Award details

Functional proteomics of the neuronal membrane complex enclosing normal and infectious prion proteins

Reference	BB/C506680/1
Principal Investigator / Supervisor	Professor Roger James Morris
Co-Investigators / Co-Supervisors
Institution	King's College London
Department	Wolfson Centre for Age Related Diseases
Funding type	Research
Value (£)	203,463
Status	Completed
Type	Research Grant
Start date	01/10/2004
End date	30/09/2007
Duration	36 months

Abstract

The critical infectious event in the prion diseases (or transmissible spongiform encephalopathies: in man, CJD; cattle, BSE; sheep, scrapie) is believed to be the conversion of a normal cellular protein, prion protein, into an altered, extraordinarily stable, conformation. Normal cellular prion protein is readily degraded (it has a half-life of hours), has little beta-pleated sheet and does not aggregate; the pathogenic form strongly resists proteolysis and almost all forms of denaturation, readily aggregates to form amyloid fibrils, and appears to be quasi-immortal. Although it is not clear why the pathogenic form kills neurons, it is easy to see that the remorseless accumulation of non-degradable material in the brain is ultimately harmful. That this is necessary and sufficient to sustain prion infection should be readily testable: since the pathogenic conformation is so stable, it must be possible to convert pure, recombinant prion protein into this conformation in the test tube, inject this in vivo and induce an infectious prion disease. All attempts to produce in vivo infection by this method over the past 20 years have failed, leading to the suspicion that other membrane components make an obligatory contribution to the infectious conversion process. In addition, analyses of the infection process, such as by Prusiner¿s group studying the basis for the species barrier to transmission in transgenic mice, have concluded that other components (termed protein X by Prusiner) are necessary for infectious conversion. Knowing the protein and other membrane components that surround cellular prion protein, and which influence its interaction with infectious prions, has been a major goal in research in this area for the past decade. The trouble is that prion protein is remarkably difficult to study. We have recently shown that this is due, at least in part, to the fact that on neurons prion protein is trafficking remarkably rapidly, leaving specialist sphingolipid rafts in which (as a GPI-anchored protein) it should normally reside, to cross normal surface membrane, enter coated pits and recycle through early endosomal compartments back to the cell surface. This poses a problem ¿ the protein occupies in quick succession a number of membrane environments; but also a solution ¿ no GPI-anchored protein can traffic like this by itself, it has to bind to other, transmembrane proteins that are constitutively endocytosed. We further identified the amino acid motif on prion protein that mediates this trafficking (i.e. that binds to the transmembrane partner). We have gone on to show that the raft environment of prion protein can be reliably isolated by its insolubility in the non-ionic detergent Brij 96, followed by immunoprecipitation. We have used ESI-MS/MS analysis to quantitatively identify the lipid composition of these rafts. This project is to isolate PrP rafts for protein (rather than lipid) analysis so as to identify the proteins with which normal cellular prion protein interacts. This will be complemented by chemical cross-linking, to give a complementary view of the molecular environment of prion protein. With this background, the environment of prion protein in infected cells and brains will also be identified. Identification will be by MS/MS at the new Proteomics Unit at the Institute for Animal Health laboratories in Compton, headed by Dr Andrew Gill. The picture that emerges of the proteins associated with prion protein on neurons, will be tested using high resolution fluorescent and EM imaging. Overall, this work couples the ability of the Morris lab to dissect out the different environments of prion protein, with the state of the art Proteomics Unit at IAH Compton, to answer a major question about the mechanism by which the infectious conformation of prion proteins is produced.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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