March 18, 2004
Experiments Establish "Protein-Only" Nature of Prion Infections
Two independent research groups have established conclusively that
prions are proteins, and that they do not depend on genes or other
factors for transmission of their traits. According to the scientists,
the studies answer a nagging question that had raised doubts among some
researchers about the validity of the so-called
“protein-only” hypothesis of prion infectivity.
Scientists have grappled for years with one of the central tenets of
the protein-only hypothesis, namely, that a single prion protein, when
unaltered by genetic mutation, can give rise to different strains of
prions with varying infectivity and other properties. The two research
groups established that the strains could be accounted for by different
misfolded conformations of the same protein. The researchers say this
finding could contribute to better understanding of the functioning of
disease-causing prions in animals and humans.
“I would say this puts to rest any question about whether the protein-only prion hypothesis as a general principle is true.”
Jonathan S. Weissman
Both groups published their findings in the March 18, 2004, issue of
the journal Nature. Howard Hughes Medical Institute investigator
Jonathan
S. Weissman at the University of California at San Francisco led
one group. The other effort was led by Chi-Yen King at Florida State
University.
Both groups worked with yeast prions, which are similar to the
mammalian prions known to cause fatal brain-destroying human diseases
such as Creutzfeldt-Jakob disease and kuru, and the animal diseases
bovine spongiform encephalopathy (“mad cow disease”) and
scrapie.
Scientists theorize that both yeast and mammalian prions transmit
their characteristics via protein-protein interactions, in which an
abnormally folded prion influences its normal counterpart to assume an
irregular conformation.
In mammalian prion infections, abnormal, insoluble shapes trigger
protein clumping that can kill brain cells. In yeast cells, the
insoluble prion protein is not deadly; it merely alters a cell's
metabolism.
Both the mammalian and yeast prions adopt similar infectious
conformations characterized by a high content of beta-sheet structures.
These beta-sheet-rich aggregates, commonly referred to as amyloid, are
also associated with a number of noninfectious neurodegenerative
diseases including Alzheimer's disease and Parkinson's disease. In both
yeast and mammalian prions, the generation of different strains can
sometimes enable prions to jump the “species barrier” — to
infect a species other than the one originally infected.
While considerable research had indicated that amyloids were a key
component of prions, many researchers had suggested that other
components, including perhaps RNAs, might underlie the differences in
the various prion strains.
“I would say this puts to rest any question about whether the
protein-only prion hypothesis as a general principle is true,”
said Weissman of his group's findings. “And it also establishes
that prion strains can be accounted for solely by the ability of the
protein to misfold into more than one conformation. There might be
other factors that influence it in mammalian prions, but at this point
people have to prove that there are; there is no reason to suspect that
there need be.”
The researchers from Florida State conducted experiments
demonstrating that different strains of yeast prions can transmit their
strain-specific characteristics simply through “seeding” by
a prion protein.
“What we were looking for was a smoking gun,” Weissman
said of the experiments in his laboratory. “We wanted to be able
to take one protein, misfold it into two different self-propagating
infectious conformations and show that you get two different strains,
with no possibility of there being another molecule there at
all.”
To do so, the lead author, Motomasa Tanaka, developed a technique to
generate specific strains of yeast prion proteins simply by varying the
temperature at which the newly produced proteins folded into their
infective shapes.
“The use of temperature to influence folding was an elegant
approach, because once you've changed the temperature, it leaves no
trace in the solution,” said Weissman. “There are no other
molecules that it might be argued are contributing to the
differences.”
In test tube experiments, the researchers demonstrated that the
protein conformations produced at different temperatures propagated
themselves as distinct strains — providing templates for the folding
of other proteins into the same shapes. Further structural analyses of
two of the strains confirmed that the proteins were, indeed, folded
differently.
When the researchers introduced the differently folded proteins into
yeast cells, they found that inside cells, these proteins did indeed
produce different prion strains that passed their properties from
generation to generation. Finally, they showed that extracting prion
protein from subsequent generations of yeast cells yielded protein with
the same properties as the strain with which the cells had originally
been infected.
Weissman said that the ability to generate, manipulate and study
distinct prion strains in yeast should lead to more detailed studies of
how amyloid proteins form and propagate, which will be useful in
guiding future studies of strain properties of the disease-causing
mammalian prions.
“Clearly, it's technically much harder to work with mammalian
prions, in large part because they are dangerous and because they take
much more time to cause the disease,” said Weissman.
“Nonetheless, I think some of what we are learning about how to
make proteins misfold into different conformations will be directly
relevant to understanding mammalian prions, and perhaps even to trying
to understand the strain phenomenon in mammalian prions. This includes
how strains can affect the virulence of a disease or how likely it is
to jump a species.”
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