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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 1 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 2 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, 3 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 4 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 5 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. 6 MANOUSIS, T., S. Verghese-Nikolakaki, P. Keyes, M. Sachsamanoglou, M. Dawson, O. Papadopolous, T. Sklaviadis. 2000. Characterization of the murine BSE infectious agent. Journal of General Virology. 81: 1615-1620. MOUILLET-RICHARD, S., M. Ermonval, C. Chebassier, J. Laplanche, S. Lehmann, J. Launay, and O. Kellermann. 2000. Signal transduction through prion protein. Science. 289: 1925-1928. PAN, K., M. Baldwin, J. Nguyen, M. Gasset, A. Serban, D. Groth, I. Mehlhorn, Z. Huang, R. Fletterick, F. Cohen, and S. Prusiner. 1993. Conversion of α-helices into βsheets features in the formation of the scrapie prion proteins. Proceedings of the National Academy of Sciences, USA. 90: 20962-10966. PRUSINER, S. 1982. Novel Protinaceous Infectious Particles Cause Scrapie. Science 216: 136-144. PRUSINER, S. 1994. Biology and Genetics of Prion Diseases. Annual Review of Microbiology. 48: 644-686. PRUSINER, S. 1995. The prion diseases. Scientific American. 272: 48-57. STAHL, N., M. Baldwin, D. Teplow, L. Hood, B. Gibson, A. Burlingame, and S. Prusiner. 1993. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry. 32, 1991-2002. WILL, R., J. Ironside. 1999. Oral infection by the bovine spongiform encephalopathy prion. Proceedings of the National Academy of Sciences, USA. 96: 4738-4739. WONG, B. C. Clive, S. Haswell, I. Jones, and D. Brown. 2000. Copper has differential effect on prion protein with polymorphism of position 129. Biochemical and Biophysical Research Communications. 269, 726-731. 7