Prion protein library of recombinant constructs for structural biology

Hornemann, Simone; Christen, Barbara; Schroetter, Christine von; Pérez, Daniel R.; Wüthrich, Kurt

doi:10.1111/j.1742-4658.2009.06968.x

Cited by 24 publications

(20 citation statements)

References 41 publications

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“…Cells were further incubated for 1h at 37°C and induced by addition of 1 mM IPTG. After 6h cells were harvested and rmPrP was purified according to our standard protocol 29,43 .…”

Section: Protein Expression and Isotopic Labellingmentioning

confidence: 99%

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Sonati

Reimann

Falsig

et al. 2013

Nature

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Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the 1 and 3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides. (Fig. 1b). None of three high-affinity antibodies to the octapeptide repeats (OR, residues 50-90 embedded within the FT) were neurotoxic (Fig. 1b). Antibodies POM3 and D13, which bind the "charged cluster-2" 11 (CC2, residues 95-110), were innocuous at 67 nM but neurotoxic at 200 nM (Fig. 1b). None of the tested antibodies were toxic to Prnp o/o COCS ( Supplementary Fig. 2a). The identity of the targeted epitopes appeared to be a better predictor of PrP C antibody toxicity than their affinity to PrP C , suggesting that neurotoxicity resulted from the interaction of antibodies with specific PrP C domains (Supplementary Table 2).The mechanisms of neurotoxicity were further explored using POM1, a highly toxic antibody targeting the GD. Wild-type (wt) and tga20 COCS lost most granule cells (CGC) within 28 and 14 days post-exposure (dpe) to POM1, respectively (Fig. 2a-c). Controls included POM1-treated Prnp o/o COCS 12 , t...

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“…Cells were further incubated for 1h at 37°C and induced by addition of 1 mM IPTG. After 6h cells were harvested and rmPrP was purified according to our standard protocol 29,43 .…”

Section: Protein Expression and Isotopic Labellingmentioning

confidence: 99%

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Sonati

Reimann

Falsig

et al. 2013

Nature

Self Cite

195

289

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“…Three new variants of mPrP(121-231) were cloned by introducing the single-residue substitutions F175L and F175A and the two-residue substitution Y169A and F175A. All four proteins were expressed in Escherichia coli and purified using our standard protocol for PrP purification 3,[25][26][27] ; that is, the proteins were expressed as N-terminal Histagged constructs with intervening thrombin cleavage site, isolated from the resulting inclusion bodies by guanidinium chloride denaturation and bound to a Ni 2+ -nitrilotriacetic acid column. To avoid intermolecular interactions, we performed oxidation and refolding with the protein bound to the column, followed by elution with imidazole-containing buffer, thrombin cleavage, removal of thrombin with paminobenzamidine-celite and dialysis of protein fractions into NMR buffer.…”

Section: Preparation Of Mprp(121-231) Variants With Substitution Of F175mentioning

confidence: 99%

Prion Protein mPrP[F175A](121–231): Structure and Stability in Solution

Christen

Hornemann

Damberger

et al. 2012

Journal of Molecular Biology

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“…Preparation of Recombinant Mouse mPrP and Fibril Formation-Recombinant mouse PrP comprising residues 23-231 was expressed and purified as described elsewhere (43)(44)(45). mPrP(23-231) fibers were produced by incubating the protein in 50 M Tris-HCl, 1 M GdnHCl, 150 mM sodium chloride (pH 7.5), for 48 at 37°C h and shaking at 600 rpm (46).…”

Section: ϫ4mentioning

confidence: 99%