Poster Session PPo1: Protein Misfolding

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Erratum to: Poster Session PPo1: Protein Misfolding

PPo1-1: Exposure of Protein Core in the Human Prion Protein H187r Mutant

Linghao Zhong

Pennsylvania State University; Mont Alto, Pennsylvania USA

Key words: human prion protein, H187R, molecular dynamics, conformational change, pathogenesis

Structural effect of H187R mutation on human prion protein (huPrP) has been investigated by molecular dynamics modeling. In the wild type huPrP, the protein core is well protected by Arg156 side chain while it is kept in place by a hydrogen bond with H187. However, the introduction of the positively charged Arg187 leads to the disruption of this hydrogen bond. The side chain of Arg156 is driven away from its native position due to the strong electrostatic repulsion, leaving the protein hydrophobic core to be more solvent exposed. The same conformational effect has also been observed in the T188R mutant of huPrP. It is proposed that this destabilizing factor could lead to further conformational change in huPrP and eventually pathogenesis.

PPo1-2: A Vertically Transmissible Amyloid Proteinopathy (Prionoid) in Bacteria

Rafael Giraldo,^1,* M. Elena Fernández-Tresguerres,¹ Susana Moreno-Díaz de la Espina² and Fátima Gasset-Rosa¹

¹Department of Chemical and Physical Biology; and ²Department of Cell Proliferation and Development; Centro de Investigaciones Biológicas; CSIC; Madrid, Spain

Key words: bacterial amyloid proteinopathy, ligand(DNA)-promoted amyloidogenesis, prionoid

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β-sheet backbone. Amyloid aggregates are able to replicate by templating the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self-replicative macromolecular assemblies. As mammalian PrP does (Deleault et al. PNAS 2007; 104:9741–6; Wang et al. Science 2010; 327:1132–35), the WH1 domain of the bacterial, plasmid-encoded protein RepA (Fernández-Tresguerres et al. Plasmid 2004; 52:69–83) can assemble into amyloid fibers upon binding to short, defined DNA sequences in vitro (Giraldo PNAS 2007; 104:17388–93; Gasset-Rosa et al. NAR 2008; 36:2249–56). Exploiting our current understanding of ligand (DNA)-induced RepA-WH1 amyloidosis, here we report that its hyper-amyloidogenic functional variant (A31V), fused to a red fluorescent protein, causes a synthetic amyloid proteinopathy in Escherichia coli (Fernández-Tresguerres et al. submitted): (i) In the presence of multiple copies on the specific DNA sequence opsp, WH1(A31V) accumulates as cytoplasmatic inclusions seggregated from the nucleoid. (ii) Such aggregates have amyloid nature. (iii) Bacteria carrying the amyloid inclusions age, exhibiting a five-fold expanded generation time. (iv) Before cytokinesis, small inclusions are assembled de novo and transferred to the daughter cells. Transmission failures cure amyloidosis in the progeny. (v) In the absence of inducer DNA, purified cellular WH1(A31V) inclusions seed amyloid fiber growth in vitro from soluble protein. The RepA-WH1 prionoid (Aguzzi, Nature 2009; 459:92–925) is a potential bacterial model system for transmissible amyloid proteinopathies.

PPo1-3: Design and Syntheses of Peptides Focusing on Detection of the Structural Change of Prion Proteins

Kiyoshi Nokihara,¹ Akiyoshi Hirata,¹ Kazuo Kasai,² Shunsuke Yajima,³ Takafumi Ohyama,¹ Takashi Yokoyama² and Shiro Mohri²

¹HiPep Laboratories; Kamigyo-ku; Kyoto, Japan; ²Prion Disease Research Center; National Institute of Animal Health; Tsukuba, Ibaraki Japan; ³Faculty of Applied Bioscience; Tokyo University of Agriculture; Setagaya-ku, Tokyo Japan

Key words: structural conversion, self-assembly, peptide-peptide interactions, bio-detection system, fluorescent label, structured peptide libraries, recombinant bovine PrP

Introduction. Although detailed mechanisms of the structural conversion and self-assembly of prions remain elusive, these diseases are associated with the conversion of the normal cellular protein, PrPC, into the b-sheet rich form, PrPSc, and may involves specific peptide-peptide interactions. We have been developing a bio-detection system utilizing structural

Results. Although several peptides designed have difficult sequences, improved solid-phase syntheses and the use of the recently developed HPLC column, HiPep-Intrada, allowed production of high quality peptides. The fluorescent intensity change of the resulting peptides interacting with normal and scrapie-affected mouse brain homogenates were measured to discriminate structural differences between PrPC and PrPSc. Some designed peptide seems to promote structural conversion of rbPrP.

Conclusion. We have developed a novel biodetection concept involving labeled structured peptides as capture molecules. In this concept the differential structural recognition of analysts gives a ‘protein-fingerprint’ affording a barcode-like visualization. In the present study peptides that recognize PrPSc were found in the designed peptide libraries. It is proposed that these peptides can be used as sensor elements to identify PrPSc.

Materials and Methods. Recognition modules of fluorescent labeled structured peptide libraries, comprising human prion protein fragment peptides, have been designed and chemically synthesized. The purified and characterized peptides were used to discrimination infected/normal mouse-brain homogenate. In order to detect peptides responsible for structural conversion, the monitoring system was constructed using recombinant bovine PrP (rbPrP) with synthetic peptides carrying thioflavin T.

PPo1-4: Role of Polybasic Domains in Prion Propagation

Michael B. Miller, James C. Geoghegan and Surachai Supattapone

Department of Biochemistry; Dartmouth Medical School; Hanover, NH USA

Key words: prion, PrP, polybasic domain, PMCA

Prions are largely composed of PrP^Sc, a misfolded conformer of the cellular protein PrP^C. PrP^Sc appears to propagate by binding to PrP^C and inducing conformational change, but the mechanism of this interaction is poorly understood. Peptides and motif-grafted antibodies containing PrP polybasic domains bind to PrP^Sc. We tested if these polybasic domains constitute required sites for full-length PrP^C to bind PrP^Sc. Using a novel in vitro binding assay with recombinant myc-tagged PrP as bait, we found that deletion of either polybasic domain in PrP impaired its interaction with PrP^Sc. We also tested if this binding impairment caused functional consequences for conversion of PrP^C to nascent PrP^Sc. To assess conversion, we used recombinant PrP^C molecules expressed in Chinese hamster ovary (CHO) cells as substrate in serial protein misfolding cyclic amplification (sPMCA) reactions reconstituted with Prnp^-/- brain homogenate. In contrast to wild type PrP^C controls, mutant substrates lacking either the N-terminal or central polybasic domain showed no PrP^Sc propagation for initial 3-round sPMCA experiments. However, upon performing additional sPMCA rounds, one mutant (c-PBD) showed conversion to protease-resistant form, which propagated indefinitely. Like wild-type prions, c-PBD PrP^Sc propagation required the presence of a cofactor provided by the Prnp^-/- homogenate. These results suggest that PrP^C polybasic domains mediate interaction with PrP^Sc for normal prion conversion. However, when polybasic domains are unavailable, PrP^Sc may promiscuously utilize alternate mechanisms for propagation.

