Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.
PMID:_12817476 [PubMed - in process]
Pol J Pathol. 2003;54(1):39-47. Immunohistochemical investigations of the prion protein accumulation in human spongiform encephalopathies. Special report II.
Zaborowski A, Kordek R, Botts GT, Liberski PP.
I Psychiatric Clinic, Lodz.
Creutzfeldt-Jakob disease (CJD) in a proportion of cases may have nonspecific clinical signs and symptoms and no characteristic neuroimaging and EEG picture. Thus, neuropathological studies are mandatory for a diagnosis. However, spongiform change, neuronal loss and astrocyte proliferation--the hallmarks of prion diseases, may also be absent or variable. In such cases, the diagnosis should be supported by the detection of prion protein (PrP) by Western blotting or immunohistochemistry (ICC). PrP may not be visualised under "regular" conditions, but it is unmasked following pretreatment procedures: incubation in formic acid or guanidine thiocyanate, microwave treatment, and hydrated or hydrolytic autoclaving, and these methods were included in standard diagnostic procedures in several different protocols. The aim of this study was to compare the effectiveness of these pretreatment methods and to introduce an optimal protocol for our laboratory. For this purpose, we used brain sections of 11 cases of CJD, 1 case of Gerstmann-Straussler-Scheinker syndrome (GSS), 1 case of kuru and 3 control brains. For pretreatment we used the hydrated and hydrolytic autoclaving and incubation with formic acid. Immunostaining was performed with monoclonal 3F4 antibody against PrP. The best results were achieved with hydrolytic autoclaving. By this procedure we were able to detect the "synaptic" type of PrP accumulation in all CJD cases, as well as in GSS and kuru, while with other two methods the signal was weaker or even absent.
PMID:_12817879
Biochemistry. 2003 Jul 1;42(25):7675-81. Generation of hydrogen peroxide from mutant forms of the prion protein fragment PrP121-231.
Turnbull S, Tabner BJ, Brown DR, Allsop D.
Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK.
By means of electron spin resonance spectroscopy, in conjunction with the spin trapping technique, we have shown previously that Abeta and alpha-synuclein (aggregating proteins that accumulate in the brain in Alzheimer's disease, Parkinson's disease, and related disorders) both induce the formation of hydroxyl radicals following incubation in solution, upon addition of Fe(II). These hydroxyl radicals are apparently formed from hydrogen peroxide, via Fenton's reaction. An N-terminally truncated fragment of the mouse prion protein (termed PrP121-231) is toxic to cerebellar cells in culture, and certain human mutations, responsible for inherited prion disease, enhance this toxicity. Here we report that PrP121-231 containing three such mutations (E200K, D178N, and F198S) also generated hydroxyl radicals, upon addition of Fe(II). The formation of these radicals was blocked by catalase, or by metal chelators, each of which also reduced the toxicity of the PrP121-231 fragments to cultured normal mouse cerebellar cells. Wild-type PrP121-231, full-length cellular PrP, and its homologue doppel did not generate any detectable hydroxyl radicals. We conclude that the additional cytotoxic effects of the mutant forms of PrP121-231 could be due to their ability to generate hydrogen peroxide, by a metal-dependent mechanism. Thus, one effect of these (and possibly other) prion mutations could be production of a particularly toxic form of the prion protein, with an enhanced capacity to induce oxidative damage, neurodegeneration, and cell loss.
PMID:_12820876
Brain. 2003 Sep;126(Pt 9):2065-73. Epub 2003 Jun 23. Regional heterogeneity of cellular prion protein isoforms in the mouse brain.
Beringue V, Mallinson G, Kaisar M, Tayebi M, Sattar Z, Jackson G, Anstee D, Collinge J, Hawke S.
CNS Infection and Immunity Group, and Department of Neurogenetics, Imperial College, Norfolk Place, London W2 1PG, UK.
Prion diseases are a group of invariably fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle. The infectious agent or prion is largely composed of an abnormal isoform (PrPSc) of a host encoded normal cellular protein (PrPc). The conversion of PrPc to PrPSc is a dynamic process and, for reasons that are not clear, the distribution of spongiform change and PrPSc deposition varies among prion strains. An obvious explanation for this would be that the transformation efficiency in any given brain region depends on favourable interactions between conformations of PrPc and the prion strain being propagated within it. However, identification of specific PrPc conformations has until now been hampered by a lack of suitable panels of antibodies that discriminate PrPc subspecies under native conditions. In this study, we show that monoclonal antibodies raised against recombinant human prion protein folded into alpha or beta conformations exhibit striking heterogeneity in their specificity for truncations and glycoforms of mouse brain PrPc. We then show that some of these PrPc isoforms are expressed differentially in certain mouse brain regions. This suggests that variation in the expression of PrPc conformations in different brain regions may dictate the pattern of PrPSc deposition and vacuolation, characteristic for different prion strains.
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