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/Source:/       *Howard Hughes Medical Institute*       
/Date:/         2004-09-28
/URL:/  http://www.sciencedaily.com/releases/2004/09/040921084443.htm

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   Amyloid Fibers Sprout One Step At A Time

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.

*Editor's Note:* The original news release can be found here 
<http://www.hhmi.org/news/weissman4.html>.

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/This story has been adapted from a news release issued by Howard Hughes 
Medical Institute./