Quantitative investigations into the molecular mechanisms of amyloid fibril fragmentation

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

Abstract

Amyloid fibrils are forms of protein that have received much recent attention through their association with numerous devastating human brain diseases. Examples include Alzheimer, Creutzfeldt-Jakob (CJD), Huntington and Parkinson diseases. Furthermore, the unusual physical characteristics of amyloid fibrils mean that they have the potential to become strong and stable engineered nanomaterials. Breaking amyloid fibrils into smaller pieces is a key process that must be fully understood if we are to understand how amyloid fibrils normally function in nature, and how they are involved in diseases so we can develop effective therapies against the amyloid-associated diseases. Nevertheless, the causes and consequences of amyloid fibril fragmentation remain a largely unexplored area of research. The long-term goal of this project is to resolve the molecular and cellular mechanisms of fibril fragmentation.

Amyloid fibrils are assembled from whole or parts of normal proteins and the devastating human diseases associated with amyloid are linked to the way the amyloid fibrils are assembled and deposited in the brain or in other parts of the human body. However, amyloid fibrils have also been recognised as a class of natural protein forms, so-called 'functional amyloids'. Functional amyloids can play a number of important roles in bacteria, yeast and even humans. A sub-class of amyloids can spread between organisms by forming small seeds through the fragmentation of larger fibrils. This sub-class is referred to as prions and they exist in humans where they cause diseases such as CJD. In baker's yeast, they confer special cellular properties on the cells that are passed on from generation to generation; a form of 'protein gene'. To fully understand how prions are formed and transmitted requires that we understand how the prion seeds (which we call propagons) are generated through amyloid fibril fragmentation. Detailed characterisation of amyloid fibrils of different origins has revealed incredibly strong structures that are commonly only tens of nanometres thick but many micrometers long. The fragmentation property of amyloid fibrils is, however, the dominating factor for their stability. Fibril fragmentation is also an important factor in amyloid-associated disease because it influences the size and shape of the disease-associated forms of the fibrils, which are typically large clumps of aggregates. How easy do disease-associated prions spread, how fast are amyloid aggregates assembled and deposited, and how toxic are these aggregates to cells are important disease properties that are influenced by amyloid fibril fragmentation.

To address these points, our goal is to answer the following questions: how are amyloid fibrils fragmented, how fast can they fragment, and how is fragmentation linked to their properties in living cells. Using a combination of experimental, theoretical and computational approaches, the fragmentation properties of three different amyloids will be studied: one that forms a prion in yeast, one that is associated with human disease, and the third is an artificial model system. The insights gained from this project will also be critical for further exploring amyloid fibrils as potential nanomaterial in technological applications, and will provide new insights that will facilitate the future development of therapeutic strategies against amyloid associated disease.

Technical Summary

In order to characterise the molecular mechanisms of amyloid fibril fragmentation, to quantify the rates of fibril fragmentation, and to determine the influence of fragmentation on cellular processes, a combination of experimental, theoretical and computational approaches will be employed. The fibril fragmentation properties of three amyloid models: the yeast prion protein Sup35, human alpha-synuclein, and hen egg white lysozyme, representing a functional, a disease-related and an artificial model system, respectively, will be examined. Firstly, their fibril stability towards fragmentation will be characterised using tapping-mode atomic force microscopy. Image data collected during fragmentation facilitated by stirring will be analysed through the application of statistical image analysis methods in order to quantify changes in fibril dimensions. Secondly, models of how fibril fragmentation might occur will be developed and computer simulations of fragmentation processes will be performed in parallel with the experiments. Modelling strategies, through a systems approach with large-scale population balance simulations will be developed to tackle the complexities of heterogeneous mixture of species involved in fibril fragmentation. Potential models will be quantitatively tested against the data using regression and model comparison methods to delineate models that can describe and predict fibril fragmentation. Thirdly, the in vitro measurements of fragmentation properties will be applied to the quantitative characterisation of the biological consequence of Sup35 fibril fragmentation in yeast cells. How changes in the fragmentation properties of Sup35 fibrils affect the resulting [PSI+] prion phenotype in vivo will be accessed using mutants of Sup35. This application of quantitative fibril fragmentation analysis will test the hypothesis that enzyme controlled fibril fragmentation is an integral part of functional amyloid in biology.

