Researchers have combined sophisticated biochemical and imaging
techniques to get a glimpse of the stepwise assembly of amyloid fibers
in a yeast prion protein. Their findings suggest that these structured
fibers form in competition with the amorphous globules that some believe
may cause toxicity in amyloid diseases such as Alzheimer's and
Parkinson's. The researchers say this may have important implications
for those designing drugs to prevent formation of the brain-damaging
proteins in those diseases.
The researchers reported their findings in the October 2004 issue of the
Public Library of Science Biology. They were led by Howard Hughes
Medical Institute investigator Jonathan S. Weissman at the University of
California, San Francisco. HHMI investigator Ronald D. Vale, also of
UCSF, was a co-author of the article.
Working in yeast, Weissman and his colleagues investigated the mechanism
by which a prion protein assembles individual polypeptides into long
amyloid fibers. These fibers are similar to the amyloid plaques that
clog the brains of patients with Alzheimer's or Parkinson's disease.
Unlike bacteria and viruses, prions consist only of aberrant proteins
that misfold into forms that, in turn, induce normal proteins to
misfold. In mammalian prion infections, these abnormal, insoluble
proteins trigger protein clumping, producing a plaque that can kill
brain cells. In humans, clumping causes fatal brain-destroying diseases
such as Creutzfeldt-Jakob disease and kuru; in animals it causes bovine
spongiform encephalopathy (mad cow disease) and scrapie.
In the yeast cells Weissman and his colleagues used as research models,
however, the insoluble prion merely alters a cell's metabolism. Besides
offering a model for studying prions, the yeast system also provides an
excellent model for the growth and aggregation of amyloid protein, said
Weissman. Studying this process could have important implications for
understanding amyloid diseases, he said.
Initial efforts to understand amyloid formation compared the process to
the formation of the cell's cytoskeleton, – a better understood
mechanism known as nucleation-polymerization, in which the cytoskeletal
proteins (actin and tubulin) coalesce into long fibers. Experiments from
a number of labs, however, revealed that this process could not explain
amyloid formation.
Amyloid formation was also associated with the transient accumulation of
intermediate molecules that have been implicated in causing disease.
"The process of forming amyloids seems to be implicated in disease
perhaps as much as the actual aggregates themselves," said Weissman. "So
understanding why some proteins form amyloids and aggregate, and under
what conditions that occurs, and the intermediate processes involved, is
critical in determining what is toxic about amyloid and how it might be
possible to affect the pathology it causes. Yet despite the importance
of this process, we know little about the underlying mechanism by which
amyloid forms and grows."
For example, said Weissman, evidence is accumulating that it is not the
plaque itself that is toxic, but rather the smaller and more amorphous
oligomers that typically accompany plaque formation. But why such
oligomers form and what role they play in making amyloid plaques was
unknown.
Weissman and his colleagues sought to understand the dynamics of how the
amyloid puzzle pieces assemble themselves. They analyzed the timing of
the yeast prion protein assembly, and how that varied with different
concentrations of the fiber and of the individual units, or monomers,
that add to the growing fibers. The researchers also explored a
particularly puzzling feature of amyloid formation: the fact that
agitation dramatically accelerates the process.
Drawing on expertise in the Vale laboratory, the scientists complemented
these indirect studies with "single-molecule fluorescence technology" to
observe fiber growth directly. In this technique, an immobilized fiber
is first tagged with one fluorescent molecule. When shorter amyloid
segments or monomers are tagged with a fluorescent molecule of a
different color and added to the immobilized fiber, researchers can
watch the growth of the fiber.
The analytical approaches revealed that the yeast amyloid fibers grow by
the addition, one by one, of individual monomers -- rather than assembly
of amorphous, globular oligomers. Thus, said Weissman, if the oligomeric
globules are, indeed, the toxic molecules, they form in competition with
the structured fibers, rather than being key intermediates in fiber
formation. Such a possibility could have implications for treating
amyloid diseases such as Alzheimer's and Parkinson's, he said.
"Investigators are now screening for drugs that would prevent amyloid
from forming, to treat these disorders," said Weissman. "While it is
quite speculative at this point, if such drugs favored the production of
more oligomers, which are toxic, then those drugs could actually have
the opposite effect than was intended. Conversely, drugs that encourage
the rapid formation of a relatively inert and stable amyloid might
deplete the toxic oligomers and therefore be beneficial," he said.
Weissman emphasized that basic studies of amyloid formation must be
extended beyond the yeast prion model before the monomer-addition
mechanism can be considered a general one. Thus, he and his colleagues
are now studying the mechanism of formation of other amyloid proteins,
including the molecular details of how individual monomers bind to a
growing fibril.