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Jacob Corn
Prions: New Incongruities in the Protein-Only Hypothesis
Prions are perhaps most famous for their role in bovine spongiform
encephalopathy, or the infamous “mad cow disease” that plagued Britain. Sheep also
carry a well-known type of prion disease known as scrapie. In humans, however, there
exist four main “strains” of prion diseases: kuru, Creutzfeldt-Jakob Disease (CJD),
Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI)
(Wong, Clive, Haswell, Jones, and Brown, 2000). Kuru, also known as the “laughing
death” due to the uncontrollable laughter exhibited by its victims, was the first human
prion disease described, and was prevalent among the Fore Highlanders of Papua New
Guinea who honored the deceased by eating their brains. The cause of death, as in all
prion diseases, was large holes present in the victim’s brain, creating a sponge-like
appearance. This symptom of prion infection has led prion diseases to be termed
Transmissible Spongiform Encephalopathies, or TSEs (Prusiner, 1995).
Prion diseases are incurable and ultimately fatal, with death occurring anywhere
between 300 days to decades after infection. Surprisingly, prion diseases do not fit into
any class of currently known pathogens. The presence of foreign particles in infected
brain tissue and species barriers to prion infection (for example, humans can be infected
by exposure to spongiform bovine brain extracts, but not by spongiform mouse brain
extracts) seem to fit a viral model of transmission. However, In 1972 Dr. Tikvah Alper
discovered that ultraviolet or ionizing radiation, which should degrade any nucleic acid
present in a sample, had no affect upon prion infectivity (Prusiner, 1995). Later work by
Dr. Stanley Prusiner also showed that infectivity was not decreased under conditions
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even more damaging to nucleic acid, such as heating in the presence of zinc ions and
treatment with psoralens or hydroxylamine (Prusiner, 1982). Taken alone, these results
could have meant that the nucleic acid portion of the infecting agent was somehow
shielded from radiation and harmful reagents. However, Prusiner also found that prion
infectivity was temporarily decreased under acid conditions and permanently decreased
under alkaline conditions or in the presence of a detergent such as SDS, all of which are
environments known to damage proteins. In conjunction with the nucleic acid data, this
suggested that prion diseases were actually transmitted via proteins, leading Prusiner to
propose the now generally accepted name “protinaceous infectious particles,” or “prions”
(Prusiner, 1982). The hypothesis that a protein alone could be infectious was met with
great skepticism, since it suggested a non-nucleic acid vehicle of information. According
to the Central Dogma of Biology, nucleic acid in the form of DNA or RNA, is the
cellular “data molecule” through which information is replicated and passed on. The
prion hypothesis began to gain respect, however, as inheritable components to GSS, FFI,
and familial CJD were discovered, corresponding to a gene coding for the PrP protein.
Furthermore, brain extracts from diseased mice that had inherited a familial prion disease
were able to infect healthy mice (Prusiner, 1994).
Once sequenced, it was discovered that all mammals carry the PrP gene,
regardless of whether they show signs of encephalopathy, suggesting the existence of a
non-infective prion (now known as PrPC) (Stahl, Baldwin, Teplow, Hood, Gibson,
Burlingame, and Prusiner, 1993). The normal PrP gene product was quickly sequenced,
leading to a hypothesis of how a protein can display virus-like species barriers to
infection. In mammals that could infect each other, the amino acid sequences of PrPC
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were almost completely identical. Where a species barrier existed, the primary sequence
differed by several amino acids (Cohen, Pan, Huang, Baldwin, Fletterick, and Prusiner,
1993). However, it wasn’t until 1993 that the primary structure of a putative infectious
protein (known as PrPSc) from infected brain tissue was determined, and a lack of
posttranslational modification differences between PrPC and PrPSc noted (Stahl et al.,
1993). Therefore, it seemed the only major difference between infectious prions and
normal prions was secondary and tertiary structure.
The infectious protein hypothesis gained significant respect when circular
dichroism and Fourier-transform infrared spectroscopy analyses of PrPC and PrPSc
indicated major differences in secondary structure between normal and infectious prions.
While PrPC is composed of mostly α-helices (42%) and almost no β-sheets (3%), PrPSc is
mostly β-sheets (43%) with lower amounts of α-helices (30%) than PrPC. Additionally,
isolated PrPSc exhibited aggregation into rod-shaped amyloids that were very similar to
protein plaques observed in the brains of infected organisms, suggesting that it was
important for the formation of TES symptoms (Pan, Baldwin, Nguyen, Gasset, Serban,
Groth, Mehlhorn, Huang, Fletterick, Cohen, and Prusiner, 1993). These findings strongly
implicated conversion of α-helices in PrPC to β-sheets in PrPSc as the primary event in the
formation and propagation of infectious prions.