PPo1-5: Spontaneous Amyloidosis in Mice Expressing Anchorless Prion Protein

Jan Stöhr,¹ Joel C. Watts,¹ Giuseppe Legname,^1,2 David W. Colby,¹ Azucena Lemus,³ Hoang-Oanh B. Nguyen,¹ Joshua Sussman,¹ Holger Wille,^1,2 Stephen J. DeArmond,^1,3 Stanley B. Prusiner^1,2 and Kurt Giles^1,2

¹Institute for Neurodegenerative Diseases; and Departments of ²Neurology and ³Pathology; University of California; San Francisco, USA

Key words: spontaneous disease, anchorless, PrP, gerstmann sträussler scheinker, amyloidosis, amyloid, PrP interaction

PrP^C is a neuronal glycoprotein that is tethered to the cell membrane by a glycosylphosphatidyl inositol (GPI) anchor and is a precursor to PrP^Sc, the disease-causing isoform. To investigate the role of the GPI anchor in the cellular physiology of PrP^C, we established transgenic (Tg) mice expressing various levels of PrP lacking its GPI anchor (PrP-ΔGPI). Expression of PrP-ΔGPI at ∼2x the levels of PrP^C found in wild-type (wt) mice led to the development of a late-onset, spontaneous neurological illness caused by PrP amyloidosis in the brain. Neuropathological analysis of these spontaneously sick animals showed severe amyloidogenic PrP deposits throughout all brain areas. Proteinase K digestion of brain tissue revealed the presence of C- and N-terminally truncated, protease-resistant PrP fragments of 10 kDa, reminiscent of those found in human Gerstmann-Sträussler-Scheinker disease. Remarkably, coexpression of GPI-anchored PrP and PrP-ΔGPI hastened the onset of illness in Tg mice in a dose-dependent manner. Subsequent bioassays showed that this phenomenon is not based upon the conversion of PrP^C into PrP^Sc because no infectivity was found in symptomatic animals. In contrast to these findings, coexpression of N-terminally truncated PrP had no effect on the course of amyloidosis, arguing for an interaction site of these PrP amyloids within the N-terminal region of PrP^C. This study clearly shows that expression of non-anchored PrP results in severe amyloidosis in Tg mice and that coexpression of PrP^C accelerates the onset of clinical signs without being converted into an infectious isoform.

PPo1-6: Molecular Interactions Between Prions as Seeds and Recombinant Prion Proteins as Substrates Resemble the Biological Interspecies Barrier in vitro

L. Luers, G. Panza, D. Riesner, D. Willbold and E. Birkmann

Heinrich Heine Universität Düsseldorf; Forschungszentrum Jülich; Düsseldorf, Germany

Key words: species barrier, seeded in vitro conversion, sds, fibrillization

Background. The main event in prion disease is the conversion of the cellular form of the prion protein (PrPC) to its pathogenic form (PrPSc). As shown before, this conversion could be mimicked by fibrillization of recombinant PrP and low amounts of SDS. Fibrillization could be accelerated by seeding with prepurified PrPSc from brain tissue.1 Prion diseases are transmissible within one species and certain different species, but nor between others.

Objectives. Is the species barrier based on the moleculare interaction of soluble PrP as substrate and prepurified PrPSc as seed?

Results. We adopted the SDS-based conversion system to different species: hamster,2 cattle,3 human4 as well as sheep and mice (unpublished data) and studied intraspecies transmission by seeding recPrP with PrPSc. To analyze whether an interspecies transmission or barrier could be shown by the kinetic of fibril formation we combined substrate and seeds from different species. We showed that in vitro simulation of the species barrier is in complete agreement with the experimental data from in vivo transmission studies.

Discussion. We established an interspecies in vitro seeding assay with exclusively proteinacious components. A partly denatured intermediate structure of substrate PrP is prerequisite for fibrillisation. But critical for transition its potency of the substrate to refold into a conformation well adapted to the PrPSc-seed.

Methods. Amyloid fibril formation was monitored by ThT-fluorescence and electron microscopy. The SDS-based in vitro seeding assay2 consists of recombinant PrP as substrate and PrPSc prepurified by NaPTA-precipitated from brain tissue.

References

1. Birkmann, prion.

2. Stöhr, PNAS.

3. Panza, BBRC.

4. Luers, Rej Res.

PPo1-7: Studies of PrP/Polyanion Interactions in Infectious Prions Generated by PMCA

Justin Piro, James Geoghegan, Surachai Supattapone

Dartmouth Medical School; Hanover, NH USA

Key words: prion, polyanion, PMCA, photocleavable

Prion diseases require the conversion of host encoded protein, PrP^C, to the pathogenic isoform, PrP^Sc. We previously showed that purified hamster PrP^C requires the presence of polyanionic molecules to convert to the pathogenic isoform in vitro. Using an in vitro enzymatic reaction to radioactively label poly(A) RNA we show this RNA becomes selectively incorporated into stable complexes with PrP and is protected from nuclease digestion during the protein misfolding cyclic amplification (PMCA) assay. Furthermore, by using a fluorescently labeled oligonucleotide and fluorescent confocal microscopy we demonstrate that Poly(A) DNA molecules are incorporated into aggregates of hamster prions produced in vitro. In an attempt to analyze the role of the polyanion on prion infectivity a photocleavable (PC) nucleic acid was synthesized. This nucleic acid supports propagation in the PMCA assay using purified hamster PrP^C as a substrate. Similarly to previous results, the PC nucleic acid forms a complex with purified hamster PrP^C as well as recombinant PrP. When in complex this nucleic acid is protected from nuclease digestion. However, the PC nucleic acid can be completely degraded when treated with light at 315 nm both by itself and in complex with recombinant PrP allowing us to determine the role the polyanion plays in prion infectivity.

PPo1-8: Insertion of Glutamines into the Rigid Loop Promotes PrP Conversion

Matevž Avbelj, Iva Hafner-Bratkovic, Jernej Gašperšic and Roman Jerala

National Institute of Chemistry Slovenia; Department of Biotechnology; Ljubljana, Slovenia

Transmissible spongiform encephalopathies (TSE), also known as prion diseases, are associated with conformational conversion of cellular prion protein (PrP^c). In prion diseases PrP^C undergoes conformational transition into a beta-sheet-rich form, PrP^Sc, which is thought to be the infective agent responsible for TSE. Different mechanisms for prion propagation have been proposed, but the exact molecular mechanism is still unknown. In PrP from certain animals the loop between B2 and H2, called rigid loop, represents a structurally less defined region of the C-terminal folded domain and plays an important role in species barrier. Another neurodegenerative disease, Huntington’s disease, is associated with expansion of glutamine repeats in a protein Huntingtin. Poly-Gln is prone to aggregation and formation of a polar zipper. Rigid loop of PrP contains high fraction of Gln and Asn residues. Previously we identified, using engineered covalent tethers, that opening of structured domain of PrP at the rigid loop is essential for PrP conversion. We wanted to assess if the replacement or insertion of Gln residues into the rigid loop of PrP is compatible with conversion of PrP, where it could enhance the intermolecular interactions between folding intermediates of PrP compact domain. Experiments have shown that introduction of additional Gln residues indeed decreases the delay for in vitro formation of prion fibrils. We propose a model for the conformational changes in C-terminal part of PrP, which includes PrP dimer as the basic building unit.

