July 28, 2000 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 process.
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 chemistry.
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.
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