Quantitative analysis of the operation and control of oxidative protein folding in the yeast endoplasmic reticulum

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

Abstract

All living cells possess the ability to secrete proteins from their interior to their exterior environment. This ability serves a variety of purposes, including the transformation of nutrients into a form where they can be taken up by the cell, communication with other cells, and the formation of scaffold structures on which cells can grow. The ability of cells to secrete proteins is also exploited by the bioprocessing industry, which re-programs cells to make and secrete protein-based frontline drugs against debilitating diseases like cancer, multiple sclerosis and arthritis.
Part of the secretion processes in higher (eukaryotic) cells is to ensure that secreted proteins adopt a structure in which they have optimal activity. Proteins are polymeric strings of amino acids, and their folded structure depends on interactions between individual amino acids within them. A specific type of interaction that is critical to the activity of many proteins occurs when two cysteine amino acids form bonds between the sulphur atoms they contain: this is known as a disulphide bond. Disulphide bond formation occurs as an integral part of the secretion process, and involves a cascade of specific enzymes. These enzymes remove an electron from the interacting cysteines, allowing them to form a bond between them that determines the affected protein's shape. The electron is then passed between different enzymes and ultimately onto an oxygen atom, which reacts with water to form hydrogen peroxide. Since the latter is toxic if present in large amounts, it has to be removed in a further series of reactions. The entirety of these reactions is called the oxidative protein folding (OPF) pathway and is the focus of this project.
There are fundamental differences between the OPFs of different types of higher cells. We will explore differences between the OPFs of two specific cell types (simple yeast cells and complex human cells) to improve our understanding of the molecular machinery involved in oxidative folding. Such knowledge will also improve our ability to manipulate the pathway by genetic engineering in order to generate better producing cells for the bioprocessing industry.
Yeast cells only secrete relatively small amounts of proteins, and their OPF machinery therefore evolved to operate on a minimal enzyme set. In contrast, many human cells are prolific secretors, due to their need to communicate extensively with other cells in the body, to produce enzymes for the digestion of food, or to produce molecules of the immune system. Human cells therefore have a much more complex OPF, with different forms of the OPF enzymes that are only act on specific types of target proteins. Interestingly, human cells are also able to use the toxic hydrogen peroxide to drive the OPF reactions, whereas yeast cannot do this.
We will use a three-pronged strategy to exploit these differences: 1), we will isolate the enzymes of the OPF machinery from yeast and human cells and will study their detailed properties in test tubes; 2) we will use the information from these experiments to generate a computational model that can predict properties of the OPF pathways inside cells; and 3) we will use predictions made with the computational model to change properties of the yeast OPF enzymes, and to mix them with human enzymes, in living yeast cells. Overall, this strategy will enable us to better understand how the OPF machinery functions, and in the longer term will enable us to engineer yeast cells that are better suited for use in bioprocessing applications.

Technical Summary

The oxidative protein folding (OPF) pathway in the ER generates disulphide (SS)-bonded native proteins, yet we know surprisingly little about quantitative aspects of the pathway, how it is regulated or how it links to endogenous reducing processes. By combining experimental in vivo, in vitro and modelling work on the yeast and human OPF pathways, we will provide important new insights into the operation and control of SS-bond formation in eukaryotic cells. The focus will be on processes that convert reduced proteins into native, export-competent SS-bonded proteins. Our study will be aided by development of a computational model, which will initially focus on the simpler yeast pathway but subsequently extended to include heterologous components.
Yeast strains expressing homologous and recombinant human proteins will be used to determine i) the levels of secretion of the target proteins, ii) the abundance and redox status of Ero1p and Pdi1p, iii) the intracellular levels of reduced, native and intermediate forms of the target proteins, and iv) the extent of ER stress responses. In parallel, we will analyse the pathway in vitro using purified components (e.g. Ero1p and Pdi1p) with reduced ribonuclease as substrate. We will extend the assay by inclusion of recently-discovered components which use H2O2 to reoxidise reduced PDI and thus derive kinetic parameters and identify stoichiometries of the various catalysts that optimise production of active folded RNAase.
The computational model will be used to make quantitative predictions which will suggest in vivo manipulations of yeast. The yeast OPF pathway will engineered by varying the levels of components, by introducing novel capabilities for using H2O2 generated in the core pathway, and by adding the capabilities of various human PDI isoforms to facilitate OPF in both the in vitro and in vivo systems with the long term aim of engineering yeast strains optimally designed to produce high-value biopharmaceuticals.

Planned Impact

This project will have a significant impact on UK industry, the UK economy and in the longer term the wider population through medical benefits.

Impact on Industry.
Within the biopharmaceutical industry, the need for the cost-effective and robust production of pharmaceutical grade recombinant proteins is now a major priority. Improving the fold, solubility and purity of high value rPs remains a major challenge for this sector, in particular post-translational modifications such as glycosylation and disulphide bond formation that are prevalent in many of the high value target proteins such as recombinant antibodies. Our study will deliver new knowledge on the pathway that ensures the correct formation of disulphide bonds i.e. the oxidative protein folding pathway. This knowledge will be of benefit to end users who seek to rationally engineer improved eukaryotic host cells that are optimised in this pathway. A number of recent reviews (e.g. Idiris et al. 2010, Appl Microb Biotech 86: 403) have highlighted disulphide bond formation as the most promising target for improving key parameters that will increase the efficacy and accuracy of protein secretion.

