Structural Studies of Prion Proteins and their ligand interactions

Lead Research Organisation: University College London

Abstract

The central basic research aim of the Unit remains determining of the atomic structures of the infectious agent. Prions consist of abnormal forms of one of the bodys own proteins, the prion protein, which has changed its shape and which then clumps together to form aggregates that build up in the brain. Highly specialised facilities and expertise have been developed at the Unit to study prion structure. Genetic engineering of bacteria has been used to make large quantities of various prion proteins to study their structure and also to learn how infectious prions can be made synthetically. Understanding the molecular mechanism of prion replication will be of major importance well beyond prion biology and disease. Such mechanisms will be of wide interest with respect to a range of diseases know to involve the bodys proteins changing their shape and forming aggregates, which includes most of the commoner degenerative brain diseases (such as Alzheimers disease and Parkinsons disease).

Technical Summary

Prion diseases such as variant CJD and BSE involve accumulation of misfolded forms of normally benign host protein (PrPC) in the tissues of the brain. These misfolded, disease-associated forms are known as PrPSc. To understand this switch in structure and to generate material for work on diagnostic and therapeutic aspects of the disease, we have developed recombinant methods for expressing PrP in E. coli. The production of large quantities of protein have allowed us to make made significant progress in studying the folding and unfolding pathways of human prion protein leading to the identification of several conformational isomers of the protein, in particular ?-PrP, which shares many biophysical characteristics with PrPSc. When ?-PrP is presented as an antigen for the creation of anti-PrP monoclonal antibodies, the resultant antibodies have affinity for native PrPSc. Defining the folding pathway of PrP is crucial to understanding the events leading to the formation of PrPSc. In the case of other amyloidogenic proteins partially structured folding intermediates have been implicated in the destructive aggregation reactions leading to disease. Using NMR techniques to define backbone amide exchange rates we have demonstrated that PrP, unlike other amyloidogenic proteins, does not populate any folding intermediates. This finding portends that native PrPC must unfold before rearrangement to the PrPSc conformation can occur. This finding has been of particular importance in the design of therapeutic strategies for prion disease. Our efforts in screening for potential anti-prion drugs have focused upon the identification of compounds that stabilise PrPC, and hence reduce the pool of unfolded molecules available for conversion to PrPSc.The biological function of PrP remains a subject of much debate; with suggestions that it may play a role in protection from oxidative stress or participate in copper homeostasis. The hypothesis that PrP may be a cupro-protein in vivo has lacked convincing support due to the apparent low affinity with which the protein binds divalent metal ions. Using a combination of fluorescence and NMR techniques we have fully characterized the affinity and location of such sites. A key objective for the future is to establish a synthetic source of prion infectivity. Although current work indicates that recombinant material may be rendered infectious, the titre appears low suggesting only a minor component is responsible. To mimic the situation in vivo more closely, mature, eukaryotically expressed PrP possessing a GPI anchor and carbohydrate moieties is required. The conversion of this material into a form that possesses high titre prion infectivity would represent the essential model by which the molecular pathogenesis of prion disease could be dissected. The determination of ?-PrP structure is a key element of furthering our understanding of the conformational events that underlie prion diseases. In addition to the solution structure of soluble ?-PrP we wish to map the interaction surfaces that dictate the ordered packing of PrP into fibrillar structures. Structural information on the nature of sub-unit interfaces in fibrillar PrP may assist in the rational design of therapeutics.

Publications

10 25 50
 
Description Amyloid super-resolution microscopy (TAB) 
Organisation Washington University in St Louis
Country United States 
Sector Academic/University 
PI Contribution Development of techniques and sample preparation for TAB microscopy
Collaborator Contribution Development of microscopy techniques and data analysis algorithms for TAB microscopy
Impact Outcomes: 1 research paper 29953718, 3 published conference abstracts this is a multi-disciplinary collaboration between Dr MD Lew (WUSTL), Department of electrocal Engineering - discipline: advanced optics and the Bieschke group (UCL Institute of Prion Diseases) - discipline: biophysics and neurodegenerative disease
Start Year 2017
 
Description Analytical Ultracentrifugation 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to relevant data, intellectual input into collaborators' research.
Collaborator Contribution Intellectual input, Training of staff, Publications
Impact none
Start Year 2008
 
Description Electron microscopy 
Organisation Birkbeck, University of London
Department Institute of Structural and Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to relevant data, intellectual input into collaborators' research.
Collaborator Contribution Intellectual input, Access to EM equipment and facilities, Training of staff in EM data acquisition, Publications
Impact none
 
Description Lipid / membrane Asscociations 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to relevant data, intellectual input into collaborators' research.
Collaborator Contribution Intellectual input, Access to equipment and facilities, Training of staff in lipid reconstution, Publications
Impact none yet
Start Year 2012
 
Description Molecular Modelling 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to relevant data, intellectual input into collaborators' research
Collaborator Contribution Provisiion of computing hardware and software, MD modelling experiments, intellectual input, publications
Impact none yet
Start Year 2010
 
Description Muscle amyloid 
Organisation Washington University in St Louis
Department School of Medicine
Country United States 
Sector Academic/University 
PI Contribution Biophysical and biochemical experiments characterizing the mechnism of prion replication in muscle amyloid proteins
Collaborator Contribution Cell biology and in vivo experiments characterizing the mechnism of prion replication in muscle amyloid proteins
Impact no outcomes yet
Start Year 2017
 
Description Protein crystallography 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to relevant data, intellectual input into collaborators' research.
Collaborator Contribution Intellectual input, Access to equipment and facilities, Training of staff, Publications
Impact none
 
Description biomolecular NMR 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input into collaborators' research. Access to AUC facilities. Laboratory space, supervision / funding for PhD students. Data acquisition and data analysis of structural NMR experiments on prion protein.
Collaborator Contribution Access to relevant data. Access to NMR facilities and data acquisition. Intellectual input into collaborators' research.
Impact none
 
Description synuclein structural study 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution preparation and characterization of amyloid seeds from Lewy body disease brain
Collaborator Contribution structural characterization of amyloid seeds from Lewy body disease brain
Impact no output yet
Start Year 2018