Structural Properties of Gerstmann-Straussler-Scheinker Disease Amyloid Protein

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Structural Properties of Gerstmann-Straussler-Scheinker Disease Amyloid Protein
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  Structural Properties of Gerstmann-Stra¨ussler-Scheinker Disease Amyloid Protein* Received for publication, July 8, 2003, and in revised form, September 5, 2003Published, JBC Papers in Press, September 11, 2003, DOI 10.1074/jbc.M307295200 Mario Salmona‡§, Michela Morbin ¶ , Tania Massignan‡, Laura Colombo‡, Giulia Mazzoleni ¶ ,Raffaella Capobianco ¶ , Luisa Diomede‡, Florian Thaler‡, Luca Mollica‡, Giovanna Musco  ,Joseph J. Kourie**, Orso Bugiani ¶ , Deepak Sharma‡‡, Hideyo Inouye‡‡, Daniel A. Kirschner‡‡§§,Gianluigi Forloni‡, and Fabrizio Tagliavini ¶  From the  ‡  Istituto di Ricerche Farmacologiche “Mario Negri,” Via Eritrea 62, 20157 Milan,  ¶  Istituto Nazionale Neurologico “Carlo Besta,” Via Celoria 11, 20133 Milan,    Dulbecco Telethon Institute, Neurobiologia Cellulare e Molecolare c  /  o DIBIT, Via Olgettina 58, 20132 Milan, Italy,  **  Membrane Transport Group, Department of Chemistry, Science Road, The Australian National University, Canberra City, ACT 0200, Australia, and  ‡‡  Biology Department, Boston College, Chestnut Hill, Massachusetts 02467-3811 Prion protein (PrP) amyloid formation is a centralfeature of genetic and acquired forms of prion diseasesuch as Gerstmann-Stra¨ussler-Scheinker disease (GSS)and variant Creutzfeldt-Jakob disease. The major com-ponent of GSS amyloid is a PrP fragment spanning res-idues   82–146. To investigate the determinants of thephysicochemical properties of this fragment, we synthe-sized PrP-(82–146) and variants thereof, including en-tirely and partially scrambled peptides. PrP-(82–146)readily formed aggregates that were partially resistantto protease digestion. Peptide assemblies consisted of 9.8-nm-diameter fibrils having a parallel cross-  -struc-ture. Second derivative of infrared spectra indicatedthat PrP-(82–146) aggregates are primarily composed of   -sheet (54%) and turn (24%) which is consistent withtheir amyloid-like properties. The peptide induced a re-markable increase in plasma membrane microviscosityof primary neurons. Modification of the amino acid se-quence 106–126 caused a striking increase in aggrega-tion rate, with formation of large amount of protease-resistant amorphous material and relatively fewamyloid fibrils. Alteration of the 127–146 region hadeven more profound effects, with the inability to gener-ateamyloidfibrils.Thesedataindicatethattheintrinsicproperties of PrP-(82–146) are dependent upon the in-tegrity of the C-terminal region and account for themassive deposition of PrP amyloid in GSS. The molecular signature of prion diseases is a post-transla-tional modification of the prion protein (PrP) 1 from a normalcellular isoform (PrP C ) to disease-specific species (PrP Sc ) (1, 2).The transition from PrP C to PrP Sc involves conformationalchanges with decrease in   -helical secondary structure andsignificant increase in   -sheet content (3–5). This rearrange-ment is accompanied by the acquisition of abnormal physico-chemical properties including insolubility in non-denaturing detergents and partial resistance to proteinase K (PK) diges-tion (1). In the presence of detergents, the protease-resistantcore of PrP Sc assembles into insoluble fibrillar structures withthe tinctorial and ultrastructural properties of amyloid (6). PrPamyloidogenesis occurs consistently in genetic forms of disease,such as Gerstmann-Stra¨ussler-Scheinker (GSS) disease andPrP cerebral amyloid angiopathy (7, 8), and in the new variantof Creutzfeldt-Jakob disease that is causally linked to bovinespongiform encephalopathy (9, 10). In all these conditions,amyloid fibrils are associated with PrP aggregates that aredevoid of the tinctorial and ultrastructural properties of amy-loid (11), suggesting that different PrP peptides or proteinconformers may trigger fibrillar or non-fibrillar aggregates.Biochemical studies have shown that amyloid fibrils purifiedfrom GSS brain contain a major PrP fragment of    7 kDa,spanning residues 81–82 to 144–153 of PrP (12, 14). Thisfragment is very similar in patients with different mutations( i.e.  