Modelling yeast prion dynamics in the living cell

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

The last decade has seen a new challenge to the dogma that inheritance is based exclusively on DNA. This has come in part from the discovery of prions in yeast and other fungi. Historically, prions have been intensively studied in relation to their ability to cause diseases such as 'Mad Cow Disease' and the human form, Creutzfeldt-Jakob Disease (CJD). However the discovery of prions in fungi that can potentially be of benefit to the host organism suggests that prions may actually represent an entirely new form of protein-based heredity. One such prion-based 'epigenetic' determinant in Baker's Yeast (Saccharomyces cerevisiae) will be studied in this project; the so-called [PSI+] prion. This prion can significantly alter the way that the yeast cell responds to certain toxic chemicals for example. The approach we are planning to take is a novel one, integrating biological experiments with mathematical modelling to study the behaviour of prions in a living yeast cell. This is an approach we first developed on a previous BBSRC-funded project and this new project builds on that, broadening the approach to model the process by which prions are replicated in an asymmetrically dividing yeast cell. Our long-term goal is to understand fully the molecular details of exactly how prions are so effectively generated and then passed on to other yeast cells. We already know that this requires cells to produce prion 'seeds' which can be passed from the mother cell to the daughter cell (a process we refer to as prion transmission). These seeds - which we refer to as propagons - are generated by breaking up much larger aggregated forms of the prion protein that are found in yeast cells. By employing a multidisciplinary approach we aim to establish the mechanism that operates in the growing yeast cell to ensure that new prion seeds are efficiently generated and transmitted to a new daughter cell. The research project will be built upon an iterative process of model improvement through biological experiments conducted to allow estimation of model parameters and then assessment of the adequacy of the new models that emerge. This approach will provide us, for example, with the ability to assess and ultimately predict the way in which changes in the cell's make up or function may impact on prion replication and/or transmission. The outcome will be a new understanding of how this important class of 'epigenetic' elements - some 20 of which have now been described - are replicated in the living cell, and how that process can be perturbed. The findings will guide future studies on the processes both in fungi and in mammals. A longer term outcome of the research will be identifying components of the cell that can be targeted by new drugs to block the generation and/or transmission of prion seeds.

Technical Summary

The stable propagation of yeast prions requires the continued generation of new prion seeding molecular entities (propagons) and their efficient transmission to daughter cells at cell division. We have developed a novel, stochastic modelling approach to establishing the key molecular events in the propagation and transmission of the [PSI+] prion. Our current models provide an important phenomenological description of how the [PSI+] prion is eliminated in the absence of a mechanism to generate new propagons. In this new project we will develop new mechanistic models that not only take into account the kinetics of prion protein polymerisation but also polymer fragmentation in the growing yeast cell. To achieve this we will generate stochastic simulation models for yeast prion polymer kinetics and support this model building by experimentally establishing the values of the important model factors including the cellular levels of Sup35p and the key chaperone proteins Hsp104 and Sis1p, the number and sizes of Sup35p prion polymers and the rate of synthesis and turnover of Sup35p in its various cellular forms. To evaluate the emerging simulation models we will assess the in vivo consequences of modulating levels of Sup35p, Hsp104 and Sis1p all of which are essential for [PSI+] propagation. One crucial parameter in our models is pi, the proportion of propagons transmitted to the daughter cell. We will identify factors that lead to a change in pi, particularly in relation to the number and length of Sup35p polymers in the cell. Throughout the project we will carry out model sensitivity and validation studies in the light of experimental data obtained in order to generate a definitive stochastic simulation model for yeast prion polymer kinetics in the dividing cell. This project involves a close interplay between bioscience-led research and integrated mathematical modelling with the aim of shedding new light on how infectious amyloids are generated and transmitted in vivo.