Impact on the Economy.
Global sales of recombinant therapeutic proteins including monoclonal antibodies (mAbs) were estimated to be $160 billion in 2013 (BCC Research 2008). The bioprocessing industry is thus a major economic factor world-wide and the UK is a major player in the sector, with nearly all international companies having a UK base. In addition, numerous UK-based SMEs are highly active in this sector. Even a minor improvement in host cell capability will translate into a measureable impact on this sector. The systems level information we obtain with yeast will provide a platform on which step changes in production platforms can be made - both yeast and mammalian cells - that will have long-lasting economic benefits.

Impact through Medical Benefits.
Biopharmaceuticals are state-of-the-art treatments for many high-impact diseases including multiple sclerosis, cancer, and inflammatory diseases. These diseases have adverse impact beyond the affected individuals themselves including carers and dependent family members. Improvements in the production of recombinant proteins thus have high potential for positive impact felt in wider society. In the longer term, more efficient production of higher quality drugs will benefit the Health Service provision and patient.

Publications

10 25 50
 
Description A poster presented by Dr Emma Bastow at the Cold Spring Harbor Laboratory Conference on "Protein Homeostasis". Poster title: "The biological consequences associated with defects in protein disulphide isomerase (Pdi1p) in S. cerevisiae". 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presenting the results of our project to a large international of experts raising the profile of the research undertaken.
Year(s) Of Engagement Activity 2018
 
Description An invited talk by Dr Dave Beal at the conference on "Protein disulphide bonds-biochemistry, biotechnology and biomedical impact" organised by the Biochemical Society. Title of the talk was "Modelling the process and selectivity of oxidative folding in the yeast endoplasmic reticulum" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This conference brought together the communities working on the biochemistry and cell biology of the oxidative protein folding pathway, cell manipulation for production of high-value, disulphide-bonded protein products, and the contribution of protein misfolding to human disease, highlighting recent advances and to stimulate new research. In so doing the conference also celebrated the work of Robert Freedman, a pioneer in this field and a co-grant holder of this grant.
Year(s) Of Engagement Activity 2018
 
Description Invited talk given by Mick Tuite at conference on "Protein disulphide bonds-biochemistry, biotechnology and biomedical impact" organised by the Biochemical Society. Title fo the talk was "In vivo and in vitro analyses of the operation and control of yeast oxidative protein folding" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This meeting brought together the communities working on the biochemistry and cell biology of the oxidative protein folding pathway, cell manipulation for production of high-value, disulphide-bonded protein products, and the contribution of protein misfolding to human disease, highlighting recent advances and to stimulate new research. In so doing the conference celebrated the work of Robert Freedman, a pioneer in this field and co-grant holder of this grant.
Year(s) Of Engagement Activity 2018
 
Description Involvement and co-organisation of an event at the Canterbury Festival entitled The Beer Lab and held October 17th 2017. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This event was set up as part of the science strand of the annual Canterbury Festival, to explore the links between science and the art of brewing. A Canterbury-based microbrewery The Foundry hosted the Beer Lab, providing a tasting menu of selected beer styles and an insight into the brewing process, while members of the Kent Fungal Group (including Professor Mick Tuite) explored the use of yeast in biology, from producing alcohol, to understanding human diseases. The event was sold out.
Year(s) Of Engagement Activity 2017
 
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 Poster presented by Dr Dave Beal at British Yeast Group meeting, Sept 2017 "Quantitative analysis of the operation and control of yeast oxidative protein folding" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Poster presented by Dr D M Beal at British Yeast Group meeting, Canterbury, Sept 2017. Topic of poster was "Quantitative analysis of the operation and control of yeast oxidative protein folding"
Year(s) Of Engagement Activity 2017
 
Description Presentation by Dr Dave Beal at the Molecular Chaperone Club meeting, December 2016. Talk title: "Optimisation of the Ero1/PDI interaction for improved protein folding and secretion in yeast." 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Optimisation of the Ero1/PDI interaction for improved protein folding and secretion in yeast
Speaker: Dave Beal, School of Biosciences, University of Kent
Co-authors: Gemma Staniforth, Tobias von der Haar, and Mick Tuite, School of Biosciences, University of Kent
Robert Freedman, School of Life Sciences, University of Warwick
Year(s) Of Engagement Activity 2016
 
Description Presentation of a poster by Dr Emma Bastow at the British Yeast Group meeting in Sept 2017, entitled The biological consequences associated with defects in protein disulphide isomerase (PDI) in Saccharomyces cerevisiae 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presentation of a poster by Dr Emma Bastow at the British Yeast Group meeting in Sept 2017, entitled "The biological consequences associated with defects in protein disulphide isomerase (PDI) in Saccharomyces cerevisiae".
Year(s) Of Engagement Activity 2017