Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity

Benkemoun, Laura; Sabaté, Raimon; Malato, Laurent; Reis, Suzana Dos; Dalstra, H.J.P.; Saupe, Sven J.; Maddelein, Marie‐Lise

doi:10.1016/j.ymeth.2006.04.006

Cited by 26 publications

(30 citation statements)

References 17 publications

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“…Briefly, cells were lysed by sonication and the protein was extracted from the insoluble pellet fraction with 8 M guanidine-HCl, purified on a Talon (Clontech) column, and eluted in 8 M urea. Denaturant was removed using a HiTrap column GE Healthcare) in 175 mM acetic acid (37). The eluate containing 1.4 mM protein was neutralized with Tris base and incubated at 4°C without agitation for 7 days to allow fibril formation.…”

Section: Methodsmentioning

confidence: 99%

Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy

Chen

Thurber

Shewmaker

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

118

131

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We demonstrate that accurate values of mass-per-length (MPL), which serve as strong constraints on molecular structure, can be determined for amyloid fibrils by quantification of intensities in dark-field electron microscope images obtained in the tilted-beam mode of a transmission electron microscope. MPL values for fibrils formed by residues 218 -289 of the HET-s fungal prion protein, for 2-fold-and 3-fold-symmetric fibrils formed by the 40-residue ␤-amyloid peptide, and for fibrils formed by the yeast prion protein Sup35NM are in good agreement with previous results from scanning transmission electron microscopy. Results for fibrils formed by the yeast prion protein Rnq1, for which the MPL value has not been previously reported, support an in-register parallel ␤-sheet structure, with one Rnq1 molecule per 0.47-nm ␤-sheet repeat spacing. Since tilted-beam dark-field images can be obtained on many transmission electron microscopes, this work should facilitate MPL determination by a large number of research groups engaged in studies of amyloid fibrils and similar supramolecular assemblies.Alzheimer's disease ͉ molecular structure ͉ prions ͉ solid state NMR T he mass-per-length (MPL) of an amyloid fibril is an important constraint on its molecular structure. Given that amyloid fibrils contain ␤-sheets with cross-␤ alignment relative to the long fibril axis (1, 2) and that the spacing between ␤-strands in ␤-sheets is always 0.47 Ϯ 0.01 nm, one expects the MPL to be nearly ϫMW/0.47 kDa/nm, where MW is the amyloid-forming polypeptide's molecular weight and is the number of molecules in each ␤-sheet spacing. MPL measurements were the first indication that fibrils formed by the 40-residue ␤-amyloid peptide (A␤ 1-40 ) might have both 2-fold-symmetric ( Ϸ 2) and three-fold-symmetric ( Ϸ 3) polymorphs (3-6). MPL measurements support a two-fold-symmetric structural model for amylin fibrils with a specific morphology (7) and provide evidence for polymorphism similar to that of A␤ 1-40 fibrils (8). MPL measurements (9) provide support for a ␤-helix-like structure (10, 11) for fibrils formed by residues 218-298 of the HET-s prion protein (HET-s 218 -289 ), with each molecule spanning two turns of the ␤-helix ( Ϸ 0.5). MPL measurements on fibrils formed by the prion domains of the yeast prion proteins Ure2p (12) and Sup35 (13) support in-register parallel ␤-sheet structures (14-16), with backbone hydrogen bonds in the ␤-sheets being purely intermolecular ( Ϸ 1). Molecular models corresponding to these various values of are given in the relevant papers (4,7,10,16).MPL data in these cases comes from quantification of intensities in images obtained with scanning transmission electron microscopy (STEM). STEM images of unstained samples are dark-field images, in the sense that the background has low intensity (arising from weak electron scattering when the rastering electron beam strikes the thin carbon film on which fibrils are adsorbed), while the fibrils have higher intensity (arising from stronger electron scattering when the ...

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Section: Methodsmentioning

confidence: 99%

Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy

Chen

Thurber

Shewmaker

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

118

131

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“…The purified material was then used to infect prion-free P. anserina strains by using a protein transfection method as previously described. [16] While insoluble extracts from an E. coli control strain containing only the empty vector or IBs of a different protein (human NDP kinase) did not induce the appearance of the prion form [Het-s] (Table 1), strains transfected with HET-s(218-289) IBs acquired [Het-s] at a frequency comparable to or even higher than that found for transfection with HET-s(218-289) amyloids assembled in vitro. We conclude Next, a set of experiments was performed in which further properties of the HET-s IBs and amyloid fibrils assembled in vitro were compared.…”

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confidence: 95%

“…Herein we describe a structural and functional characterization of HET-s(218-289) IBs produced in E. coli by using electron microscopy (EM), NMR spectroscopy to determine H/D exchange, solid-state NMR spectroscopy, and in vivo prion infectivity assays as described earlier. [16] HET-s(218-289) was expressed in E. coli cells and located as typical angular-shaped inclusions with high electron density and a cross-section of 100-400 nm preferentially at the cellular poles ( Figure 1 and Figure S1 in the Supporting Information). The IBs were partially purified (see the Supporting Information) and this material was shown by EM (Figure 1 b) to consist of fibrillar structures.…”

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Solid‐State NMR Spectroscopy Reveals that E. coli Inclusion Bodies of HET‐s(218–289) are Amyloids

et al. 2009

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“…Die IBs wurden teilweise gereinigt (Hintergrundinformationen), woraufhin in weiteren EM-Bildern (Abbildung 1 b) bereits fibrilläre Strukturen sichtbar wurden. Das gereinigte Material wurde auch verwendet, um prionenfreie P.-anserina-Stämme mithilfe einer Protein-Transfektionsmethode [16] zu infizieren. Während unlösliche Extrakte von E.-coli-Kontrollstämmen, die nur einen leeren Vektor oder IBs eines anderen Proteins [9,17] Außerdem verhalten sich die HET-s(218-289)-IBs und -Amyloide bei chemischer Denaturierung ähnlich: Beide sind im neutralen pH-Bereich, jedoch nicht bei pH 2 resistent gegen Denaturierung durch Harnstoff (Abbildung S2B, Hintergrundinformationen).…”

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Festkörper‐NMR‐Spektroskopie zeigt: Die Einschlusskörperchen von HET‐s(218–289) in E. coli sind Amyloide

et al. 2009

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Während der heterologen Expression von Proteinen in E. coli tritt häufig eine Proteinablagerung in Einschlusskörperchen (IBs) auf. Die Struktur der E.‐coli‐IBs der prionbildenden Domäne des Pilzprions HET‐s wurde analysiert, und es wurde nachgewiesen, dass diese IBs die gleiche Struktur wie In‐vitro‐Fibrillen haben und eine Prioneninfektiosität zeigen. Die Resultate belegen, dass die IBs von HET‐s(218–289) Amyloide sind.magnified image

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Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity

Cited by 26 publications

References 17 publications

Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy

Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy

Solid‐State NMR Spectroscopy Reveals that E. coli Inclusion Bodies of HET‐s(218–289) are Amyloids

Festkörper‐NMR‐Spektroskopie zeigt: Die Einschlusskörperchen von HET‐s(218–289) in E. coli sind Amyloide

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