Planned Impact

In terms of research findings: the primary beneficiaries will be researchers in the academic and pharmaceutical sectors who are exploring both the underlying molecular mechanism of amyloid formation and the resulting disease states. A further dimension of the impact of the research proposed comes from the realisation that yeast prions represent a completely novel class of epigenetic regulator are likely to exist in other organisms and have a major impact on cellular functions and development. Kent Innovation & Enterprise (KIE), the university's dedicated business development unit, will provide full support and guidance on any business opportunities that may arise from research findings made. In terms of staff training; the project will provide the two appointed PDRAs with an opportunity to develop multidisciplinary skills combining experimental biology and mathematical modeling techniques. Training will be provided for the PDRAs to develop these new skills through the project and its investigators, through courses available within the host Departments and also through the Faculty-run 'Researcher Skills Training' programmes at the University of Kent. The training of a new cohort of young scientists with a combination of biological and mathematical skills is important if we are to make effective use of mathematical models to describe complex biological systems, that is increasingly becoming the approach of choice. In terms of dissemination: in addition to the usual publication of methods and results in a range of academic, peer-reviewed journals (including top international journals for statistical methods as well as for novel developments in biology), a variety of strategies will be used to ensure that others have the opportunity to benefit from this research without the delays naturally arising from the peer-reviewed publication route. For example, as part of our previous BBSRC-funded project (96/E18382) we established a web site dedicated to our collaborative project, the methods developed and its scientific outputs, including computer software. As part of this new project we would continue to exploit this dissemination route but also increase the outreach component of the web material. These strategies will be coupled with a wide range of poster presentations and seminars at national and international conferences.

Publications

10 25 50
 
Description The last decade has seen a new challenge to the dogma that inheritance is based exclusively on DNA. This has come from the discovery of prions in yeast and other fungi. Historically, prions have been intensively studied in relation to their ability to cause diseases such as 'Mad Cow Disease' and its human form, Creutzfeldt-Jakob Disease (CJD). However, the discovery of prions in fungi that can potentially be of benefit to the host organism suggests that prions may actually represent an entirely new form of protein-based heredity. One such prion-based 'epigenetic' determinant in the yeast Saccharomyces cerevisiae was the focus of our project; the so-called [PSI+] prion. This prion can significantly alter the way that the yeast cell responds to certain toxic chemicals for example.

Our long-term goal was to unravel the complex mechanism by which this prion (and other yeast prions) is able to multiply in the growing cell. We already knew some of the major players in this, in particular proteins known as chaperones. Our approach was a novel one: to integrate biological experiments with mathematical modelling to interrogate the behaviour of prions in a living yeast cell. This is an approach we first developed in a previous collaboration (funded by the BBSRC: ref no. E18382) in which we explored the impact of inhibiting the ability of a cell to propagate the prion by preventing one of the chaperones (called Hsp104) from functioning as it should. By modelling this we were able to make a number of predictions that have subsequently been followed up by biochemical and genetic experiments.

In this new project we built on this by broadening the approach to modelling the process by which prions are replicated in an asymmetrically dividing yeast cell. Key to this understanding was the recognition that we must understand fully the molecular details of exactly how prions are so effectively generated and then passed on to other yeast cells. We already knew these required cells to produce prion 'seeds' which can be passed from the mother cell to the daughter cell (a process we refer to as prion transmission). In this project we developed for the first time, analytical methods to identify and characterise these entities and found that they consist of a large number of molecules of the prion strung together. By employing a multidisciplinary approach we went on to gain new insights into the mechanism that operates in the growing yeast cell to ensure that new prion seeds are efficiently generated and transmitted to a new daughter cell.

The research programme was built upon an iterative process of model improvement through biological experiments conducted to allow estimation of model parameters and then assessment of the adequacy of the new models that emerge. This approach provided us with the ability to assess and ultimately predict the way in which changes in the cell's make up or function impacted on prion replication and/or transmission. We also made a number of unexpected observations including the apparent dynamic behaviour of these aggregates when one modulates either the levels of key proteins such as chaperones, or when the cell is asked to grow in a different environment; for example using a different carbon source to glucose.
Exploitation Route The main outcome of our project has been a new understanding of how this emerging class of 'epigenetic' elements are so effectively replicated in the cell, but also what changes can lead to it being perturbed. The findings will guide our future studies on the processes both in fungi and in mammals. A longer term outcome of the research will be identifying components of the cell that can be targeted by new drugs to block the generation and/or transmission of prion seeds.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The main outcome of our project has been a new understanding of how this emerging class of 'epigenetic' elements are so effectively replicated in the cell, but also what changes can lead to it being perturbed. The findings will guide future studies on the processes both in fungi and in mammals. A longer term impact of the research will be in identifying components of the cell that can be targeted by new drugs to block the generation and/or transmission of prion seeds. This will likely take several years before the impact is felt.
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
 