Planned Impact

The findings of this proposed project will not only benefit researchers exploring the basic mechanisms of amyloid formation or prion propagation, they will likely have a number of important applications. Using amyloid fibrils in novel bio-nanomaterials, and developing therapies targeting amyloid disease are examples of the many potential applications of the findings of this project. These potential applications will revolutionise a variety of industries in the medical, tele-communication and manufacturing sectors, as well as improve life in general in an aging population. In addition, this project will provide a postdoctoral researcher with an unique training opportunity. The postdoctoral scientist will develop skills to apply mathematics, chemistry and physics methods to solve important biological problems, and learn to communicate ideas across disciplinary boundaries. The findings of this project will be of interest to a wide group of researchers in diverse disciplines through the means of publications in open access multidisciplinary peer-reviewed journals and presentations in international conferences covering topics ranging from biology, physics, chemistry to material science. Any potential commercial applications arising from this proposal will be explored with the help and support from the Kent Innovation and Enterprise business development unit within the University of Kent.
 
Description - Developed a robust experimental methodology that produces high quality image and kinetic data on the fragmentation of amyloid fibrils
- Developed a generic theory that can explain the division of amyloid particles.
- Established the molecular mechanisms and to quantified the rates of fragmentation for three types of amyloid fibril models.
Exploitation Route The experimental approach and the theory and model can be used by research to explain the potential capacity of amyloid and prion propagation, such as in the case of recently reported possible spreading of Alzheimer's Abeta aggregates.
Sectors Agriculture, Food and Drink,Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description ?Prediction of amyloid behavior? ? through single-particle analysis? of amyloid assembly and disassembly kinetics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited talk at Biophysical Colloquium, Cambridge, UK.

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2014
 
Description ?Prediction of amyloid behavior? ?through analysis of amyloid formation kinetics ? and fibril length quantification by Atomic Force Microscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited talk at Université Pierre et Marie Curie, Paris, France

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2014
 
Description ?Prediction of amyloid behavior? through fibril length quantification? by Atomic Force Microscopy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at ADAM2013, 4th Scandinavian Meeting on Amyloid Proteins and Disease, Lund, Sweden

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2013
 
Description ?The Mechanism of? Fibril Fragmentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at Conferences Jacques-Monod - Protein misfolding in disease, Roscoff, France

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2013
 
Description A systems approach to fibril fragmentation - Experiments, Models and Predictions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Conference talk (academic audience) at the "Biomolecular Thermodynamics" conference, a two day conference organised by the IOP Biological Physics Group, 26-27 November 2012, Institute of Physics, London, UK

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2012
 
Description Amyloid proteins: from Alzheimer's disease, spider silk to evolution of early life 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact BioSoc society Research talk event
Year(s) Of Engagement Activity 2015
 
Description Atomic force microscopy imaging of the dividing amyloid particles 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk, Lund OMM Meeting talk, Sweden
Year(s) Of Engagement Activity 2015
 
Description Importance of size and long-range structure in the prediction of the biological behavior of amyloid 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at Wash U, St Louis, MO, USA
Year(s) Of Engagement Activity 2015
 
Description Modelling amyloid protein self-assembly - Bridging experiments and theory 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk, 10th ECMTB and SMB 2016 conference, Nottingham, UK
Year(s) Of Engagement Activity 2016
URL http://www.ecmtb2016.org
 
Description Nano-scale Properties of the Amyloid Life-cycle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited research talk, TU Delft, Delft, Netherlands
Year(s) Of Engagement Activity 2016
 
Description Prediction of disease-associated amyloid properties and behaviour 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Chemical Biology Club (CBC) seminar, Kingston
Year(s) Of Engagement Activity 2015
 
Description Quantitative characterization of The Mechanism of Fibril Fragmentation and The Fibril's Stability Toward Breakage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at FASEB - Molecular Mechanisms and Physiological Consequences of Protein Aggregation, Big Sky, MT, USA

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2013
 
Description Seeing nanometre sized protein structures associated with neurodegenerative diseases 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact National Science and Engineering Week open lecture at Beaney Institute, Canterbury (non-academic audience)

My talk informed the public about my research and generated interest and further discussions.
Year(s) Of Engagement Activity 2013
 
Description Systems modelling of Protein self-assembly in the Amyloid life-cycle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk, Centre for Mathematical Medicine and Biology Seminar Series, Nottingham, UK
Year(s) Of Engagement Activity 2016
 
Description The division of amyloid particles put under the microscope: Comparing the stability of amyloid fibrils toward breakage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at FASEB Molecular Mechanisms and Physiological Consequences of Protein Aggregation, West Palm Beach, FL, USA
Year(s) Of Engagement Activity 2015
 
Description The mechanism of amyloid fibril fragmentation and the stability of amyloid fibrils toward breakage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk at British Biophysical Society Meeting, Warwick 2014.

My talk influenced views and provoked discussions in the field.
Year(s) Of Engagement Activity 2014
 
Description The properties of nano-scale amyloid fibril fragments in vitro and in vivo 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk, Biophysical Seminar, Cambridge Chemistry Department, Cambridge, UK
Year(s) Of Engagement Activity 2016