PPo1-9: Study of the Molecular Mechanism Responsible of the Conformational Changes Induced by E200K and D202N Pathological PrP Mutation

Alessandro Corsaro,¹ Stefano Thellung,¹ Tonino Bucciarelli,² Luca Scotti,² Katia Chiovitti,² Valentina Villa,¹ Cristina D’Arrigo,³ Antonio Aceto² and Tullio Florio¹

¹Laboratory of Pharmacology and Neuroscience; Dept. Oncology Biology and Genetics; University of Genova; Genova, Italy; ²Section of Biochemistry; Dept. Biomedical Sciences; University G. D’Annunzio; Chieti, Italy; ³Institute for Macromolecular Studies-Division of Genova (ISMac-CNR); Genova, Italy

Key words: hPrP90-231, E200K prion mutation, D202N prion mutation, hPrP90-231 cellular internalization

To study the conformation-dependent neurotoxicity of Prions, we develop an experimental model to purify recombinant hPrP90-231 (hPrP) preserving its native conformation (Corsaro, Neurochem Int 2002; 41:55–63). By controlled thermal denaturation, we can convert the native non-toxic hPrP90-231 in a PrP^Sc-like conformer able to induce apoptosis in SH-SY5Y cells (Corsaro, Int J Immunopathol Pharmacol 2006; 19:339–56). In this study, in virtue of the evidence that most of the pathogenic mutations in PRNP, is located at the border of alpha3-helices in PrP, we generated hPrP90-231 carrying E200K and D202N, CJD and GSS-linked mutations respectively. We evaluated the role of these mutations on protein three-dimensional structure, aggregation process, cell viability, internalization and sub-cellular compartmentalization, in vitro. We demonstrated that E200K mutant, differently from wt and D202N hPrP, was spontaneously refolded in a PrP^Sc-like conformation. By cell viability experiments we observed that, differently from wt or D202N hPrP, hPrP-E200K was highly toxic already in its native conformation. By internalization assays we established that E200K mutation favors the intracellular accumulation of the native protein while cellular internalization of wt or D202N prion fragment is related to the structural changed dependent on thermal denaturation procedure. Moreover we demonstrated, utilizing 2-ptoluidinylnaphthalene-6-sulfonate assay, that exposure to the solvent of wt and D202N hPrP peptides hydrophobic amino acid residues occurred in a denaturation dependent manner, while hPrP E200K natively expose to the solvent hydrophobic amino acid residues. In conclusion we demonstrated that structural destabilization induced by E200K substitution in hPrP90-231 sequence, favor its conversion towards toxic PrP^Sc-like conformation.

Acknowledgements

This work was supported by grants from Italian Ministry of University and Research (MiUR-PRIN2008) and Compagnia di San Paolo to T.F.

PPo1-10: Insights into Prion Protein Stability

Federico Benetti,^1,2 Luca Pastorino,¹ Francesco Attanasio,³ Enrico Rizzarelli⁴ and Giuseppe Legname^1,2,5,*

¹Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies (SISSA-ISAS); Trieste, Italy; ²Italian Institute of Technology; SISSA-ISAS Unit; Trieste, Italy; ³National Research Council (CNR); Institute of Biostructures and Bioimaging; Catania, Italy; ⁴Department of Chemical Sciences; Università degli Studi di Catania; Catania, Italy; ⁵ELETTRA Laboratory; Sincrotrone Trieste S.C.p.A; Trieste, Italy

Key words: prion protein stability, folding/unfolding process, fluorescence spectroscopy, circular dichroism, calorimetry, dielectric constant, disulfide bridge, ionic strength, transition metals

The molecular mechanisms by which PrP^C is converted into its pathological isoform have not yet been established. While point mutations and seeds represent a trigger to cross the energy barriers for genetic and infectious forms, respectively, there are no indications regarding sporadic forms. Conversely by Anfinsen’s principle, which foresees only one conformation per protein, many proteins have been found to adopt two or more conformations. In this work we investigated the intra-molecular forces devoted to stabilize both mouse (Mo) prion protein (PrP) forms: MoPrP^23-231 and MoPrP^89-231. Using spectroscopic techniques and differential scanning calorimetry, we evaluated the contribution of hydrophobic effect, electrostatic interactions, disulfide bond and transition metals on folding and stability of both full-length and truncated MoPrP. We found that MoPrP^23-231 is more stable than MoPrP(89–231), probably because of stabilizing interactions between the unfolded N-terminal domain and the globular C-terminal domain. MoPrP(89–231) presents a three-state unfolding process with a non-native state more prone to form aggregates than the native state. This intermediate, mainly characterized by alterations of its tertiary structure, seems to be stabilized at low temperatures. The dielectric constant of the environment seems to be crucial for the stabilization of either the native state or intermediate.

Since the MoPrP folding-unfolding process is fully reversible, we calculated the thermodynamic parameters of every conformation.

Our study revealed that intra-molecular forces, as well as the environmental changes responsible of alterations of the PrP tertiary structure, cause the transition from the native state into a non-native/aggregating state.

PPo1-11: An Investigation into the Subcellular Localisation of Co-factors that Stimulate Prion Protein Conversion

James F. Graham,¹ Sonya Agarwal,¹ Dominic Kurian,² Louise Kirby,¹ Teresa JT,¹ Pinheiro³ and Andrew C. Gill¹

¹The Roslin Institute & R(D)SVS; Neuropathogenesis Division; The Alexander Robertson Building; School of Vet Studies; University of Edinburgh; Easter Bush Vet Centre; Roslin, Midlothian USA; ²Institute for Animal Health; Compton, RG USA; ³Department of Biological Sciences; University of Warwick; Coventry, CV USA

Key words: prion, misfolding, conversion, strain, co-factor

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders affecting humans and animals. Different TSE strains are thought to be encoded by different conformations of the prion protein and strain specific co-factors may aid the conversion of the host protein (PrP^C) to a strain-specific misfolded form, PrP^Sc. We investigated the subcellular localisation of molecules enhancing prion conversion in TSE-susceptible cell lines and in scrapie associated fibrils (SAF) from TSE-infected mouse brains, assaying for their presence by use of a cell free conversion assay (CFCA) and an oligomerisation assay. Plasma membrane enriched fractions from the LD9 cell line enhanced the conversion efficiency of ME7 and 79A mouse scrapie in the CFCA yet inhibited in vitro oligomerisation of recombinant prion protein, suggesting the existence of separate, competing mechanisms of specific and non-specific misfolding in vivo. Mass spectrometric analysis of SAF from three different strains of mouse scrapie showed different proteinaceous constituents of fibrils. These initial experiments provide the basis for the identification of the molecular make-up of different prion strains through subsequent fractionations of plasma membrane derived fractions or supplementation of these misfolding assays with SAF-derived molecules.

PPo1-12: Enantiospecific Elimination of Prion by Poly-D-lysine: H2H3 Domain Under the Spotlight

Zhou Xu, Stéphanie Prigent, Franck Mouthon, Emmanuel Comoy, Human Rezaei and Jean-Philippe Deslys

Institute of Emerging Diseases and Innovative Therapies; CEA; Fontenay-Aux-Roses, France; Institut National de la Recherche Agronomique (INRA); Virologie et Immunologie Moléculaires; Equipe Biologie Physico-chimique des Prions; Jouy-en-Josas, France

Key words: prion, amyloid, H2H3 domain, poly-D-lysine, PrP conversion, enantiospecificity

The misfolding of PrP is the central feature of prion diseases. The conversion of normal α-helix-structured PrP^C into a pathological β-sheet-enriched PrP^Sc constitutes an early event in the infectious process. Several hypotheses, involving different regions of the protein, endeavour to delineate the structural mechanism underlying this change of conformation. However, these are mainly based on biophysical and modelling studies and their biological relevance still needs to be assessed in order to propose specific targets for drug design.

Our data show that poly-D-lysine drastically removes proteinase K resistant PrP from a prion-infected SN56 neuroblastoma cell model. This result is enantiospecific as it is not observed with poly-L-lysine, albeit at a fainter level. In vitro, an interaction between poly-D-lysine and H2H3 domain of PrP is evidenced by cross-linking. Further structural investigations suggest that this specific interaction can block the pathological conformational conversion of full-length PrP.

These results strongly implicate the H2H3 domain in the conversion into a β-sheeted structure and the replication of PrP in a cellular model, therefore supporting the role of H2H3 in the pathogenesis of prion diseases. Furthermore, they highlight the H2H3 region as a potential target for rational drug design against conformational conversion.

