a number of potential industrial partners for future collaboration. |
First Year Of Impact | 2021 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal,Economic |
Description | Designing targeted protein degradation tools for future therapeutics |
Amount | £21,000 (GBP) |
Funding ID | M785 |
Organisation | Rosetrees Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2018 |
End | 09/2021 |
Description | Optimizing allosteric inhibitors of the AURKA mitotic kinase to challenge its non-mitotic roles in oncogenesis |
Amount | £10,500 (GBP) |
Funding ID | IES\R3\170195 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2018 |
End | 07/2020 |
Description | Understanding targeted protein degradation for design of optimized therapeutic strategies |
Amount | £443,082 (GBP) |
Funding ID | BB/X007499/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2023 |
End | 03/2026 |
Title | Characterization of novel small molecule PROTACs targeting AURKA |
Description | In collaboration with AstraZeneca. Publication and further funding applications in preparation for 2020. |
Type Of Material | Technology assay or reagent |
Year Produced | 2020 |
Provided To Others? | No |
Impact | There is acute interest in PROTAC technology for development of new therapeutics within the pharmaceutical industry. This has not yet been matched by an interest in use of PROTACs as experimental research tools. Our study will be one of the first to show that PROTACs can be used for acute down regulation of cellular proteins, and that conformation-specific targeting will enable dissection of protein function in a more elegant way than RNA-mediated downregulation of protein expression. |
Description | Characterising the canonical degrons of AURKA |
Organisation | Sapienza University of Rome |
Country | Italy |
Sector | Academic/University |
PI Contribution | Experimental work (cell biology) measuring localization and degradation of different versions of the protein. |
Collaborator Contribution | Experimental work (in silico) to propose structures for different versions of the protein. Tools and reagents (purified proteins). |
Impact | Publications. Review article under revision. Funding application to WCR (failed). |
Start Year | 2018 |
Description | Designing peptide tools to target AURKA |
Organisation | University of Cambridge |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Developing cell-based functional assays to test peptide tools |
Collaborator Contribution | Peptide design and synthesis |
Impact | Multi-disciplinary collaboration. Protein chemistry and cell biology. |
Start Year | 2018 |
Description | Influence of deubiquitinases (DUBs) on PROTAC-mediated target degradation |
Organisation | AstraZeneca |
Department | Research and Development AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | AstraZeneca-funded PhD studentship |
Collaborator Contribution | AstraZeneca-funded PhD studentship |
Impact | no outcomes yet |
Start Year | 2021 |
Description | Role of deubiquitinases in regulating mitotic targets |
Organisation | Free University of Brussels |
Country | Belgium |
Sector | Academic/University |
PI Contribution | Providing imaging facilities for cell-based assays of a DUB targeting Aurora kinases |
Collaborator Contribution | Providing molecular reagents for cell-based assays of a DUB targeting Aurora kinases |
Impact | Very exciting preliminary data |
Start Year | 2016 |
Description | Study of mitotic chromosome attachment errors in oesophageal cancer |
Organisation | University of Cambridge |
Department | Department of Pathology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We shared the imaging platform acquired with funds from this grant with the lab of Dr D'Avino, Department of Pathology |
Collaborator Contribution | PhD student from the D'Avino lab carried out timelapse imaging experiments with our supervision |
Impact | Co-authored paper with Dr D'Avino and Professor Fitzgerald |
Start Year | 2018 |
Description | Study of mitotic chromosome attachment errors in oesophageal cancer |
Organisation | University of Cambridge |
Department | Department of Pathology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We shared the imaging platform acquired with funds from this grant with the lab of Dr D'Avino, Department of Pathology |
Collaborator Contribution | PhD student from the D'Avino lab carried out timelapse imaging experiments with our supervision |
Impact | Co-authored paper with Dr D'Avino and Professor Fitzgerald |
Start Year | 2018 |
Description | Subcellular parameters regulating efficacy of targeted protein degradation drugs |
Organisation | AstraZeneca |
Department | Research and Development AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | Student supervised in my research team. |
Collaborator Contribution | PhD studentship provided by AZ, synthesis of small molecules, biological assays. |
Impact | PhD student recruited, work on the project not yet started. |
Start Year | 2021 |
Description | Targeting Aurora A stability in cancers through its interaction with TPX2 |
Organisation | National Research Council |
Country | Italy |
Sector | Public |
PI Contribution | We have exchanged reagents & ideas & conducted experiments towards joint publications, with Dr Giulia Guarguaglini and her lab. We are planning further publications together in the next 3 years. |
Collaborator Contribution | We have exchanged reagents & ideas & conducted experiments towards joint publications. An ex-student from Dr Guarguaglini's lab came to work as my research assistant when my postdoc left after working out her maternity leave. |
Impact | http://dx.doi.org/10.1016/j.bbcan.2010.08.001 http://dx.doi.org/10.1242/jcs.075457 |
Start Year | 2009 |
Description | Testing a novel FRET-based AURKA biosensor |
Organisation | Gustave-Roussy Institute |
Country | France |
Sector | Academic/University |
PI Contribution | Providing a biological question as context for testing a new research tool. Plus benchtime of PhD student who carried out experiments. |
Collaborator Contribution | R&D to create the new research tool. |
Impact | We have a manuscript currently under revision at J Cell Science. |
Start Year | 2018 |
Description | Using repeat protein scaffolds to understand APC/C degrons |
Organisation | University of Cambridge |
Department | Department of Pharmacology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Cell biology assays to test function of new repeat protein constructs. |
Collaborator Contribution | Expertise in designing constructs and testing them with biophysical assays. |
Impact | One research publication in preparation. One review article in preparation. |
Start Year | 2018 |
Description | Running Public Engagement event at a Music Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | One of the presenters on stall hosting a developmental biology engagement event over 4 days at the Green Man Festival. Event well attended by wide range of age groups. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.gurdon.cam.ac.uk/public-engagement/greenman |