Title YeastSim++ 
Description Prion models need to simulate the dynamics of polymer growth and fragmentation within the many individual cells in a growing cell population. Amongst the features incorporated within YeastSim++ are asynchronous and asymmetrical cell division, cell expansion and volume dependent rate parameters, and threshold sizes to inhibit polymers from passing to daughter cells. The modular nature of the code allowed the model to incorporate interactions with molecular chaperones such as Hsp104. YeastSim++ and its applications are fully described in a School of Mathematics and Actuarial Science Technical Report (Outline of YeastSim++: Tech Rep. No UKC/SMSAS/11/016) and has been made available to other researchers upon request. The YeastSim++ library provides an abstract simulation environment which can be used to develop a variety of stochastic models in biology. It was used in the project to implement several different prion models from the recent literature. It was also used to simulate the well-known Lotka- Volterra model of predator-prey dynamics, for which there are numerous existing implementations, allowing YeastSim++ to be tested thoroughly. 
Type Of Technology Software 
Year Produced 2011 
Impact None as yet but currently being exploited by the applicants with a view to generating second generation models of prion behaviour in dividing yeast cells. 
URL http://www.kent.ac.uk/smsas/publications/2011techreps.html
 
Description Cafe Scientifique presentations 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Tuite presented a talk in a local Café Scientifique entitled "Senile yeast" in which he described the use of yeast to explore
the underlying basis of amyloid diseases including prions.

A discussion following the talk indicated a wide interest in the science under-pinning dementias in humans.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
URL http://www.cafescientifique.org/?id=239:canterbury
 
Description Dragons Den 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact The presentation was "highly commended" and discussions were held to see if funding could be found to develop the project idea - but this failed.

Raised awareness among students and staff of the School of Biosciences as to how one's research can be used to underpin ideas for commercialisation and exploitation.
Year(s) Of Engagement Activity 2013
 
Description Involvement in the Authentic Biology Project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Throughout the period of this grant co-Is Tuite, von der Haar and postdoctoral researcher supported by this grant were involved with Authentic Biology, a Wellcome Trust-funded outreach project led by the Simon Langton Grammar School for Boys in Canterbury. This involved 2-3 days per person per year visiting the School and participating in activities such as laboratory classes. This ongoing novel project involves years 12 and 13 school students carrying out research into multiple sclerosis by expressing myelin basic protein in yeast and characterising the purified protein (see www.mbp-squared.org). In 2012 the project was rolled out nationally, again with Wellcome Trust support and badged as the 'Authentic Biology' project that now engages some 1000 year 12 and 13 students a year in a variety of research projects across the UK (see www.authenticbiology.org). Naeimi acted as a technical aid and teacher, demonstrating techniques at workshops and providing project support.

The Authentic Biology has a national profile, has generated in excess of £500,000 funding from the Wellcome Trust and the project has and continues to receive wide coverage over the last three years from the local and national press, the scientific press (e.g. a Nature podcast; www.nature.com/nature/podcast/index-2013-11-21.html) and TV and radio interviews. This project has had a significant impact on the Langton School resulting in a doubling of the number of years 12/13 studying biology.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
URL http://www.authenticbiology.org
 
Description Oral presentation by Mick Tuite at the annual Molecular Chaperone Club, Canterbury December 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Title: The consequences of overexpression of the molecular chaperone Hsp104 on prion propagation in Saccharomyces cerevisiae
Presenter:Mick F Tuite,
Co-authors: Frederique Ness, Brian S Cox, Jintana Wongwigkarn, Wesley R Naeimi, School of Biosciences, University of Kent
Year(s) Of Engagement Activity 2016
 
Description UCAS Talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Tuite presents 2-3 talks per year to parents and year 13 students on Prions: infectious agents or a new kind of protein
based inheritance. This is part of the School of Biosciences 'UCAS Day' during which potential students and their parents visit their Universities of choice.

Invariably the talk provokes questions from both the prospective students and also their parents. On several occasions, prospective students who attended the talk returned as undergraduates at Kent and in at least two cases, carried out a research project on prions as part of their undergraduate programme.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010