PPo1-13: Recovery of Small Infectious Prpres Aggregates from Prion-infected Cultured Cells

Zaira E. Arellano Anaya, Jimmy Savistchenko, Véronique Massonneau, Caroline Lacroux, Olivier Andréoletti and Didier Vilette

UMR INRA ENVT 1225; Interactions Hôte Agent Pathogène; Ecole Nationale Vétérinaire de Toulouse; Toulouse, France

Key words: cell assay, PrP aggregates, soluble prions

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Abnormally-folded PrP may assume various aggregation states and a major challenge is to decipher which types of aggregate play important roles in prion-induced neuronal cell death and/or in prion propagation. Physical and/or chemical treatments can disrupt large PrPres aggregates and generate smaller, more infectious particles. While these studies raised the possibility that small PrP aggregates might be more infectious, the biological significance of these experimental treatments remained an open question and firm evidence for the presence of small PrPres aggregates in infected tissues or cells was lacking. In this study, we have fractionated detergent lysates from prion-infected cultured cells (including neurons) by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical highly aggregated PrPres, remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cut-off. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrPres aggregates can be recovered in the absence of any strong in vitro treatments gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

PPo1-14: Prion Protein Interaction with Low Molecular Weight Heparin

Tuane C.R.G. Vieira,¹ Mariana M.P.B. Gomes,¹ Daniel P. Reynaldo,¹ Marcius M.S. Almeida,¹ Yraima Cordeiro² and Jerson L. Silva¹

¹Instituto de Bioquímica Médica; ²Faculdade de Farmácia; Universidade Federal do Rio de Janeiro; CEP; RJ, Brazil

Key words: prion, conversion, glycosaminoglycan

Conversion of cellular PrP into the pathological conformer involves contact between both isoforms and probably requires a cellular factor, such as a glycosaminoglycan. Though direct interaction between prion protein and heparin has been reported, little is known about the structural features implicit in this interaction. In the present work, we found that Hep interacts with rPrP23-231 inducing its oligomerization/aggregation. These aggregates presented no toxicity to cells or amyloid characteristics. Following the binding kinetics and secondary structure content we found that this oligomerization is mostly transient. After reaching equilibrium, NMR HSQC spectra showed that the prion protein bound to Hep has the same general fold of the free protein, although displaying some chemical shifts changes. We also investigated the interaction of Hep with other PrP constructs (rPrPΔ51-90 and rPrPΔ32-121). Heparin did not bind these constructs at pH 7.4 but was able to interact at pH 5.5, indicating that heparin interacts with the octapeptide repeat region at pH 7.4, but can also interact with another region of the protein at pH 5.5. This interaction at pH 5.5 was dependent on histidine residues. Despite little changes on prion protein structure induced by heparin binding, the complex formed was less susceptible to aggregation by temperature or RNA binding. It also lost its intrinsic propensity to convert into amyloid forms in an in vitro conversion reaction.

PPo1-15: Conversion of Syrian Hamster Prion Protein to Beta Oligomer and Fibril Forms Under Physiological PH Conditions

Valentyna V. Semenchenko, Trent C. Bjorndahl, Xuehui Liu, Guo-Ping Zhou, Fozia Saleem, Sandipta Acharya, Adina Bujold, Connie Sobsey and David S. Wishart

Departments of Biological Sciences and Computing Science; University of Alberta; Edmonton, Alberta, Canada; NanoLife Sciences Group; National Institute for Nanotechnology; Edmonton, Alberta, Canada

Key words: prion, conversion, oligomer, fibril, NMR

Prions are believed to spontaneously convert from a native, monomeric highly-helical form (PrP^c) to a largely beta-strand, multimeric and insoluble aggregate (PrP^sc). This conformational change is believed to be the cause of various prion diseases, but the cause of conversion is unknown and the conversion process is not well understood. Because of its large size and insolubility, characterization of PrP^Sc is difficult and there are several contradictory or incomplete models of the PrP^Sc structure. A soluble intermediate, PrP beta, exhibits many of the same features as PrP^Sc and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP^C to PrP beta conversion process and of the PrP beta folding intermediate may provide insights into the structure of PrP^Sc. Using a truncated, recombinant version of Syrian hamster PrP (shPrP(90-231)), we have used NMR spectroscopy, in combination with other biophysical techniques (CD, electron microscopy, dynamic light scattering, fluorescence and proteinase K digestion) to characterize the pH-driven PrP^C to PrP beta conversion process in detail. Our findings show that conversion is preceded by slow/intermediate conformational dynamic changes in core residues of the protein that propagate outward as the pH is reduced below 3.2. Conversion to the oligomeric state occurs rapidly below pH 3.0, while the formation of fibrils is slow (taking days to weeks). Additional studies of the acid conversion process with both oxidized and reduced states of the protein were performed to assess differences in fibril morphology and stability.

PPo1-16: Lipopolysaccharide Interacted with Prion Protein and Converted it into a β-sheet-rich Isoform Resistant to Proteinase K

Ametaj BN,¹ Saleem F,^1-3 Semenchenko V,³ Sobsey C² and Wishart DS^2,3

¹Department of Agricultural, Food and Nutritional Science; ²Departments of Biological Sciences and Computing Science; University of Alberta; Edmonton, Alberta Canada; ³NanoLife Sciences Group; National Institute for Nanotechnology; Edmonton; Alberta, Canada

Prions are believed to spontaneously convert from a native, monomeric highly α−helical form, known as prion protein (PrP^C), into a largely β-strand, multimeric and insoluble aggregate known as scrapie prion protein (PrP^Sc). This conformational change is believed to be the cause of various prion diseases; however, the exact mechanism underlying the conversion process is unknown, particularly with respect to the putative role of other cellular co-factors in prion replication. We used electron microscopy (EM), nuclear magnetic resonance (NMR) and Circular Dichroism (CD) spectroscopy to evaluate whether lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is able to bind to PrP^C, and convert it into a β-rich isoform. Data showed that LPS bound directly to recombinant Syrian Hamster shPrP^C 90–232 under physiological conditions (saline solution and at 37°C) and was able to convert the protein into a β-rich oligomer that could form amyloid-like fibrils. Also, to test whether LPS would help in self-propagation of the conversion of PrP^C into a β-sheet-rich isoform we conducted a PMCA-like (no sonication) technique. Results showed successful serial propogation using only recombinant SHPrP^C as the protein seed and catalytic amount of LPS (< 1:10,000). The SDS-page demonstrated that the β-rich isoform obtained was resistant to digestion by proteinase K . In conclusion, our data showed that LPS interacted with the PrP^C and converted it into a β-rich isoform with characteristics similar to PrP^Sc. More research is warranted to test whether the converted isoform would cause infectivity in experimental animals.

PPo1-17: Propagating Artificial Amyloid Strains of Recombinant Human Prion Protein with Mutations in Position 129

Sofie Nyström, Peter Nilsson and Per Hammarström

IFM-Chemistry; Linköping University; Linköping, Sweden

The influence of the polymorphism M129V in the human PrP gene is well documented. Most cases of sporadic CJD afflict homozygous individuals. Differences in codon 129 genotype give rise to differences in phenotype regarding plaque and clinical symptoms. Despite this, little is known about the molecular background to this phenomenon.

To study this phenomenon in greater detail we employed recombinant human prion protein. Using several artificial mutations allowed us to study the influence of different amino acid properties on the formation of amyloid prion protein. The variants used were 129A, 129V, 129L, 129M, 129W, 129P, 129E and 129K. Three mutants were chosen to vary the hydrophobicity, the tryptophan mutant was chosen due to its bulkiness and the proline for its constraint of the polypeptide backbone. 129E and 129K may give information regarding the effect of charge in this position.

