UCSF scientists report first direct demonstration that deformed prion protein shape alone causes inf

By Wallace Ravven

Scientists have shown for the first time that pure prion proteins can trigger
normal proteins to change shape and become infectious. The case for this novel
form of infection has been established over the past 20 years, but researchers
have been unable to directly demonstrate infection by pure prion proteins. The
finding by scientists at the University of California, San Francisco uses a new
system to introduce prions into yeast and eliminates the possibility that
non-prion proteins, sugars and other molecules contribute to the infection

The demonstration of pure prion infectious activity is reported in the July 28
issue of the journal Science.

Prions are able to replicate, aggregate and cause deadly infections in humans
and cattle without employing genes and DNA, the first infectious agents known
to do so. They are also linked to neurodegenerative diseases including
Alzheimer’s. Stanley B. Prusiner, MD, UCSF professor of neurology and
biochemistry and biophysics, won the 1997 Nobel Prize in Physiology and
Medicine for discovery of this entirely new category of disease-causing agent
and the elucidation of its mode of action.

Prions’ ability to make copies of themselves by inducing other proteins to take
on the deformed prion shape is not only a novel form of infection, but at least
in yeast constitutes a new mode of inheritance, says Jonathan Weissman, PhD,
UCSF associate professor of cellular and molecular pharmacology and senior
author on the Science paper.

Over the last few years, Weissman and his colleagues have taken advantage of a
powerful genetic system in yeast for rapidly testing the ability of a protein
to change shape into a prion and to propagate this form. The system can also
test for related protein changes involved in Alzheimer’s, Parkinson’s and other
human diseases caused by malformed aggregating proteins.

In studies with rodents and other mammals scientists have repeatedly shown that
when prions are introduced into animals or cell cultures, infection follows.
But no one had introduced pure prions in these experiments. Instead, the prions
were derived from previously infected animals and carried with them other
molecules that could not be completely ruled out as causing the infection,
Weissman says.

“Although the evidence that prions cause proteins to change shape and become
infectious is quite strong, there always remained the possibility that other
molecules introduced at the same time - sugars, other proteins, lipids—
contributed to the infection,” he explains.

But working with yeast, the researchers faced the opposite problem: They could
readily create pure infectious prions, but there was no clear way to introduce
them into the thick-walled yeast.

They adapted a delivery system that had been developed for studies of
mammalian cells by Francis Szoka, Jr., PhD, UCSF professor of biopharmaceutical

The new approach largely mimics the strategy viruses employ to introduce their
DNA into hosts. Where viruses employ a two-layered conformation known as the
lipid bilayer to encapsulate infectious DNA on its journey to its target, the
UCSF researchers used synthetic spheres called liposomes to encase the prions
for delivery into the yeast. And while viruses use co-proteins and receptors to
direct their protein packets into the right host cells, the liposome strategy
uses a small molecule called biotin to bind the liposomes and their prion cargo
to the yeast.

Finally, in place of the viral use of so-called fusion proteins to pierce the
host’s cell membrane, the UCSF strategy employed an alcohol molecule to breach
the defensive yeast cell wall.

“One molecule gets you in, and another breaks it down,” Weissman characterizes
his team’s approach.

With the artificial delivery system in place, the researchers were able to show
that pure prions could propagate indefinitely once they infected the yeast
cells. The team used fluorescent proteins to track changes in shape of the
infected yeast proteins, showing that the pure prions did in fact induce the
yeast protein to aggregate and adopt the prion shape.

The research also demonstrated that prions from one yeast species could not
infect other species. At least in yeast, the species barrier prevents
cross-species infection. The barrier has been thought to prevent transmission
of scrapie and mad cow disease from livestock to humans, but some recent
studies have found alarming evidence that in some cases prions from cattle may
infect other species, including humans.

First author on the paper is Helmut E. Sparrer, PhD, post-doctoral researcher
in Weissman’s lab. Lipsome strategist Frank Szoka is also a co-author, as is
Alex Santoso, PhD,  postdoctoral fellow in the Weissman lab.

The research was supported by grants from the National Institutes of Health,
the Searle Scholars program, the David and Lucile Packard Foundation, and the
European Molecular Biology Organization.