Since prion diseases are invariably fatal, there has been some confusion as to why
the PrP gene, which codes for a protein that invariably causes death when turned into
PrPSc, has not been modified or removed due to evolutionary pressure. In fact,
recombinant mice totally lacking the PrP gene are able to develop normally, but express
minor defects depending on the null strain (Mouillet-Richard, Ermonval, Chebassier,
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Laplanche, Lehmann, Launay, and Kellermann, 2000). The first evidence signifying a
critical normal biological function of PrPC was the appearance of neuronal degradation in
mice expressing an N-terminally truncated version of PrPC. This suggested that if
completely removed, the body is able to compensate for the lack of PrPC, but if PrPC is
present an N-terminal feature is crucial to neuronal homeostasis. Unfortunately, the Nterminal structure of PrPC has not yet been determined, but recent research has shown
that copper binds to PrPC with a 4:1 stoichiometry somewhere within a series of Nterminal octarepeats (Baskakov, Aagard, Mehlhorn, Wille, Groth, Baldwin, Prusiner, and
Cohen, 2000). When bound with copper PrPC acquires some superoxide dimutase
activity, and therefore may prevent neuronal cell death due to oxidative stress. Of
considerable note, however, was a shift towards increased β-sheet structure within PrPC
upon copper binding (Wong et al., 2000). This result suggested that the greatly increased
β-sheet content of PrPSc may be a part of the normal function of PrPC that has proceeded
faultily, due to either a mutation within the PrP gene (causing inheritable spongiform
encephalopathy) or after exposure to a “template” PrPSc (causing infectious spongiform
encephalopathy).
All of the above evidence lent credence to the idea that a protein could act as an
information-carrying molecule. However, recent research has found several
inconsistencies in the theory that PrP proteins are the infectious molecule in prion
diseases. Since the discovery of prion diseases, it had been noted that the prion agent
caused infection when taken in orally, but the protein-only hypothesis made no attempt to
address how a foreign pathogenic protein could be transported intact from the stomach to
the brain. Emerging studies have shown that prion infections spread from the alimentary
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tract to the lymphatic system, and finally into the spinal cord and brain through the
autonomic nervous system. However, if the spleen of an organism is removed prior to
infection, the appearance of disease symptoms is markedly delayed (Will and Ironside,
1999). The increase in incubation time implies that the infectious agent may initially
replicate in the lymphatic system and then migrate to the nervous system, a model of
infection similar to that found in some viruses, but should be impossible with a
protinaceous pathogen.
Additionally, three findings published within the last two years almost definitely
invalidate the protein-only hypothesis, but unfortunately confuse the nature of prion
infection even further. First, while initially unfolded protein fragments similar in
sequence to the beta-sheet rich portions of PrPSc exhibit spontaneous self-assembly into
beta-sheet structures (Baskakov et al., 2000), full-length PrPSc produced in vitro is not
sufficient to cause prion infection (Hill, Antoniou, and Collinge, 1999). Second, early
studies separating out infected brain isolates had shown that fractions containing protein,
not nucleic acid, caused prion diseases when introduced into healthy animals. However,
more precise separation of infectious isolates showed that fractions containing more than
90% of the PrPSc present did not cause prion diseases when introduced into healthy
animals. The most infectious fraction was, in fact, an extremely low-density fraction of
unknown composition containing only trace amounts of PrPSc (Manousis, VergheseNikolakaki, Keyes, Sachsamanoglou, Dawson, Papadopoulos, and Sklaviadis, 2000).
Finally, the most confusing piece of evidence came in the form of heat resistance studies.
While it has been known for years that prion diseases remain infectious after heating to
temperatures over 150°C, it was only in mid-2000 that research tested the extent of prion
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heat resistance. After completely ashing infected brain samples at 600°C, infectivity was
still present after reconstitution in saline. Further, infectivity was only totally abolished
after exposure to 1,000°C (Brown, Rau, Johnson, Bacote, Gibbs, and Gajdusek, 2000).
These extreme temperatures, which are more than sufficient to destroy all organic matter,
suggest an inorganic agent that can act as a template for biological replication of prion
diseases.
Researchers are currently reluctant to venture new theories about the origin of
prion diseases. Just when evidence seemed to suggest that Dr. Stanley Prusiner’s radical
protein-only hypothesis was feasible, new research has shown that prions may be
something even more unusual than infectious proteins. As inorganic models of
replication are examined, the nature of prion diseases may become better understood, and
some defense from infection discovered.
Literature Cited
BASKAKOV, I., C. Aagard, I. Mehlhorn, H. Wille, D. Groth, M. Baldwin, S. Prusiner,
and F. Cohen. 2000. Self-assembly of recombinant prion protein of 106 residues.
Biochemistry. 39: 2792-2804.
BROWN, P., E. Rau, B. Johnson, A. Bacote, C. Gibbs, D. Gajdusck. New studies on the
heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600
degree C suggests an inorganic template of replication. Proceedings of the National
Academy of Sciences, USA. 97: 3418-3421.
COHEN, F., F. Pan, Z. Huang, M. Baldwin, R. Fletterick S. Prusiner. 1994. Structural
clues to prion replication. Science. 264: 530-532.
HILL, A., M. Antoniou, J. Collinge. 1999. Protease-resistant prion protein produced in
vitro lacks detectable infectivity. Journal of General Virology. 80: 11-14.
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MANOUSIS, T., S. Verghese-Nikolakaki, P. Keyes, M. Sachsamanoglou, M. Dawson,
O. Papadopolous, T. Sklaviadis. 2000. Characterization of the murine BSE infectious
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PAN, K., M. Baldwin, J. Nguyen, M. Gasset, A. Serban, D. Groth, I. Mehlhorn, Z.
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PRUSINER, S. 1982. Novel Protinaceous Infectious Particles Cause Scrapie. Science
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PRUSINER, S. 1994. Biology and Genetics of Prion Diseases. Annual Review of
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