The protein was expressed in Escherichia coli, purified and subjected to agitation at 37°C at physiological pH and salt concentration (Almstedt et al. Prion 2009). All mutants formed congophilic and Thioflavine T positive aggregates within hours. Fibrillar morphology was also confirmed using transmission electron microscopy.

Seeding the mutant proteins with preformed fibrils of the mutant itself or of wild type protein revealed differences in seeding efficiency for the different mutants. By monitoring the fibrils resulting from the seeded fibrillation reactions using luminescent conjugated polymers, a templating effect was seen. This strain-like behavior was followed through several generations of fibrils. The fragility of the seeding fibrils was taken under consideration and was analyzed using urea denaturation.

Almstedt, Nyström S, Nilsson P, Hammarström P. Prion 2009; 3:224-35.

PPo1-18: In vivo Transmission of Type-2 Diabetes by a Prion-like Mechanism

Natalia Salvadores, Diego Morales-Scheihing and Claudio Soto

Department of Neurology; University of Texas Medical School; Houston, USA

Key words: type-2 diabetes, amylin, misfolding

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia due to insulin resistance and impaired insulin secretion. Histopathologically is associated with pancreatic islet amyloid deposits, and progressive β-cell loss. T2D is the most prevalent protein misfolding disorder, with an estimated number of patients of more than 200 million people worldwide. The 37-amino-acid polypeptide amylin is the main component of amyloid in pancreas of T2D patients. Compelling evidence suggest that amylin misfolding and oligomerization plays an important role in the disease pathogenesis by inducing β-cell damage. Amylin aggregation follows a seeding-nucleation mechanism, similar to other protein misfolding disorders and in particular to the mechanism of prion replication. Our work aims to study the putative transmissibility of T2D. We used transgenic mice expressing human amylin which develop over time diabetes signs including hyperglycemia, reduced insulin secretion and amylin deposition. Intra-peritoneal injection of diluted pancreas homogenate derived from old transgenic mice containing abundant amyloid aggregates dramatically accelerates amyloid accumulation and hyperglicemia. Control groups including transgenic mice inoculated with wild type mice pancreas homogenate did not show any change on the disease onset. Also, wild-type animals inoculated with pancreas homogenates from old transgenic mice did not show any protein aggregates or hyperglicemia, indicating that acceleration of diabetes signs in the experimental group depended on the misfolding and aggregation of host amylin. Our results suggest that under these experimental conditions T2D pathology can be accelerated by a prion-like mechanism of transmission of protein misfolding.

PPo1-19: Oxidation of Helix-3 Methionines Precedes the Formation of Pk Resistant Prp^sc

Tamar Canello, Katy Frid, Ronen Gabizon, Silvia Lisa, Assaf Friedler, Jackob Moskovitch, Maria Gasset and Ruth Gabizon

Hadassah University Hospital; Jerusalem, Israel; Instituto Química-Física Rocasolano; Madrid, Spain; Hebrew University; Jerusalem, Israel; University of Kansas; Lawrence, KS USA

Key words: peptides, prions, antibodies, oxidation

While elucidating the peculiar epitope of the α-PrP mAb IPC2, we found that PrP^Sc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the α-fold, suggesting oxidation may facilitate the conversion of PrP^C into PrP^Sc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs which epitope comprise Met residues readily detected PrP^C, but could not recognize most PrP^Sc bands unless they were vigorously reduced. Next, we showed that the α-Met pAbs did not recognize newly formed PrP^Sc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized.

We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrP^C to PrP^Sc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

PPo1-20: Polymorphism/Species Specific Protein Misfolding Cyclic Amplification (PMCA) using PrP^C from Cell Lines Expressing Different Full Length Prp Variants

Jan Priem, Kirti Banwari, Fred G. van Zijderveld and Alex Bossers

Department of Bacteriology and TSEs; Central Veterinary Institute of Wageningen UR; Lelystad, The Netherlands

Key words: PMCA, cell lines, specificity, standardization

Polymorphisms in the prion protein (of both host and donor) are known to influence TSE susceptibility and transmissibility in vivo. The modulating effects on the underlying molecular conversion of prion proteins can be readily assessed using cell-free conversion systems. Cell-free conversions assessing the (potential) species/polymorphism barriers support these findings on the molecular level and these conversion tools have been used widely to assess virtually every species/polymorphism/strain barrier.

We previously used the Protein Misfolding Cyclic Amplification (PMCA) technique to study species/polymorphism barriers in sheep genetic variants using natural and experimental sheep scrapie and several BSE sources. As substrate we needed to use negative brain homogenates which made us dependent on the availability of “fresh” negative brain, preferentially from PrP homozygote genotypes. Some of these negative brain materials from other species (like some PrP variants of goats and humans) are hard or impossible to get in fresh condition and other sources are welcomed.

Here we present the results of our efforts to replace negative brain homogenates as a PrP^C source by partially purified lysates from eukaryotic cell lines expressing different PrP variants/genotypes. We show that using PrP^C from cell lines gives comparable conversion specificity and efficiency as when using default negative brain homogenates as conversion substrate. The ability to use cell lysates as a substrate will strengthen batch-to-batch reproducibility as well that it will enable us to assess virtually every species’ PrP for linkage on the molecular level with susceptibility and transmissibility, including the barriers to humans.

PPo1-21: Structural Investigation of PrP^C/PrP^Sc Conversion Using MD Simulations and X-Ray

Nesrine Chakroun,^1,2 Stéphanie Prigent,³ Marc Malfois,⁴ Franca Fraternali,² Human Rezaei³ and Cécile A. Dreiss¹

¹King’s College London; Molecular biophysics Group; Pharmaceutical Science Division; London, UK; ²King’s College London; Randall Division of Cell and Molecular Biophysics; London, UK; ³INRA; Molecular Virology and Immunology; Physico-chemical biology of Prion group; Jouy-en-Josas, France; ⁴Diamond Light Source; Non-crystalline diffraction beam line; Didcot, UK

Key words: prion conversion, MD simulations, SAXS

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of extracellular beta-rich fibrillar deposits of a structurally modified form (PrP^Sc) of the cellular prion protein (PrP^C). Despite the increasing interest for PrP diseases, the mechanism of PrP^C/PrP^Sc conversion is still unknown. Various Studies on PrP diseases increasingly suggest that neurotoxicity arises from small soluble pre-fibrillar oligomers. We have used Molecular Dynamics simulations (MD) in combination with Small Angle X-Ray Scattering (SAXS) to resolve the early oligomerization pathways of recombinant sheep PrP (sPrP). Under well established conditions, sPrP oligomerizes into three distinct oligomers, which form in parallel (Eghiaian F, et al. PNAS 2007; 104:7414–9). We have identified the minimal region of sPrP, namely H2H3, which leads to the same oligomerization profile as the entire sPrP. The conversion of sPrP^C into sPrP^Sc was investigated at the molecular scale using MD. Atomistic simulations of the H2H3 region reproducing experimental conditions revealed a complete unfolding of H2 and H3 helices and the formation of a stable beta-rich refolded “double hairpin” structure, a potential nucleus for oligomerization (Chakroun, et al. FASEB 2010). It has been shown that single point mutations in H2 and H3 present structural polymorphisms and oligomerization properties that could constitute the basis of species or strain variability. We have also investigated the structural arrangement of the oligomers by determining the low resolution shapes of sPrP, H2H3 and the resulting oligomers using SAXS. Time-resolved studies have been used to follow the oligomerization of sPrP and H2H3 monomer into the final oligomers.

