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Mechanism of prion generation in vitro
A selection of talks on Biochemistry
The ERK1/2 MAPK cascade
- Prof. Melanie H. Cobb
- University of Texas Southwestern Medical Center at Dallas, USA
Amino acid conjugation: mechanism and enzymology
- Dr. Kathleen Knights
- Flinders University, Australia
My name is Surachai Supattapone and I'm from Dartmouth Medical School. The subject of this talk will be the mechanism of prion generation in vitro.
According to the protein-only hypothesis, prion diseases are associated with the conformational change of a normal cellular protein known as the prion protein (or PrPc) into an abnormal disease-associated conformer known as PrPScrapie (or PrPSc). Whereas the normal PrPc structure is non-infectious, sensitive to protease digestion and has a structure which has been solved by NMR techniques, the PrPSc isoform has an unknown structure and is resistant to proteases, which is a very useful property for experimental detection of this isoform, as will be shown in subsequent slides. Furthermore, it is postulated that PrPSc is in fact the infectious entity of prion diseases. This talk will primarily concern recent discoveries that have been made in studying the mechanism of conformational change of PrPc to PrPSc in in vitro systems.
There have been two important biochemical models of PrPSc formation which have been developed. In 1994 Byron Caughey and his colleagues at Rocky Mountain Laboratories developed the cell-free conversion assay, in which a radioactive PrPc substrate was mixed together with purified PrPSc template. In this assay, it was observed that the radioactive PrPc substrate was converted into a protease-resistant PrPSc conformation in a species- and strain-specific manner. In the second assay, known as the 'protein misfolding cyclic amplification' (PMCA) assay developed by Claudio Soto and his colleagues in 2001, normal brain homogenates were mixed with prion-infected brain homogenates, and it was observed that the PrPc present in the normal brain homogenate was converted into the protease-resistant PrPSc conformation. Unlike the cell-free conversion assay, small amounts of PrPSc present in the infected brain were able to cause the autocatalytic transformation of larger amounts of PrPc into PrPSc. Furthermore, it could be shown that this technique generates infectious prions.