A117V, F198S, Q217R) and is derived from mutant PrP,although in GSS F198S and Q217R it does not contain theamino acid substitution (13). Evidence suggests that N- andC-terminal cleavage of abnormal PrP isoforms generating amy-loid peptides occurs before rather than after fibril formation(15). Western blot analysis of total brain extracts from F198Spatients has revealed three major protease-resistant PrP frag-ments of 27–29, 18–19, and 8 kDa. The 18–19- and 8-kDapeptides are N- and C-terminal truncated, as deduced by theirantigenic profile, and are unglycosylated, likely representing amyloid protein precursors (15). Similar low molecular weightfragments have been detected in GSS patients with other  PRNP  mutations and are also present in areas without amyloiddeposits, suggesting that regional factors feature in amyloido-genesis (15–17). Of note, the PrP region spanning residues89–140 is an integral part of the minimal sequence whichsustains prion replication (18, 19), suggesting that it plays acentral role in the conformational transition of PrP C into PrP Sc and in PrP Sc propagation.To investigate the contribution of different regions of theGSS amyloid protein to the physicochemical properties of dis-ease-specific PrP isoforms and fibrillogenesis, we synthesized apeptide homologous to the smallest amyloid subunit purifiedfrom GSS brains (PrP-(82–146) wt ) and PrP peptides withscrambled sequences thereof. In particular, we generated an * This work was supported in part by Italian Ministry of HealthGrant RF 2001.96, Italian Ministry of University and Research GrantPRIN 2001, European Union Grant QLRT 2001-00283, an Alzheimer’s Association/T.L.L. Temple Discovery Award (to D. A. K.), and by insti-tutional support from Boston College. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “ advertisement ” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed. Tel.: 39-02-39210812; Fax: 39-02-3546277; E-mail: salmona@marionegri.it.§§ Recipient of a Fulbright Senior Research Scholar award from theBinational United States-Italian Fulbright Scholar Program. 1 Theabbreviationsusedare:PrP,prionprotein;FTIR,Fouriertrans-form IR; GSS, Gerstmann-Stra¨ussler-Scheinker; PK, proteinase K;HPLC, high pressure liquid chromatography; EM, electron microscopy;FP, fluorescence polarization; DPH, 1,6-diphenyl-1,3,5-hexatriene. T HE  J OURNAL OF  B IOLOGICAL  C HEMISTRY   Vol. 278, No. 48, Issue of November 28, pp. 48146–48153, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.  Printed in U.S.A. This paper is available on line at http://www.jbc.org 48146  entirely scrambled PrP-(82 – 146) (PrP-(82 – 146) scr ) and partiallyscrambled PrP-(82 – 146), where only the sequence 106 – 126 (PrP-(82 – 146) 106 – 126scr ) or 127 – 146 (PrP-(82 – 146) 127 – 146scr ) was altered(Fig.1).Theselectionofthesequencestobemodifiedwasbasedonprevious observations that the region spanning residues 106 – 147is central to amyloid fibril formation (20). In particular, a peptidecomprising residues 106 – 126 showed high propensity to adoptstable   -sheet secondary structure and to assemble into straight,unbranched amyloid fibrils, similar in ultrastructure to those ob-served in GSS patients (20 – 22). Also, a peptide spanning PrPresidues 127 – 147 (PrP-(127 – 147)) was able to generate amyloid-like fibrils, resembling scrapie-associated fibrils isolated from sub- jectswithtransmissiblespongiformencephalopathies(20).Herewereport that PrP-(82 – 146) wt  has high intrinsic ability to form amy-loid fibrils indistinguishable from those observed in GSS diseaseand that the formation of ordered structures having a parallel  -sheet organization is dependent upon the integrity of the C-terminal region. MATERIALS AND METHODS  Synthesis and Purification of PrP Peptides  — The following peptideswere chemically synthesized: PrP-(82 – 146) wild type (PrP-(82 – 146) wt ,GQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVG-GLGGYMLGSAMSRPIIHFGSDYE); PrP-(82 – 146) with a scrambledsequence in the region spanning residues 106 – 126 (PrP-(82 – 146) 106 – 126scr , GQPHGGGWGQGGGTHSQWNKPSKPNGAKALM-GGHGATKVVGAAAGYMLGSAMSRPIIHFGSDYE); PrP-(82 – 146)with a scrambled sequence in the region spanning residues 127 – 146(PrP-(82 – 146) 127 – 146scr , GQPHGGGWGQGGGTHSQWNKPSKPKTN-MKHMAGAAAAGAVVGGLGSMYPASHGLMEDFYGIGSIR); PrP-(82 – 146) with a totally scrambled sequence (PrP-(82 – 146) scr , EADQF- ALGGSKHGNGMQQVAGHGGSMGAKAWGANGHPSGTGIPTAKM- VPYKIYGGGWAGMGRPSS). In some experiments, the shorter pept-ides PrP-(106 – 126) wt  (KTNMKHMAGAAAAGAVVGGLG) and PrP-(127 – 146) wt  (GYMLGSAMSRTIIHFGSDYE) were also used. Peptideswere prepared by solid-phase synthesis on a 433A synthesizer (AppliedBiosystems, Foster City, CA) purified by reverse phase-HPLC and verified by amino acid sequencing (46600 Prosequencer, Milligen,Bedford, MA) and electrospray mass spectrometry (model 5989A,Hewlett-Packard, Palo Alto, CA) as described previously (22, 23).  Hydropathic Profile and Transmembrane Helix Prediction of PrP Peptides  — PrP-(82 – 146) wt  and its analogues were analyzed by a programdevised by Kyte and Doolittle (24) that progressively evaluates the hydro-philicity and hydrophobicity of a protein along its amino acid sequence(www.expasy.ch/cgi-bin/protscale.pl). The common hydropathy value, de-fined at one specific position in a sequence, is the mean value of thehydrophobicity of the amino acids within a window around each position.In hydrophobic regions the hydropathy value is high for a number of consecutive positions in the sequence. The grand average of hydropathic-ity (GRAVY) is defined as the average value of the hydropathy value ateach position (24). For prediction of membrane protein topology theTMHMM method, based on a hidden Markov model developed by Kroghand co-workers, was applied (25) (www.cbs.dtu.dk/services/TMHMM/).  Preparation of PrP Peptide Stock Solutions  — Peptides were dissolvedin sterile deionized water at 5 mg/ml. Under these conditions they weresoluble, as deduced by the absence of a visible pellet after centrifugationat 13,000   g  for 10 min. The stock solutions were stable for 2 weeks at  80  ° C, as determined by reverse phase-HPLC. For all the experimentsaliquots of stock solutions were diluted with different buffers as speci-fied below.  Sedimentation Experiments  — The study was performed with 0.5 and1 m M  peptide in 20 m M  Tris-HCl, pH 7.0. Aliquots of 30   l wereincubated at 37  ° C for 1, 4, 8, 24, 48, 72, and 168 h and then chilled onice and centrifuged at 13,000   g  for 10 min at 4  ° C. Supernatants wereanalyzed by reverse phase-HPLC, and peptide concentrations at differ-ent times were expressed as percentages of the corresponding valuesdetermined at zero time (26).  PK Digestion  —  Aliquots of 1 m M  peptides in 100 m M  Tris-HCl, pH 7.0,containing 1 m M  CaCl 2  were incubated at 37  ° C for 96 h and thendigested with PK for 30 min at 1:20 (w/w) enzyme-to-substrate ratio.Proteolysis was terminated by the addition of EGTA at a final concen-tration of 5 m M . After centrifugation at 13,000   g  for 10 min at 4  ° C,the pellets were dissolved in 30   l of 97% formic acid, and 20   l wereanalyzed by reverse phase-HPLC. Control samples were processed un-der the same conditions in the absence of PK. The extent of the prote-olysis was calculated as the percentage of the peptide present in thepellets compared with undigested controls (23).  Light and Electron Microscopy  — Samples of 0.5, 0.1, and 0.05 m M peptides in 50 m M  Tris-HCl, pH 7.0, were incubated at 37  ° C for 1, 4, 8,24, 48, 72, and 168 h. At each time point, sample aliquots were analyzedby light and electron microscopy. For light microscopy, 10   l of suspen-sion were air-dried on poly- L -lysine-coated slides (Bio-Optica, Milan,Italy), stained with thioflavine S and Congo Red, and viewed underfluorescent or polarized light, respectively (Nikon Eclipse E-800, Kyoto,Japan). For ultrastructural examination, 5   l of suspension were ap-plied to Formvar-carbon 200-mesh nickel grids for 5 min, negativelystained with