Modelling yeast prion dynamics in the living cell

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.

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.

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.