Biochemistry. 2002 Oct 15;41(41):12277-83. Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?
Kuwata K, Li H, Yamada H, Legname G, Prusiner SB, Akasaka K, James TL.
Department of Biochemistry and Biophysics, School of Medicine, Gifu University, 40 Tsukasa-machi, Gifu 500-8705, Japan.
A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.
PMID:_12369815
BMC Infect Dis. 2002 Oct 8;2(1):23. Epub 2002 Oct 08. A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies.
Polymenidou M, Verghese-Nikolakaki S, Groschup M, Chaplin MJ, Stack MJ, Plaitakis A, Sklaviadis T.
Laboratory of Pharmacology, Department of Pharmaceutical sciences School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece. magdalini.polymenidoty.usz.ch
BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases. METHODS: TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. RESULTS: Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. CONCLUSION: The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment.
PMID:_12370086
FEBS Lett. 2002 Oct 9;529(2-3):256-60. Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant beta-sheet structure.
Jamin N, Coic YM, Landon C, Ovtracht L, Baleux F, Neumann JM, Sanson A.
CEA-Saclay, DBJC and URA CNRS 2096, Bat. 532, 91191 Gif sur Yvette Cedex, France. jamisvidf.cea.fr
The conversion of the cellular prion protein into the beta-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C-terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined. Our results show that all these fragments, excepted that strictly encompassing helix 1, have a very high propensity to form structured aggregates with a dominant content of beta-sheet structures.
PMID:_12372610
J Biol Chem. 2002 Dec 13;277(50):49065-70. Epub 2002 Oct 07. Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form.
Vanik DL, Surewicz WK.
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.
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