uranyl acetate, and observed in an electron microscope(EM109 Zeiss, Oberkoken, Germany) operated at 80 kV at a standardmagnification, calibrated with an appropriate grid. The mean diameterof fibrils generated by PrP peptides after short (1 h) and long (1, 3, and7 days) incubation was measured on printed photos at a final magnifi-cation of   90,000 by using a computer-assisted image analyzer (Nikon,Japan) (23). At day 7, samples were centrifuged at 13,000    g  for 15 min. Thepellets were fixed in 2.5% glutaraldehyde in 50 m M  phosphate buffer,pH 7.4, post-fixed in 0.1% aqueous osmium tetroxide, dehydrated ingraded acetone, and embedded in epoxy resin (Spurr, Electron Micros-copy Science, Fort Washington, PA). Ultrathin sections (50 nm) werecollected on 200-mesh copper grids, positively stained with uranylacetate and lead citrate, and observed with the electron microscope.  X-ray Diffraction  — The peptides were analyzed under the following conditions: lyophilized, vapor-hydrated, solubilized, and then dried.Lyophilized peptide was gently packed into a siliconized thin walledglass capillary (0.7 mm diameter; Charles A. Supper Co., South Natick,MA) to form a disk. For vapor hydration, the lyophilized peptide wasequilibrated against a column of water that was sealed with it in thecapillary. For preparing solubilized/dried peptide assemblies, lyophi-lized peptide was dissolved at  10 mg/ml in water, and the solution wasaspirated into a thin walled siliconized capillary to a column height of   10 – 12 mm. A tiny hole was poked through the sealing wax at one endof the capillary, and the sample was left to dry gradually under ambienttemperature and humidity while in a 2-tesla permanent magnet(Charles A. Supper Co.) (27), which can promote fibril alignment bydiamagnetic anisotropy (28). Orientation and dehydration were moni-tored by periodic observation of sample birefringence between crossedpolarizers. When the sample had dried to a small uniform disk, thecapillary was transferred to a holder for x-ray diffraction. X-ray diffraction patterns were obtained using nickel-filtered, dou-ble-mirror focused CuK    radiation from an Elliott GX-20 rotating anodex-ray generator (GEC Avionics, Hertfordshire, UK) with a 200-  m focalspot, operated at 35 kV and 25 mA. A helium tunnel was placed in thex-ray path to reduce air scatter. Patterns were recorded on EastmanKodak DEF film. The known Bragg spacing of calcite (0.3035 nm) wasused to calibrate the specimen-to-film distance (87.6 mm). The Bragg spacings of reflections were measured with a 6   optical comparatordirectly off the films.  FTIR of Peptide Aggregates  — Samples of 0.1 m M  PrP-(82 – 146) wt  andPrP-(82 – 146) 106 – 126scr  in 20 m M  Tris-HCl, pH 7.0, were incubated at37  ° C for 24 h. Following centrifugation at 3,000   g  for 10 min, peptideaggregates were collected and gently dried in a stream of nitrogen toremove any water residue. The samples were mixed with KBr, pulver-ized, and formed into a disk-shaped pellet (29, 30). Data were collectedon the solid pellets at room temperature with a Fourier transform IRspectrum BX (PerkinElmer Life Sciences) for wavelengths in the rangeof 4000 to 400 cm  1 . The spectral processing and the determination of the secondary structures from the intensities of bands in the secondderivative amide I spectra were performed as described by Caughey  etal.  (3). As indicated by the relatively small signal of the second deriv-ative spectrum in the range 1720 – 1800 cm  1 (Fig. 8  A ,  inset ), there waslittle or no appreciable residual water vapor contribution to the spec-trum, and the signal-to-noise was sufficiently high for the secondarystructure determination.  