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0:00
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.
0:12
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.
1:16
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.
