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The bovine prion protein: from structure analysis to the molecular mechanism of conformational transitions

Objective



The main objective of the present proposal is to understand the mechanisms by which native prion proteins become misfolded in such a way as to provoke neurodegenerative diseases and become transmissible agents. In order to achieve this aim, we will take advantage of the mature bovine prion protein which can be generated de novo and in large amounts by the participants. More particularly, the topics of investigation will be:
1. Determination of the structural, biochemical and electrophysiological features of the mature bovine prion protein in its native and mutated forms
2. Structural identification of intermediate conformations
3. Search of in-vitro conditions leading to the conversion from the native to the pathological conformation of the prion protein.
4. Role of molecular chaperones in thwarting such transition and the consequent aggregation phase.

These topics are particularly pertinent within the framework of the "protein only" hypothesis of transmissible spongiform encephalopathies, which states that some abnormally folded molecules of the cellular prion isoform impose their three-dimensional aberrant characteristics on the normal protein, possibly by simple contact, thus spreading the infection within the cell and then to other cells and animals. Biochemical and spectroscopic investigations of the two isoforms have led to the conclusion that the native protein (PrPc) is rich in a helix while the pathogenic form (PrPSc), albeit with an identical primary sequence, contains almost exclusively beta-sheets. These studies, however, have been severely hampered by the paucity of expression of the native form or by insolubility of the pathogenic form, frequently aggregated in amyloid-like plaques. Although much effort has been devoted in the heterologous expression of the protein in eukaryotic cells or in bacteria, so far only parts of the prion protein have been recovered and isolated. Recently, one of these fragments (from mouse) was studied by NMR. Thus, a molecular model for the complete sequence necessary for the search of conformational transitions is still missing. In our laboratories, the full length mature bovine prion (25- 242), expressed intracellularly in, and recovered from, E. coli, is now available in a soluble form. This opens the way to projects aiming to gain detailed biochemical and 3-dimensional structural determinations of the protein as well as insight into the chemico-physical and/or biological mechanisms which are fundamental to the process of conversion from the native to the pathogenic form.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITA DEGLI STUDI DI PADOVA
Address
Viale G. Colombo 3
35121 Padova
Italy

Participants (2)

MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.
Germany
Address
Am Klopferspitz 18A
82152 Martinsreid
THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom
Address
South Parks Road
OX1 3QT Oxford