PPo1-22: Membrane Permeabilization by Purified Soluble Oligomers of Prion Protein

Sylvie Noinville,¹ Jean-François Chich,¹ Céline Chapuis,¹ Céline Henry² and Human Rezaei¹

¹INRA; Virologie et Immunologie Moléculaires; Jouy-en-Josas, France; ²INRA; BioBac; Jouy-en-Josas, France

Key words: oligomers, lipid membrane, conformational change leakage

The conversion of normal PrP^C to its pathological isoform PrP^Sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. The early events leading to the structural conversion of the PrP seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. We examined the respective actions of native monomeric PrP and β-rich oligomeric PrP on model lipid membranes (Chich, et al. J Mol Biol 2010). We also study the influence of the N-terminal flexible region by comparing full-length PrP24-234 and N-terminally truncated PrP103-234 oligomers.

Soluble 12-mers were purified by Size-Exclusion Chromatography after in vitro temperature-induced PrP conversion. We compare their structural properties when associated with lipid and study their propensities to permeabilize the membrane. The interaction of monomeric PrP with membranes entailed α-to-β conversion while the binding of β-rich oligomers caused their α-structure changes. Interaction of PrP oligomers caused a large increase in membrane permeability, whereas equivalent amounts of monomers were without any detectable effect. The interaction of 12-mers of full-length PrP or of the N-truncated form results in destabilization of both the membrane and the oligomer itself, suggesting a pore-like mechanism. The existence of transient pores mediated by soluble oligomers could be the origin of the neurotoxic mechanism.

PPo1-23: Types or Strains: What Classifies Prion Diseases?

Wiebke M. Wemheuer,¹ Sylvie L. Benestad,³ Arne Wrede,¹ Wilhelm E. Wemheuer,² Tatjana Pfander,¹ Bertram Brenig² and Walter J. Schulz-Schaeffer¹

¹Department of Neuropathology; University Medical Center; Goettingen, Germany; ²Institute of Veterinary Medicine; Georg August University; Goettingen, Germany; ³National Veterinary Institute; Oslo, Norway

Key words: prion types, prion strains, CJD, sheep scrapie, stability

In the 1950s scrapie in small ruminants was classified as a “Slow Virus Disease” (Sigurdsson, et al. Br V et J 1954; 110:314–54). Different incubation times upon transmission from scrapie to mice were thought to originate from different virus strains (Dickinson, et al. J CompPathol 1968; 78:293–9). Along with the “prion hypothesis” and the actuality that the conformation of proteins determines their properties the idea was established that strains exist due to conformational differences of the pathological prion protein (PrP^Sc). By definition, prion strains are identified after transmission of an isolate to a new host species (Aguzzi, et al. Rev Mol Cell Biol 2007; 8:552–61). However, in human Creutzfeldt-Jakob disease (CJD) the existence of types in humans as the original host has been described and conformational differences are presumably the reason for different cleavage sites of proteinase K (Parchi, et al. AnnNeurol 1996; 39:767–78). Our own evaluation of sporadic CJD cases and sheep scrapie samples provides proof that types in prion diseases are present across species, but they need to be defined and clearly separated from strains. Using the PrP^Sc deposition pattern and PrP^Sc stability against denaturation with GdnHCl as parameters we found that sporadic CJD type 1 and atypical/Nor98 scrapie as well as sporadic CJD type 2 and classical scrapie show striking similarities (Wemheuer, et al. Am J Pathol 2009; 175:2566–73). From our results and ongoing research we conclude that types with distinct conformational motives are the major determinants within a prion disease; a type may also contain different strains, but a strain cannot belong to more than one prion type.

PPo1-24: Probing Structural Differences Between PrP^C and PrP^Sc by Surface Nitration and Acetylation

Binbin Gong,^1,2 Adriana Ramos,¹ Jana Alonso³ and Jesús R. Requena¹

¹Department of Medicine; University of Santiago de Compostela; Santiago de Compostela, Spain; ²Key Laboratory for Zoonosis Research; College of Animal Science and Veterinary Medicine; Jilin University; Changchun, P.R. China; ³Laboratory of Proteomics; IDIS; Santiago de Compostela, Spain

Key words: PrP^Sc, structure, chemical derivatization, mass spectrometry

We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride, which specifically target accessible tyrosine and lysine residues, respectivelly, to modify Syrian hamster recombinant PrP(90-231) (rPrP) and PrP27-30, aiming at finding locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin, and subsequent analysis by mass spectrometry (MALDI-ToF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E221-R229 (containing Y225 and Y226) which in rPrP was much more extensively modified than in PrP27-30; peptide H111-R136, containing Y128, was also more modified in rPrP. Conversely, peptides Y149-R151, Y157-R164 and R151-Y162 suffered more extensive modification in PrP27-30. Acetic anhydride modified very extensively peptide G90-K106, containing K101, K104, K106 and the amino terminus, in both rPrP and PrP27-30. Differences were detected in peptides involving lysine residues flanking the glycosylation sites (K185, K194 and K204). These results suggest that (1) PrP^Sc exhibits important conformational differences in the C-terminal region with respect to PrP^C, resulting in loss of solvent accessibility of Y225 and Y226, very solvent-exposed in the latter conformation; since other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in PrP^Sc; (2) contrarily, tyrosines contained in the stretch spanning approximately from Y149 to R164 are more accessible in PrP^Sc, suggesting rearrangements in alpha-helix H1 and the short beta-sheet of PrP^C; (3) the amino terminal region of PrP^Sc is very accessible. These data should help validate and construct structural models of PrP^Sc.

PPo1-25: Further Characterization of Flexible Regions of PrP^Sc by Limited Proteolysis

Ester Vázquez Fernández, Gustavo Sajnani, Adriana Ramos and Jesús R. Requena

Department of Medicine; University of Santiago de Compostela; Santiago de Compostela, Spain

Key words: PrP^Sc, structure, li, ited proteolysis

Background. We previously used limited proteolysis and mass spectrometry to pinpoint flexible regions within Sha PrPSc. We detected four of such regions spanning, approximately, G86-K101, A117-G119, M129-G142 and N153-R156. Due to carbohydrate and GPI moieties, and limitations of the mass spectrometry technique, it was impossible to extend this analysis into the C-terminal region.

Objectives. To detect and locate additional flexible, proteinase K (PK) susceptible regions beyond N153-R156, towards the C-termius of PrPSc by using limited proteolysis followed by western blot-based analysis.

Results and Discussion. We detected 7 bands with apparent MWs of approximately 19.5, 17, 15, 14.3, 10.5, 7 and 7.5 kDa. Given that the 19.5 kDa band corresponds to the predominant G90-S231 peptide, whose mean MW is 16.2 kDa, the apparent mass contribution of the GPI is ∼3.3 kDa. Assuming that all bands correspond to peptides containing GPI (as only one band was detected with antibody 3F4), masses of bands >10.5 kDa fit with cleavages at previously identified flexible stretches, while the 10.5, 7.5 and 7 kDa bands would correspond to cleavages around positions 171, 197 and 201, which flank the C-terminal alpha-helices predicted to persist in PrPSc. With cautions and waiting for confrimation by sequencing, these results add to our knowledge of the structure of PrPSc.

Methods. We analyzed brain homogenates and PrPSc isolated from Syrian hamsters inoculated with the 263 K and Drowsy strains of scrapie. Samples were treated with 50 µg/ml of PK, deglycosylated, subjected to Tricine-SDS-PAGE, and probed with antibody R1, which recognizes epitope Y226-S231.

PPo1-26: Characterization of Mutant Prion Proteins Extracted from the Brains of Transgenic Mice

Laura Tapella,^1,2 Matteo Stravalaci,³ Emiliano Biasini,^1,2 Marco Gobbi³ and Roberto Chiesa^1,2

¹Dulbecco Telethon Institute; ²Department of Neuroscience and ³Department of Molecular Biochemistry and Pharmacology; Mario Negri Institute for Pharmacological Research; Milan, Italy

Inherited prion diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI) are linked to point or insertional mutations in the prion protein (PrP) gene. How different PrP mutations encode the information to specify distinct disease phenotypes is unknown. It is postulated that each mutation favors conversion of PrP into a specific misfolded structure that is selectively toxic to specific neuronal populations of the brain.