Membrane Microviscosity Determination  — Membrane microviscositywas assessed in suspensions of primary cultures of rat cortical neuronsusing DPH as a fluorescent probe as described previously (31). Thereported FP (expressed as arbitrary units) is a function of the emission(420 nm) detected through an analyzer oriented parallel (  p 1 ) and per-pendicular (  p 2 ) to the direction of the polarization of the exciting light(365 nm), according to the equation FP    (  p 2    p 1 )/(  p 2    p 1 ) (12).Membrane microviscosity (  , poise) is related to FP according to theequation      2FP/0.46    FP. Neuronal cells (2    10 6 cell/ml) weremechanically detached, gently centrifuged at 550    g  for 10 min,washedwithsaline,resuspendedin2.5mlof5m M phosphatebuffer,pH  Structural Properties of PrP-(82  – 146)  48147  7.4, containing 2   M  DPH, and incubated for 30 min at room tempera-ture. PrP peptides were dissolved in 5 m M  phosphate buffer, pH 7.4, andtested within 10 min of solubilization. The FP value was determined at25  ° C, before and 30 min after the addition of 25   M  PrP peptides to cellsuspensions (31). RESULTS  Hydropathic Profile of PrP-(82  – 146) Peptides  — The hydro-pathic profile of the peptides selected for study was evaluatedaccording to the Kyte and Doolittle scale (24). The analysisshowed that PrP-(82 – 146) wt  has an N-terminal hydrophilicregion followed by an extended hydrophobic domain spanning residues 112 – 131, with a maximum of 1.844 at Gly-119 and aminimum of 0.633 at Gly-131. PrP-(82 – 146) 127 – 146scr  exhibiteda similar profile, whereas the alteration of the 106 – 126 se-quence (PrP-(82 – 146) 106 – 126scr ) resulted in a shorter hydropho-bic region spanning residues 120 – 131, with a maximum of 1.511 at Ala-124 and a minimum of 0.633 at Gly-131. PrP-(82 – 146) scr  showed a flat profile, consistent with its disarrangedhydrophilic and hydrophobic domains compared with PrP-(82 – 146) wt  (Fig. 1).  Disarrangement of C-terminal Region of PrP-(82  – 146) Has Profound Effect on Peptide Aggregation  — To investigate theability of PrP-(82 – 146) wt  and its analogues to form macroag-gregates, we carried out sedimentation experiments. Peptideswere dissolved in 20 m M  Tris-HCl, pH 7.0, incubated at 37  ° Cfor 0 – 96 h, and then centrifuged. The supernatants were ana-lyzed by HPLC to determine the percentage of peptide still insolution after centrifugation. The aggregation kinetics of PrP-(82 – 146) wt  was substantially linear during the first 3 days,yielding   25, 40, and 60% of sedimentable peptide after 24, 48,and 72 h, respectively. Thereafter, the peptide showed an in-creased aggregation rate, and more than 90% was found in thepellet after 96 h. The modification of the amino acid sequence106 – 126 resulted in a change in the aggregation kinetics, asalmost 60% of PrP-(82 – 146) 106 – 126scr  was sedimentable after24 h; subsequently, the aggregation proceeded slowly, exceptfor an acceleration in the last 24 h leading to more than 85% of sedimentable peptide after 96 h of incubation. On the otherhand, the alteration of the 127 – 146 region remarkably reducedthe aggregation ability, as PrP-(82 – 146) 127 – 146scr  showed a lim-ited rate of assembly into sedimentable structures that reachedstatistical significance (t   0.01, Student ’ s  t  test,  n    5) incomparison to the non-sedimentable PrP-(82 – 146) scr  only after96 h (Fig. 2).  Protease Resistance of PrP Peptides Related to Their Aggre- gation Properties  — Suspensions of pre-aggregated peptideswere subjected to PK digestion at 37  ° C for 30 min at 1:20 (w/w)enzyme-to-substrate ratio. Following centrifugation, the pel-lets were dissolved in formic acid and analyzed by HPLC. Theextent of proteolysis was calculated as percentage of peptidepresent in the pellet compared with undigested controls. Underthese conditions, 47.2  3.0% of PrP-(82 – 146) wt  was protease-resistant, whereas PrP-(82 – 146) scr  was almost completely de-graded (Fig. 3). The modification of the amino acid sequence127 – 146 resulted in a decrease of the protease-resistant frac-tion to 15.4  2.3% of control values. Conversely, the change of the 106 – 126 region yielded a striking increase in PK resistance(78.3  8.2%).  