NMR characterization of the full‐length recombinant murine prion protein, <i>m</i>PrP(23–231)

Riek, Roland; Hornemann, Simone; Wider, Gerhard; Glockshuber, Rudi; Wüthrich, Kurt

doi:10.1016/s0014-5793(97)00920-4

Cited by 688 publications

(518 citation statements)

References 28 publications

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“…7). Comparisons with published structures 2,10,13,14 indicate that F(ab) 1 POM1 binding did not cause conformational distortions in the GD. We also studied the binding interface of scFv POM1 complexed with full-length recombinant mouse PrP (residues 23-230, rmPrP ) by solution nuclear magnetic resonance (NMR) spectroscopy.…”

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confidence: 81%

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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confidence: 81%

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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194

289

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“…1). The last domain or C-terminal globular domain (Donne et al, 1997;Riek et al, 1997) contains the -helix and two parallel stranded -sheets (Riek et al, 1996). PrP c presents at least three distinct topological orientations: the fully extracellular form (or (sec)PrP) (Holscher et al, 2001), and two transmembrane isoforms (called Ntm-PrP and Ctm-PrP) with opposite sequence orientations with respect to the lumen of the endoplasmic reticulum (Hegde et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Nicolas

Gavı́n

Rı́o

2009

Brain Research Reviews

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“…The three-dimensional structures of recombinant prion proteins from a number of different organisms, including mouse, hamster, and human PrP, have revealed that the entire N-terminal segment comprising amino acid residues 23-120 is flexibly disordered and that only the segment containing the C-terminal 111 residues, PrP-(121-231), possesses a defined three-dimensional structure (17)(18)(19)(20). The N-terminal flexible region appears to play an important role in transmissible spongiform encephalopathy transmission and influences the formation of proteinase-resistant conformations of PrP (e.g.…”

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confidence: 99%

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Martins

Frosoni

Martínez

et al. 2006

Journal of Biological Chemistry

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Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP C , into a misfolded, protease-resistant form, PrP Sc . Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106 -120 from the so-called amyloidogenic core of PrP (residues 106 -126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65°C. Under partially denaturing conditions (65°C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65°C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65°C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65°C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP Sc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106 -120 of PrP and does not require other cellular factors or a PrP Sc template.The conformational conversion of the normal cellular isoform of the prion protein, PrP C , 4 into an abnormal pathological isoform, PrP Sc , underlies a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases, which includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle (1). Neuropathologically, prion diseases are characterized by neuronal loss and astrogliosis and often by spongiform degeneration of the brain and deposition of amyloid plaques (1). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent. The "proteinonly" hypothesis of prion propagation postulates that the abnormal isoform, PrP Sc , acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrP C as a substrate (1).PrP Sc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrP C and most other proteins are readily degraded (2). In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3-7...

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NMR characterization of the full‐length recombinant murine prion protein, mPrP(23–231)

Cited by 688 publications

References 28 publications

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

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

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

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