Interrogating the molecular basis of aggregation inhibition by molecular chaperones

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

Proteins are the basic building blocks of all organisms. Over 100,000 different types are responsible for various functions such as moving muscles, digesting food, defending against infections or sending signals between different cells or other parts of the organism. They are all composed of only 20 different building blocks, called amino-acids, arranged in a sequential chain. The exact sequence of the protein is stored in our genes, and can be many hundreds or even thousands of blocks long, and they are produced inside cells as linear chains. In order for protein molecules to function, they must adopt a well defined 3D structure. The process by which the newly synthesised chain finds its 3D structure is called 'protein folding'. A family of proteins known as 'molecular chaperones' are present to facilitate the folding process. Biological organisms need correctly folded proteins in order to function optimally.

Rather than always adopting their correct fold, protein chains however have an intrinsic tendency to cluster together and aggregate. The build up of aggregates in tissues together with the loss of function of the aggregating protein can lead to a set of diseases known as 'mis-folding diseases'. A wide range of pathologic human conditions, including neurodegenerative disorders Alzheimer's and Parkinson's diseases as well as type II diabetes and the prion diseases are mis-folding diseases. Such diseases as well as being some of the most feared in old age, place a larger financial burden on society than cancer and heart disease combined. At present, the fundamental roots of mis-folding disease are poorly understood.

A particular family of molecular chaperones, the sHSPs are found bound to aggregates removed post-mortem from Alzheimer's disease. Strikingly, they are able to stop proteins from aggregating in vitro, but we know very little about how they are able to do this.

sHSPs naturally exist in an aggregated state that makes them intrinsically very hard to study. My proposed research is aimed at determining how they are able to prevent protein aggregation. To do this, I will take advantage of recent technological advances in three state-of-the-art methodologies - nuclear magnetic resonance spectroscopy, mass spectrometry and electron microscopy. Each is able to look at very different aspects of sHSPs, and the combination will make a powerful toolkit that I anticipate will lead to new insights into how sHSPs can prevent protein aggregation. By doing so, I hope to find new ways to look at developing therapeutic treatments for those suffering from mis-folding diseases.

The potential for these techniques to provide insights into biological systems is immense. A BBSRC David Phillips Fellowship will enable me bring the necessary expertise to conduct such studies in the UK. The research will be performed at the University of Oxford, a world leading centre of research. I am very excited to have the opportunity to enter this uniquely stimulating environment at the next stage of my career.

Technical Summary

A wide range of human disorders including Alzhemier's and Parkinson's diseases are associated with a build up of potentially pathogenic protein aggregates in vivo. At least under normal circumstances, a family of molecular chaperones, the sHSPs are able to inhibit proteins from forming potentially pathogenic aggregates. These promiscuous chaperones reduce the aggregation rates of a wide range of misfolded proteins, but do not bind functional proteins. How they do this is far from certain, as they have proven highly recalcitrant to structural characterisation, primarily due to the wide range of oligomers they populate and the local dynamics that allow individual subunits to move between oligomers. My proposal is concerned with interrogating the mechanism by which these complex molecules are able to inhibit protein aggregation.

My proposal is to take advantage of the most recent developments in NMR that allow studies of conformational dynamics and interactions of large molecular assemblies at atomic resolution. I will complement information describing secondary/tertiary structure from NMR with MS and EM, techniques able to reveal the quaternary structure, shape and size of complexes under study. This hybrid methodology forms a tool box with which I can interrogate these otherwise biophysically intractable heterogeneous assemblies.

This research will answer fundamental questions about how the chaperones recognise mis-folded protein states, and inhibit their aggregation. In doing so, it will shed light onto how biological organisms naturally hold aggregation at bay and reveal novel aspects of mis-folding diseases, the group of diseases that is rapidly becoming both the most costly in terms of health care and the most feared, in the developed world.

Planned Impact

The primary potential beneficiaries of my research in the longer term will be those in the wider public that suffer from mis-folding diseases such as senile dementia and those that live in fear that this condition will strike them in their old age. At present, there remain some very fundamental questions surrounding the process of protein aggregation and the biological mechanisms that have evolved to regulate it. Clearly the role of molecular chaperones is central to the body's aggregation defence network. The sHSPs are able to prevent and in some cases reverse aggregation in vitro. This is precisely the reason why I have chosen to study them at the start of my independent career. By delineating the molecular mechanism by which sHSPs can prevent protein aggregation I hope that my research will lead to novel insight into how we can combat, for example, neurodegenerative disorders.

Protein mis-folding diseases represent an enormous threat to our very quality of life, and the threat looms particularly as we age. One of the most feared conditions in old age is that of senile dementia, associated with neurodegenerative disorders. A recently published report from the World Alzheimer's society has revealed that there are 36 million cases worldwide of senility resulting from neurodegenerative conditions, costing the world's economy 1% of its GDP each year in care (£388 billion). By 2030 there are predicted to be 66 million, at an anticipated cost of £700 billion. The study concludes that dementia poses the most significant health and social crisis of the century. Such rapidly proliferating conditions are clearly a grave reality to an ever growing proportion of the total global population and a looming threat to an even greater proportion. Specifically in the UK, a study by Oxford University and the Alzheimer's Research Trust estimated that the total cost to our economy due to dementia is £23 billion p.a., exceeding that of £12 billion p.a. for Cancer and £8 billion p.r heart disease. Current treatments revolve around long term care, as there are no treatments at present that can deal with the cause of such conditions. Consequently, there is a clear public need for the biomedical sciences to produce quality research that can treat these disorders. I believe that in characterizing in detail how molecular chaperones are able to suppress protein aggregation in vitro, my proposed research will be helping to fulfill this need.