PrP-(82  – 146) Protofilament and Amyloid Fibril Formation Depends upon Integrity of C-terminal Region  — The staining properties and ultrastructure of aggregates generated by PrP-(82 – 146) analogues were determined at various incubationtimes ranging from 1 to 168 h. At each time point, aliquots of peptide suspensions were stained with Congo Red and thiofla- vine S and examined by polarized light and fluorescence mi-croscopy, respectively, or negatively stained with uranyl ace-tate and analyzed by EM. After 168 h, the samples werecentrifuged, and the pellets were fixed in glutaraldehyde andembedded in Spurr. Ultrathin sections were positively stainedand analyzed by EM.The analysis showed that PrP-(82 – 146) wt  readily formedamyloid fibrils, whereas the entirely scrambled peptide, PrP-(82 – 146) scr , did not generate filamentous structures even after F IG . 1.  Primary structure, hydropathy plot and position of netcharges of PrP-(82 – 146) and its analogues.  The potential trans-membrane domains, as deduced by a membrane protein topology pre-diction method (36), are  underlined. F IG . 2.  Time course of PrP peptides aggregation.  Peptides weredissolved in 20 m M  Tris-HCl, pH 7.0, at the concentration of 0.5 m M  andincubated for 0 – 96 h at 37  ° C. Following centrifugation, the superna-tants were analyzed by HPLC. The non-sedimentable peptide fractionwas expressed as percentage of the total amount of peptide at time 0.Each value is the mean  S.D. of at least five experiments.  Structural Properties of PrP-(82  – 146) 48148  a 7-day incubation. Two distinct populations of long, straight,unbranched fibrils were observed in negatively stained PrP-(82 – 146) wt  suspensions after 1 h: (i) 5.5-nm-diameter protofila-ments with high propensity to adhere to each other, organizing into bundles of various widths (Fig. 4  A ); and (ii) 9.5-nm-diam-eter fibrils generating loose meshworks (Fig. 4  B  and Table I).The first population progressively decreased, whereas the sec-ond population progressively increased with time, and only9.8-nm-diameter fibrils organized into dense meshworks weredetectable after 72 h.The fibrillogenic ability of the peptide was affected by mod-ification of the 106 – 126 region. After 1 h of incubation, PrP-(82 – 146) 106 – 126scr  generated primarily amorphous aggregatesthat were associated with few, relatively short, unbranched,irregularfilamentshavinganaveragediameterof7.3nm.Withtime, the density of fibrillar assemblies progressively increasedand more regular, straight, unbranched fibrils having a diam-eter of 7.7 nm were detected (Fig. 4 C  and Table I). These fibrilswere often paired or organized into bundles or loose meshworksand were associated with a substantial amount of amorphousaggregates. Alteration of the 127 – 146 region had an even moreprofound effect. After 1 h of incubation, negatively stainedsamples of PrP-(82 – 146) 127 – 146scr  essentially contained someamorphous material. After 24 h, a few irregular threads andfilamentous structures having a diameter of 7.1 nm were alsopresent. The filaments increased with time and tended to ad-here to each other, with formation of twisted bundles of variouswidths, depending on the number of the constituent filaments(Fig. 4  D  and Table I). However, even after 7 days, the sampleslargely consisted of amorphous material, and the overall den-sity of the aggregates was remarkably lower than observedwith PrP-(82 – 146) wt  and PrP-(82 – 146) 106 – 126scr .EM examination of the pellets obtained by centrifugation of  F IG . 3.  Protease resistance of PrP peptides.  Peptides were dis-solved in 100 m M  Tris-HCl, pH 7.0, containing 1 m M  CaCl 2  at theconcentration of 1 m M , and incubated at 37  ° C for 96 h. They were thensubjected to digestion with proteinase K for 30 min at 1:20 (w/w)enzyme-to-substrate ratio. Following centrifugation, pellets were dis-solved in 97% formic acid and analyzed by HPLC. The extent of prote-olysis was calculated as percentage of peptide present in the pellet.Each value is the mean  S.D. of at least five experiments.F IG . 4.  Electron micrographs of the aggregates generated byPrP-(82 – 146) analogues, as revealed by negative staining of pep-tide suspensions.  A  and  B  show two distinct populations of fibrilsgenerated by PrP-(82 – 146) wt  after a 1-h incubation, whereas  C  and  D correspond to PrP-(82 – 146) 106 – 126scr  and PrP-(82 – 146) 127 – 146scr  after a48-h incubation, respectively.  