We have applied a panel of biochemical assays to identify possible structural differences between three mutant PrPs expressed in transgenic (Tg) mice. We prepared detergent brain extracts from Tg mice expressing the mouse PrP homologues of a nine-octapeptide insertion associated with a mixed CJD-GSS phenotype, and the D178N/V129 and D178N/M129 mutations linked, respectively, to CJD and FFI. After ultracentrifugation to separate the detergent-insoluble proteins, we analyzed the mutant PrPs in the soluble fraction by size-exclusion chromatography to estimate their oligomeric state. This analysis showed that the mutant molecules were monomeric. To investigate possible differences in secondary/tertiary structure we analyzed the mutants by a surface plasmon resonance-based immunoassay, which uses a panel of monoclonal antibodies against well-defined epitopes of PrP. This analysis indicated possible structural differences in the C-terminal region of the protein between residues 175–194. In order to perform more detailed structural analyzes, we have set up a method for purifying mutant PrPs from the mouse brains by immuno-affinity chromatography. Our approach offers the opportunity of defining the structural diversity of mutant PrPs using molecules purified from biological sources.

PPo1-27: Structural and Physiological Aspects of PrP Interaction with RNA Molecules

M.P.B. Gomes,¹ T.C.R.G. Vieira,¹ P.S. Ferreira,¹ L.P. Rangel, M.S. Almeida,¹ J.L. Silva¹ and Y. Cordeiro ²

¹Instituto de Bioquímica Médica; ²Faculdade de Farmácia; Universidade Federal do Rio de Janeiro; Brazil

Key words: prion, RNA, aggregation, misfolding

Prion diseases occurrence is related to a misfolded isoform of PrP^C, PrP^Sc. The “protein only hypothesis” postulates that the PrP^Sc is the only agent responsible for triggering prion diseases, acting as a template to PrP^C conversion, generating more PrP^Sc. However, there are evidences that an adjuvant factor may be involved, lowering the energy barrier between the two isoforms. In this context, our interest is in the peculiar relationship between PrP protein and nucleic acids (NAs). Instead of transmitting information, NAs may be involved in PrP^Sc generation, lowering the energy barrier between the isoforms. We know that RNA interacts with PrP and potentially modulates PrP^C/PrP^Sc conversion. In this work we have investigated the interaction of recombinant full-length PrP and two PrP amino-terminal deletion mutants with distinct RNA sequences or total RNA extracted from cultured cells. The aim of the present study is to evaluate the structural aspects of murine PrP interaction with RNA using a spectroscopic approach. Our results demonstrate that incubation with RNA leads to PrP aggregation with different intensities, depending on RNA origin. The PrP:RNA complex is mainly maintained by ionic interactions and the N-terminal portion of PrP is essential to RNA binding. Cell viability assays showed that some PrP:RNA aggregates can affect cultured cells. These results indicate that RNA can act as an adjuvant in PrP conversion and maybe involved in prion diseases.

PPo1-28: The Oligomerization Properties of Prion Protein are Restricted to the H2H3 Domain

Stéphanie Prigent,¹ Nesrine Chakroun,^1-3 Cécile Dreiss,³ Sylvie Noinville,¹ Céline Chapuis,¹ Miquel Adrover,^4,5 Kris Pauwels,⁴ Cesira de Chiara,⁴ Zhou Xu,^1,6 Annalisa Pastore,^1,4 Franca Fraternali² and Human Rezaei¹

¹INRA; VIM; Prion Physico-chemical Biology; Jouy-en-Josas, France; ²Randall Division of Cell and Molecular Biophysics; and ³Pharmaceutical Science Division; King’s College London, UK; ⁴MRC National Institute for Medical Research; The Ridgeway, London UK; ⁵Department de Química; Universitat de les Illes Balears; Palma de Mallorca, Spain; ⁶Commissariat à l’Énergie Atomique; Fontenay-aux-Roses, France

Key words: polymerization pathway, intermediate, strain, oligomer

According to the ‘protein-only’ hypothesis, the key event in the pathogenesis of prion diseases is the conversion of the ­alpha-helix-rich monomer (PrP^C) into a beta-sheet-rich polymeric state (PrP^Sc). To determine the prion protein (PrP) region responsible for polymerization, we compared the polymerization of the full-length recombinant ovine PrP to the one of PrP single point mutants or truncated constructs.

Two complementary approaches were used: biophysical experiments, via size exclusion chromatography (SEC), static light scattering and circular dichroism and molecular dynamic simulations.

The H2H3 mutant, i.e., the protein containing only H2 and H3 helixes, mimicked the oligomerisation pattern of the full length PrP (oligomers O1, O2 and O3). Purified H2H3 oligomers were rich in beta-sheets and presented depolymerisation kinetics similar to the one of purified PrP oligomers. These results indicate that regions outside of H2H3 do not significantly contribute to oligomerization. Furthermore we also showed that H2H3 domain is fibrillogenic and forms amyloid fibres morphologically similar to those obtained for the full-length protein (Adrover et al. JBC 2010). Considering that H2H3 is responsible for polymerization, we designed single mutations on H2H3 which selectively affected some polymerization pathways (O1 or O3), leading to the polymerization pattern of a given species such as mouse.

By combining both experimental approaches and Molecular Dynamics, we show that single point mutations in H2H3 lead to structural polymorphism and then oligomerization properties that could constitute the basis of species or strain variability (Chakroun, et al. FASEB J 2010).

PPo1-29: Exploitation of PrP Misfolding Epitopes in Cancer Immunotherapy

Li Li, Alan Huang and Neil R. Cahsman

Brain Research Centre; University of British Columbia; Vancouver, BC Canada

Key words: PrP misfolding, cancer immunotherapy, antibody

Specific misfolded proteins are implicated in neurological diseases, such as prion protein (PrP) in Creutzfeldt-Jakob disease (CJD) and superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis (ALS). Abnormally exposed protein domains, designated disease specific epitopes (DSEs), can serve as diagnostic disease markers and treatment targets for these and other neurodegenerative diseases. The DSE concept may also be applicable to cancer, in which defects in protein folding and/or exposure to oxidative stress are likely accompanied by subtle denaturation of a proportion of molecules at the cell surface, especially for those proteins which are highly overexpressed. We have discovered that two PrP DSEs Tyr-Tyr-Arg (YYR) and Tyr-Met-Leu (YML), which are generated by exposure of PrP β-strands, selectively bind to infectious prions, not the normal PrP^C. Recently we identified that YYR and YML antibodies bind to many cancer cell lines, but not untransformed cells. Experiments are underway to test these epitopes as immunotherapeutic targets in mouse models of cancer. We conclude that PrP is partially unfolded specifically at the surface of cancer cells, but not normal cells. Exposure of PrP-DSEs may serve as a target for therapeutic vaccines and/or passive immunotherapies that spare natively folded PrP^C from autoimmune recognition on normal cells.

