Translating the ubiquitin code in mitotic cells

Lead Research Organisation: University of Cambridge
Department Name: Pharmacology

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.

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.

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.
 
Description We have furthered characterization of motifs in AURKA that are responsible for its regulated destruction by the Anaphase-Promoting Complex (APC/C), manuscript in preparation.
We have identified a deubiquitinase enzyme that regulates AURKB localization and activity during mitosis, one manuscript under revision at Oncogene, another in preparation.
We have discovered how activity and destruction of AURKA and AURKB are linked during exit from mitosis.
We have discovered why AURKA is destroyed at mitotic exit, manuscript under revision at J Cell Science.
We have characterized a novel small molecule PROTAC tool against AURKA, manuscript in preparation.
We have designed and tested targeted protein degraders for AURKA based on repeat protein scaffolds.
Exploitation Route We are preparing three manuscripts to share our findings with the field.
Sectors Pharmaceuticals and Medical Biotechnology

 
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 10/2018 
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 04/2018 
End 07/2020
 
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 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 collaboration has just started, supported by an MRes student who proposes to continue to PhD on this project.
Start Year 2018
 
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 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