The research staff working on this project will be trained in molecular biology, protein expression, biophysics and applications of solution NMR to studies of structure, interactions and dynamics of proteins as well as rigorous interpretation of quantitative data. These are skills essential in many biological laboratories and companies large and small exploiting biotechnologies. In addition, establishing the research laboratory in the UK will lead to transfer of knowledge from Canada to the UK, enabling researchers access to the methodology of solution NMR of supra-molecular protein complexes up to 1MDa (Sprangers & Kay, Nature, 2007) and studying lowly populated protein states (Korzhnev et al, Science, 2010). The transfer of methodology will start immediately. I expect that the first publications will be submitted within two years from starting the research.
 
Description We have discovered mechanisms by which molecular chaperones prevent amyloid formation.
Exploitation Route This is being used by other researchers to understand biomolecular interactions. The results are being used in industry to design and screen drugs against Alzheimers and Parkinson's disease.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

URL http://baldwinlab.chem.ox.ac.uk/publications/index.html
 
Description BBSRC Follow-on fund
Amount £350,000 (GBP)
Funding ID BB/R005516/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2018
 
Description New NMR spectrometer
Amount £100,000 (GBP)
Funding ID 151/133 
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 02/2016
 
Description New electronics for NMR spectrometer
Amount £100,000 (GBP)
Funding ID 131/011 
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 02/2013
 
Description Methyl Assignments 
Organisation Chartered Institution of Building Services Engineers (CIBSE)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are working with these companies to analyse data describing protein/drug interactions involved with amyloid diseases. The analysis method was developed by work on a BBSRC grant, that is in the process of being awarded a patent. We seek to commercialise this.
Collaborator Contribution They supply data and we analyse it.
Impact Nothing to date. This year (2016) should reveal outcomes.
Start Year 2015
 
Description Methyl Assignments 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution We are working with these companies to analyse data describing protein/drug interactions involved with amyloid diseases. The analysis method was developed by work on a BBSRC grant, that is in the process of being awarded a patent. We seek to commercialise this.
Collaborator Contribution They supply data and we analyse it.
Impact Nothing to date. This year (2016) should reveal outcomes.
Start Year 2015
 
Description Methyl Assignments 
Organisation Pfizer Inc
Country United States 
Sector Private 
PI Contribution We are working with these companies to analyse data describing protein/drug interactions involved with amyloid diseases. The analysis method was developed by work on a BBSRC grant, that is in the process of being awarded a patent. We seek to commercialise this.
Collaborator Contribution They supply data and we analyse it.
Impact Nothing to date. This year (2016) should reveal outcomes.
Start Year 2015
 
Description Shire 
Organisation Shire Pharmaceuticals
Department Shire UK
Country United Kingdom 
Sector Private 
PI Contribution By sharing materials and expertise, we have worked on investigating how one of their proteins, GCase prevents aggregation of alpha-Synuclein, a protein involved in Parkinson's disease.
Collaborator Contribution They sent us materials.
Impact A paper which is about to be submitted (Shire legal department are currently looking over it).
Start Year 2014
 
Title BIOMOLECULE SEPARATION AND MODIFICATION 
Description A method for biomolecular separation and modification using a biologically inspired set of reagents developed during a BBSRC project 
IP Reference GB1518477.3 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact None
 
Title Method for assignment of methyl NMR spectra 
Description A method for assigning methylTROSY NMR spectra. 
IP Reference GB1608969.0 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact New collaborations, and more researchers both industrial and academic using our method.
 
Title DynamXL 
Description Software to compare experimental cross-linking data to structures that takes the shortest path between two sites and takes into account the variable 3D surface of a protein. 
Type Of Technology Software 
Year Produced 2017 
Impact We currently have ca. 50 downloads from predominantly academic groups. 
URL http://dynamxl.chem.ox.ac.uk/
 
Title IMPACT 
Description Software to calculate the collision cross section of biomolecules. Useful for interpretting native ion mobility mass spectrometry data. 
Type Of Technology Software 
Year Produced 2015 
Impact Has been adopted by the community. 
URL http://impact.chem.ox.ac.uk/
 
Title MAGMA 
Description Software to assign methylTROSY NMR data. 
Type Of Technology Software 
Year Produced 2016 
Impact People have downloaded it and used it. 
URL http://magma.chem.ox.ac.uk/
 
Title PULSAR 
Description Software for analysis of mass spectrometry data. Design to show whether or not specific lipids bind membrane proteins. 
Type Of Technology Software 
Year Produced 2016 
Impact It has been widely downloaded and used. 
URL http://pulsar.chem.ox.ac.uk
 
Title Unidec 
Description Deconvolves mass spectrometry data. Highly useful for analysing complex heterogeneous mixtures. 
Type Of Technology Software 
Year Produced 2015 
Impact Has been downloaded many times. The native mass spectrometry community are giving us great feedback and are enjoying using it. 
URL http://unidec.chem.ox.ac.uk/