PPo1-30: Prion Protein Residue Substitution in a Steric Zipper Site Leads to Aggregation in vivo

C. Sigurdson,¹ S. Joshi-Barr,¹ C. Bett,¹ O. Winson,¹ G. Manco,² P. Schwarz,² S. Hornemann,² T. Rülicke,³ K. Wüthrich^4,5 and A. Aguzzi²

¹Department of Pathology; UC San Diego; La Jolla, CA USA; ²Institute of Neuropathology; University Hospital of Zürich; Zürich, Switzerland; ³Institute of Laboratory Animal Science and Biomodels Austria; University of Veterinary Medicine Vienna; Vienna, Austria; ⁴Institute of Molecular Biology and Biophysics; ETH Zürich; Zürich, Switzerland; ⁵Department of Molecular Biology and Skaggs Institute for Chemical Biology; The Scripps Research Institute; La Jolla, CA USA

Key words: aggregation, neurodegeneration, loop, prion structure, transgenic mice

Prion protein (PrP) aggregation occurs with select mutations in PRNP and can lead to the generation of infectious prions in humans and mouse models. The global PrP^C structure is highly conserved among mammals, with a flexible amino terminus and a globular C-terminus composed of three α-helices and a short β-sheet. The β2-α2 loop (amino acids 165–175) is structurally polymorphic, being either disordered and ‘flexible’, or ordered and ‘rigid’. This loop has been identified as a steric zipper region where β-sheets can interdigitate as part of the cross-β spine of an amyloid fibril. We have previously found that mutations in the β2-α2 loop, which switch the structure from a disordered loop to an ordered, ‘rigid loop’, lead to the de novo generation of PrP plaques and infectious prions in vivo. To investigate whether the loop mobility impacts PrP aggregation, we developed a second ‘rigid loop’ (RL) transgenic mouse that expresses mouse PrP but with a single amino acid residue substitution corresponding to the horse RL, D167S. The horse Prnp sequence normally encodes a serine at position 167, and both the horse PrP^C and mouse PrP^C with a D167S substitution (MoPrP167) show an ordered, rigid β2-α2 loop by solution NMR spectroscopy, indicating that the D167S confers an RL structure. We found that overexpression of the MoPrP167 leads to widespread PrP aggregation in the brain and muscle of transgenic mice, similar to that seen the original RL mice (MoPrP170, 174). Thus our data indicate that the β2-α2 loop of PrP^C is highly sensitive to mutations that lead to self-aggregation.

PPo1-31: Amyloid Features and Neuronal Toxicity of Mature Prion Fibrils are Highly Sensitive to High Pressure

Driss El. Moustaine,¹ Veronique Perrier,¹ Isabelle Acquatella,¹ Valeriy G. Ostapchenko,² Ilia V. Baskakov,² Reinhard Lange¹ and Joan Torrent¹

¹Univ Montpellier 2; Inserm; U710; Montpellier, France; EPHE; Paris, France; ²Center for Biomedical Engineering and Technology; University of Maryland; Department of Biochemistry and Molecular Biology; University of Maryland School of Medicine; Baltimore, MD USA

How amyloid fibril structures are achieved and stabilized is not entirely understood, and therefore the development of in vitro approaches to study amyloidogenesis (i.e., protein model systems together with various biophysical and biochemical techniques) represent an urgent need. However, such studies are limited due to the transient and dynamic features of the protein states involved in the aggregation reaction. We describe here the use of high pressure as a thermodynamic parameter to perturb the structure of mature amyloid fibrils prepared in vitro from recombinant prion protein (PrP). In the past, examining the reverse process of a reaction had greatly advanced the protein folding research field. In a similar way, we focus here on the pressure-induced amyloid fibril structural changes to better understand the physicochemical basis of amyloid fibril formation.

We demonstrate that the soluble monomeric state can be reached by applying pressure to mature PrP amyloid fibrils. Nevertheless, the dissociation of fibrils was incomplete, highlighting a complex conformational heterogeneity in the polymerization process. The pressure treatment resulted in a new less cytotoxic ensemble with highly reduced amyloid generic features, i.e., loss of ThT and ANS binding, diminished β-sheet integrity and altered pK resistance. The considerable decrease in the partial molar volume of PrP fibrils upon forming the activated state appears as the thermodynamic driving force for the observed pressure-induced structural changes, suggesting that promoting hydration and collapse of water-excluded cavities might be used as a potential therapeutic strategy to interfere with the amyloid aggregation process.

PPo1-32: A Pharmacological Chaperone for the Structured Domain of Human Prion Protein

Andrew J. Nicoll,¹ Clare R. Trevitt,² M. Howard Tattum,² Emmanuel Risse,¹ Emma Quaterman,¹ Amauris Avila Ibarra,⁴ Graham S. Jackson,² Richard B. Sessions,⁴ Mark Farrow,¹ Jonathan P. Waltho,³ Anthony R. Clarke^2,4 and John Collinge^1,2,*

¹Department of Neurodegenerative Disease; and ²MRC Prion Unit; UCL Institute of Neurology; Queen Square, London UK; ³Department of Molecular Biology and Biotechnology; University of Sheffield; Sheffield, TN UK; ⁴Department of Biochemistry; School of Medical Sciences; University of Bristol; Bristol, BS UK

One possible approach to combat neurodegenerative protein misfolding disorders such as Alzheimer’s, Parkinson’s and prion diseases including CJD and BSE is to stabilise the folded state of the relevant protein using pharmacological chaperones. In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP^C). Numerous ligands for PrP have been reported but none have been shown to bind to the structured region with the affinity required for a chemical chaperone, and many are known to form large colloidal aggregates that act non-specifically.

Using equilibrium dialysis, we screened a selection of compounds and demonstrated that most did not interact with PrP and some formed high molecular weight aggregates. However, one compound was shown to bind to PrP and the complex was characterised thermodynamically and structurally using ITC, NMR, AUC, CD and computational modelling. The compound forms a 1:1 complex via the structured C-terminus of huPrP with a Kd of 5 μM in a manner that was almost entirely enthalpically driven. Furthermore, the compound stabilised the prion protein against thermally-induced unfolding and aggregation, as well as curing prion-infected cells and inhibiting misfolding in the Protein Folding Cyclic Amplification Assay.

The identification of a binding site with a defined 3D structure opens up the possibility of designing small drug-like molecules that stabilise huPrP and prevent its conversion into the disease-associated form.

PPo1-33: Is a Species Specific Heparan Sulfate Sequence the Infectious Agent in Prion Related Disease?

David Cullis-Hill

Sylvan Scientific; Bondi Junction, NSW Australia

The present minimal Prion (PrP^C) infectious particle (PrP^Sc) is a “prion rod,” a mixture of PrP^Sc within a heparan sulfate (HS) scaffold. Protein-free transmission has been reported, questioning the prion-only hypothesis. HS binding to PrP^C shows an increase in beta-sheet conformation and Heparinase decreases infectivity. HS is an information rich, complex linear sulfated polysaccharide, attached to a core protein termed a proteoglycan. Specific sugar sequences have been implicated in organogenesis, vascular and neurological guidance. Glypican-1 is a proteoglycan which is co-localised with PrP^C. HS polymers are highly thermostable and chemically resistant. Aside from structural roles they bind avidly to a wide range of proteins via a specific HS binding sequence, with additional interactions by recruiting adjacent amino acids. This low range HS/protein interaction has specific effects including dimerisation (antiparallel/“head-to-tail”) and activation of function. Examples of HS induced dimerisation are PrP^C, Amyloid precursor Protein (Alzheimer’s), FGF, VEGF, PDGF, IL-8, NGF, RANTES, SDF, SOD, Spectin and TGFbeta. It is proposed that specific heparan sulfate (HS) sequences are amplified/duplicated on the PrP^C dimer, and that a ‘rogue’ sequence of HS structures is amplified in scrapie which misfold PrP^c. These HS sequences are species specific due to interspecies differences in PrP^C. Thus the HS takes up an infectious character. A highly purified PrP^Sc associated HS was amplified by PMCA. The western blot demonstrated the appearance of PrP^C in the first and second amplification cycles in two consecutive studies respectively. We conclude further in vitro and in vivo studies are indicated.