NIAID Scientists Characterize the Most Infectious Prion Protein Particles
A new study of prions—apparently malformed proteins that initiate deadly brain diseases such as Creutzfeldt-Jakob disease in humans—has yielded surprising information about how the size of prions relates to their infectivity. Scientists have found that small prions are much more efficiently infectious than large ones, yet there also is a lower size limit, below which infectivity is lost.
“Researchers developing treatments for prion diseases can now focus on the most efficient purveyors of infection,” notes Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, which funded the research. “The persistence and creativity associated with this work is admirable.” The study appears in the September 8 issue of the journal Nature.
Prions appear to consist primarily of an abnormal form of a protein molecule called PrP. The new research, led by scientists at NIAID’s Rocky Mountain Laboratories (RML) in Hamilton, MT, reveals that the most infectious prions are significantly smaller than the large thread-like deposits of PrP molecules readily seen in the diseased brains of infected individuals. Yet to be infectious, a prion must be much larger than the single malformed PrP molecule that has long been thought to be the basic unit of infectivity.
Prion diseases—also known as transmissible spongiform encephalopathies (TSEs) because the prions create holes in the brain, giving it a sponge-like appearance—include Creutzfeldt-Jakob disease in humans, mad cow disease in cattle, scrapie in sheep and chronic wasting disease in deer and elk. Scientists have known that infectious prions range widely in size, but now, for the first time, the RML team has ranked them according to their infective efficiency and their findings have placed new limits on the size of the smallest prion.
Many neurodegenerative diseases such as Alzheimer’s, Parkinson’s and TSE diseases are characterized by abnormal protein deposits in the brain. But questions abound as to what types and sizes of protein deposits are the prime causes of disease.
Prions appear to be crystal-like clusters of PrP molecules that can grab normal, dissolved PrP molecules and convert them to a solid, crystal-like state, says RML senior researcher Byron Caughey, Ph.D. “Although large prion particles can do this, and are infectious, you can infect many more individuals, or cause much more rapid disease in a single individual, with an equivalent weight of small prion particles,” says Dr. Caughey. “But our findings also suggest that if the PrP cluster is smaller than a certain minimum size, it becomes unstable and loses its infectious properties.”
Normal PrP molecules found in many animals do not cause harm. But PrP molecules can become lethal and destroy the brain when they refold and gather into precisely ordered clusters. This basic infectious process is reminiscent of disease processes seen with other prominent neurological diseases, except that in each disease a different protein is involved.
The new RML research is consistent with the recently emerging evidence that, in many of the neurological protein aggregation diseases, small, misfolded clusters are more damaging than large clusters. Indeed, some scientists have suggested that the largest abnormal protein deposits may be the brain’s attempt to sequester many small toxic particles into a relatively inert dumping ground.
Thus, Jay Silveira, Ph.D., who spearheaded the RML study, cautions that treatments designed to break apart large accumulations of prions in the brain might do more harm than good by releasing the most infectious prion particles, resulting in more widespread damage than that caused by the original large cluster.
“Large deposits, or plaques, could be an attempt by the brain to detoxify the infectivity, to protect the brain,” says Dr. Caughey, who oversaw the project. On a graph illustrating how infectivity relates to PrP particle size, he notes that “as you increase particle size steadily from single molecules to particles containing thousands of molecules, there’s a sudden jump in infectivity once you get to the minimum infectious particle size (at least six PrP molecules per particle). Soon the most infectious particles appear (equivalent in weight to 14 to 28 PrP molecules per particle), followed by larger thread-like particles that are still infectious, but less so, per unit of protein,” he explains. A graph depicting the infectious efficiency of prions with respect to prion particle size can be seen at http://www3.niaid.nih.gov/news/newsreleases/2005/prion_graph.htm
The RML group completed its work using a new particle separation method that should be of interest in studies of other protein aggregation diseases, says Dr. Caughey. The process, called flow field-flow fractionation, or FlFFF, separates particles by size.
“A key to understanding a disease,” says Dr. Caughey, “is knowing what to attack and what to ignore: Do we focus on the large clumps, as scientists initially thought, or their smaller precursors?”
The RML researchers are now trying to isolate the molecular components of the most infectious prions to analyze what else is present. “There could be unknown components in there that help the infection spread,” says Dr. Caughey.
He says other researchers may have avoided this particular project because of the great potential for failure. “This project involved about three years of arduous work and was risky for a postdoctoral researcher looking to establish a career,” explains Dr. Caughey. “The separation device we used is not common in our field of work, so we had to adapt it to fit our purposes. But the extra work paid off—now we’re providing a new technological approach to solving important questions about these diseases.”
That approach included isolating aggregates of infectious prions from the brains of scrapie-infected hamsters and dispersing them into detergents. Dr. Silveira then “fractionated” the prions, or separated them according to physical size, and inoculated them into hamsters. The RML scientists determined the masses of the prion particles and ranked their infectivity by tracking the number of days that passed until the hamsters showed symptoms of scrapie.
Dispersing and fractionating the prions were the most challenging parts of the experiment, says Dr. Caughey. “At a certain point, the particles become too small to be infectious and they can accidentally be destroyed,” he says. Dr. Silveira used a variety of protein dispersion methods, including detergents, sound waves, freezing and thawing, and chemicals, before sorting by size. “He eventually found a set of conditions that worked well to generate very small particles that were still infectious,” explains Dr. Caughey.
NIAID is a component of the National Institutes of Health, an agency of the U.S. Department of Health and Human Services. NIAID supports basic and applied research to prevent, diagnose and treat infectious diseases such as HIV/AIDS and other sexually transmitted infections, influenza, tuberculosis, malaria and illness from potential agents of bioterrorism. NIAID also supports research on transplantation and immune-related illnesses, including autoimmune disorders, asthma and allergies.
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Reference:
JR Silveira et al. The most infectious prion protein particles. Nature. DOI: 10.1038/nature03989.
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