Modelling fluorescent timers to quantify protein turnover in the cell cycle
Lead Research Organisation:
University of Cambridge
Department Name: Pharmacology
Abstract
Theme: World-Class Underpinning Bioscience
Recently described tandem fluorescent protein timer tags (tFTs) have been used successfully to report on the age of pools of tagged proteins, based on the differential maturation kinetics of a slow-folding red fluorescent protein and a fast-folding green one [1]. We propose to use tFT tags as tools to report on the stability of protein pools, looking at Aurora kinases, which are well known regulators of the cell cycle. Their targeted ubiquitination at mitotic exit has been extensively characterized in the Lindon lab [2,3]. It is not known how turnover of Aurora kinases varies through other phases of the cell cycle, or in different cellular compartments. In pilot experiments in mammalian cells, the colour of a tFT linked to Aurora kinase varied qualitatively as expected when the proteins were stabilized by siRNA-mediated knockdown of an ubiquitin ligase Cdh1, or by mutation of the Aurora degron motif. However it is not clear what can be inferred from these observations, since we do not know in which cells - if any - Aurora turnover is a steady state process. Therefore, the potential of tFT technology for quantitative measurement of protein stability in unperturbed cell populations requires mathematical modelling to underpin it, in order to understand how the determinants of the output signal (synthesis, maturation, degradation) interact with biological phenomena such as the cell cycle.
In collaboration with Dr Graham Ladds (who uses a range of computational techniques to explore biological problems [4-6]), we will develop mathematical models to describe the behaviour of tFTs in the mammalian cell cycle. We propose a collaborative studentship to involve time-resolved fluorescence imaging of tFTs, testing parameters that include rate of synthesis, maturation kinetics of different tFT pairs, and subcellular localization. Some experiments will be carried out in Schizosaccharomyces pombe, where use of cell cycle arrest mutants can generate 'steady states' in the cell cycle. We will develop models to describe how tFT turnover evolves. Quantitative wet experimental data will be fitted to computational simulation to provide the most actuate reconstruction of the biological data. We will then progressively constrain and/or perturb each model to enable us to extract quantitative information on how Aurora kinase half-life varies through the cell cycle and under different growth conditions.
Recently described tandem fluorescent protein timer tags (tFTs) have been used successfully to report on the age of pools of tagged proteins, based on the differential maturation kinetics of a slow-folding red fluorescent protein and a fast-folding green one [1]. We propose to use tFT tags as tools to report on the stability of protein pools, looking at Aurora kinases, which are well known regulators of the cell cycle. Their targeted ubiquitination at mitotic exit has been extensively characterized in the Lindon lab [2,3]. It is not known how turnover of Aurora kinases varies through other phases of the cell cycle, or in different cellular compartments. In pilot experiments in mammalian cells, the colour of a tFT linked to Aurora kinase varied qualitatively as expected when the proteins were stabilized by siRNA-mediated knockdown of an ubiquitin ligase Cdh1, or by mutation of the Aurora degron motif. However it is not clear what can be inferred from these observations, since we do not know in which cells - if any - Aurora turnover is a steady state process. Therefore, the potential of tFT technology for quantitative measurement of protein stability in unperturbed cell populations requires mathematical modelling to underpin it, in order to understand how the determinants of the output signal (synthesis, maturation, degradation) interact with biological phenomena such as the cell cycle.
In collaboration with Dr Graham Ladds (who uses a range of computational techniques to explore biological problems [4-6]), we will develop mathematical models to describe the behaviour of tFTs in the mammalian cell cycle. We propose a collaborative studentship to involve time-resolved fluorescence imaging of tFTs, testing parameters that include rate of synthesis, maturation kinetics of different tFT pairs, and subcellular localization. Some experiments will be carried out in Schizosaccharomyces pombe, where use of cell cycle arrest mutants can generate 'steady states' in the cell cycle. We will develop models to describe how tFT turnover evolves. Quantitative wet experimental data will be fitted to computational simulation to provide the most actuate reconstruction of the biological data. We will then progressively constrain and/or perturb each model to enable us to extract quantitative information on how Aurora kinase half-life varies through the cell cycle and under different growth conditions.
People |
ORCID iD |
Catherine Lindon (Primary Supervisor) | |
Richard Wang (Student) |
Publications
Wang R
(2021)
Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool.
in Communications biology
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M011194/1 | 01/10/2015 | 31/03/2024 | |||
1804956 | Studentship | BB/M011194/1 | 01/10/2016 | 31/12/2020 | Richard Wang |
Description | I have explored the use of a novel type of drug against the cell cycle protein Aurora Kinase A (AurKA). This new drug, a type of protein degrader, known as a Proteolysis Targeting Chimaera (PROTAC) utilises a bifunctional compound that can bind to AurKA, and the CRB E3 ligase, bringing the two into close proximity. This result in the ubiquitination and subsequent degradation of AurKA. In this project, I have studied at a cellular level the effects of PROTAC mediated degradation of AurKA, in particular, compared to just kinetic inhibition of AurKA. I found that degradation can offer distinct phenotypes such as shorter spindle length and the ability to rescue mitochondrial morphology. I have also begun to explore my finding that the susceptibility of AurKA to PROTAC degradation can vary on its localisation and or conformation in complex with binding partners. This finding will be of great interest as it can potentially lead to more conformation or disease-specific targeting of PROTACs. |
Exploitation Route | This finding of localisation/conformation-dependent targeting of AurKA advances our understanding of how PROTACs could work and be used. Therefore, providing a further step in their ability to be utilised from a research and therapeutic objective. |
Sectors | Pharmaceuticals and Medical Biotechnology |
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 | 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 | Working using materials provided to us by AstraZeneca |
Organisation | AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | I have carried out cell biology assays using the compounds provided to me by AstraZeneca. I have tested the compounds as new tool to modulate Aurora Kinase A stability. |
Collaborator Contribution | AstraZeneca has provided the compounds which I am using. |
Impact | I have reported back to AstraZeneca the findings of our assays which shows promise in the new compounds acting to modulate Aurora Kinase A stability. |
Start Year | 2018 |