J Mol Biol 1996 Sep 6;261(5):614-619
Institut fur Molekularbiologie und Biophysik Eidgenossische Technische Hochschule Honggerberg, Zurich, Switzerland.
Prion diseases are assumed to be caused by the infectious isoform, PrPsc, of a single cellular surface protein, PrPc. PrPsc is an insoluble form of PrPc and is believed to possess a different three-dimensional fold. It may propagate by causing PrPc to adopt its own infectious conformation by an unknown mechanism. Studies on folding and thermodynamic stability of prion proteins are essential for understanding the processes underlying the conversion from PrPc to PrPsc, but have so far been hampered by the low solubility of prion proteins in the absence of detergents. Here, we show that the amino-terminally truncated segment of mouse PrP comprising residues 121 to 231 is an autonomous folding unit. It consists predominantly of alpha-helical secondary structure and is soluble at high concentrations up to 1 mM in distilled water. PrP(121-231) undergoes a cooperative and completely reversible unfolding/refolding transition in the presence of guanidinium chloride with a free energy of folding of -22 kJ/mol at pH 7. The intrinsic stability of segment 121-231 is not in accordance with present models of the structure of PrPc and PrPsc PrP(121-231) may represent the only part of PrPc with defined three-dimensional structure.
Nature 1996 Jul 11;382(6587):180-182
Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule-Honggerberg, Zurich, Switzerland.
The 'protein only' hypothesis states that a modified form of normal prion protein triggers infectious neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), or Creutzfeldt-Jakob disease (CJD) in humans. Prion proteins are thought to exist in two different conformations: the 'benign' PrPcform, and the infectious 'scrapie form', PrPsc. Knowledge of the three-dimensional structure of PrPc is essential for understanding the transition to PrPsc. The nuclear magnetic resonance (NMR) structure of the autonomously folding PrP domain comprising residues 121-231 (ref. 6) contains a two-stranded antiparallel beta-sheet and three alpha-helices. This domain contains most of the point-mutation sites that have been linked, in human PrP, to the occurrence of familial prion diseases. The NMR structure shows that these mutations occur within, or directly adjacent to, regular secondary structures. The presence of a beta-sheet in PrP(121-231) is in contrast with model predictions of an all-helical structure of PrPc (ref. 8), and may be important for the initiation of the transition from PrPc to PrPsc.
J Neurochem 1996 Jan;66(1):277-285
Division of Medical Cell Biology, Karolinska Institutet, Stockholm, Sweden.
The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5-fold increase in both total ubiquinone and its reduced form. Furthermore, alpha-tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie-infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.
Philos Trans R Soc Lond B Biol Sci 1994 Mar 29;343(1306):447-463
Department of Neurology, University of California, San Francisco 94143.
Scrapie was thought for many years to be caused by a virus. Enriching fractions from Syrian hamster (SHa) brain for scrapie infectivity led to the discovery of the prion protein (PrP). To date, no scrapie-specific nucleic acid has been found. As well as scrapie, prion diseases include bovine spongiform encephalopathy (BSE) of cattle, as well as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS) of humans. Transgenic (Tg) mice expressing both SHa and mouse (Mo) PrP genes were used to probe the molecular basis of the species barrier and the mechanism of scrapie prion replication. The prion inoculum was found to dictate which prions are synthesized de novo, even though the cells express both PrP genes. Discovery of mutations in the PrP genes of humans with GSS and familial CJD established that prion diseases are both genetic and infectious. Tg mice expressing MoPrP with the GSS point mutation spontaneously develop neurologic dysfunction, spongiform degeneration and astrocytic gliosis. Inoculation of brain extracts prepared from these Tg(MoPrP-P101L) mice produced neurodegeneration in many of the recipient animals after prolonged incubation times. These and other results suggest that prions are devoid of foreign nucleic acid and are thus different from viruses and viroids. Studies on the structure of PrPSc and PrPC suggest that the difference is conformational. Whether one or more putative alpha-helices in PrPC are converted into beta-sheets during synthesis of PrPSc is unknown. Distinct prion isolates or 'strains' exhibit different patterns of PrPSc accumulation which are independent of incubation times. Whether variations in PrPSc conformation are responsible for prion diversity remains to be established. Prion studies have given new insights into the etiologies of infectious, sporadic and inherited degenerative diseases.
Arch Neurol 1993 Nov;50(11):1129-1153
Department of Neurology, University of California, San Francisco.
Enriching fractions from Syrian hamster (SHa) brain for scrapie prion infectivity led to the discovery of the prion protein (PrP). Prion diseases include scrapie of sheep and bovine spongiform encephalopathy of cattle as well as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS) of humans. Transgenic (Tg) mice expressing both SHa and mouse (Mo) PrP genes were used to probe the molecular basis of the species barrier and the mechanism of scrapie prion replication. Bioassays of brain extracts from two scrapie-infected Tg lines showed that the prion inoculum determines that prions are synthesized de novo, even though the cells express both PrP genes. Studies with artificial prions produced from chimeric Mo/SHaPrP transgenes underscore the concept that inoculated prion dictates which prion will be replicated. Discovery of mutations in the PrP genes of humans with GSS and familial CJD established that prion diseases are both genetic and infectious. Transgenic mice expressing high levels of MoPrP-P101L, corresponding to the GSS point mutation (P102L) in human PrP, spontaneously develop neurologic dysfunction, spongiform degeneration, and astrocytic gliosis. Inoculation of brain extracts prepared from these Tg (MoPrP-P101L) mice produced neurodegeneration in recipient animals after prolonged incubation times. These results are in accord with those of other studies and argue that prions are devoid of foreign nucleic acid. Structural investigations of cellular prion protein (PrPC) and prion protein scrapie (PrPSc) suggest that the difference may be conformational. Conditions that diminished the beta-sheet content of PrPSc were the same as those identified previously that inactivate prion infectivity. Whether prion diversity as reflected by distinct "strains" producing different patterns of PrPSc accumulation is due to different conformers of PrPSc remains to be established. Advances in the purification and characterization of both PrPC and PrPSc seem to have identified the central event in PrPSc synthesis and prion propagation, ie, the unfolding of PrPC followed by its refolding into PrPSc. These findings underscore the fundamental features of prion structure and propagation that differentiate prions from other transmissible pathogens.