Bar , 100 nm.F IG . 5.  Electron micrographs of the aggregates generatedby PrP-(82 – 146) analogues, as revealed by positive staining of ultrathin sections of the pellets obtained by centrifugationafter 1 week of incubation.  A , PrP-(82 – 146) wt ;  B  and  C , PrP-(82 – 146) 106 – 126scr ;  D , PrP-(82 – 146) 127 – 146scr .  Bar , 100 nm.F IG . 6.  Congo Red staining of PrP peptide aggregates.  Pep-tide suspensions were incubated for 48 h and then analyzed by polar-ized light microscopy.  A , PrP-(82 – 146) wt ;  B , PrP-(82 – 146) 106 – 126scr ; C , PrP-(82 – 146) 127 – 146scr .T  ABLE  I  Diameter (nm) of fibrils generated by PrP-(82-146) analogues atdifferent incubation times Results are the means  S.D. of at least 100 measurements in threedifferent experiments. Time PrP-(82-146) wt  PrP-(82-146) 106-126scr  PrP-(82-146) 127-146scr h 1 5.5  1.8 No fibrils No fibrils9.5  1.124 5.6  1.8 7.3  1.0 7.1  1.59.6  1.272 9.7  1.1 7.6  0.9 7.3  1.1168 9.8  1.1 7.7  1.1 7.4  1.0  Structural Properties of PrP-(82  – 146)  48149  peptide suspensions after a 7-day incubation enabled a betterdefinition of the fine morphology of different types of aggre-gates and the evaluation of their relative amount. Positivelystained ultrathin sections of PrP-(82 – 146) wt  contained onlylong, straight, unbranched fibrils that formed dense mesh-works (Fig. 5  A ) or, less often, star-like structures whose mor-phology was very similar to human GSS amyloid plaques cores. Also, PrP-(82 – 146) 106 – 126scr  preparations contained straight,unbranched amyloid-like fibrils that could be distinguishedfrom those of PrP-(82 – 146) wt  by being smaller in diameter andshorter. Furthermore, the fibrillar assemblies were less dense(Fig. 5  B ) and were associated with a large amount of granularand amorphous material (Fig. 5 C ). The pellet obtained fromPrP-(82 – 146) 127 – 146scr  was smaller than that generated by PrP-(82 – 146) wt  and PrP-(82 – 146) 106 – 126scr  and contained primarilyelectron dense amorphous material intermingled with a rela-tively small number of short filamentous structures. PrP-(82 – 146) 127 – 146scr  filaments did not possess the ultrastructural fea-tures of amyloid fibrils and were usually assembled intotwisted bundles (Fig. 5  D ).Macromolecular assemblies of PrP-(82 – 146) wt  showed thetinctorial and optical properties of   in situ  amyloid,  i.e.  birefrin-gence under polarized light after Congo Red staining and yel-low fluorescence after thioflavine S treatment. These proper-ties were detected even after 1 h and were more apparent afterlonger incubation times, as the number and size of the aggre-gates increased (Fig. 6  A ). In PrP-(82 – 146) 106 – 126scr  samples,birefringent and fluorescent aggregates were observed onlyafter 24 h and increased with time, without reaching the sizeand density of the wild type peptide even after 7 days (Fig. 6  B ).PrP-(82 – 146) 127 – 146scr  aggregates did not possess the tinctorialproperties of amyloid, except for a few small bundles observedafter long incubation (Fig. 6 C ).  PrP-(82  – 146) wt  Fibrils Have Cross-  -structure  —  X-ray dif-fraction patterns were recorded from PrP-(82 – 146) wt , PrP-(82 – 146) 106 – 126scr , PrP-(82 – 146) 127 – 146scr , and PrP-(82 – 146) scr  pep-tides under three different conditions,  i.e.  lyophilized (L), vapor-hydrated (VH), and solubilized/dried (S/D) (Table II).PrP-(82 – 146) wt  after the S/D treatment gave an oriented dif -fraction pattern where a sharp and strong reflection at Bragg spacing 0.477 nm was observed on the meridian (Fig. 7) andafter L and VH treatments showed a circular reflection at  0.47 nm. A similar circular reflection at   0.47 nm was ob-served for PrP-(82 – 146) 106 – 126scr  after L and S/D treatments;conversely, the vapor-hydrated peptide did not show a distinctreflection, possibly due to the short exposure time. NeitherPrP-(82 – 146) 127 – 146scr  nor PrP-(82 – 146) scr  gave any distinctreflections, even after   100 h of exposure. The spacing at  0.47 nm corresponds to the hydrogen-bonding distance be-tween parallel or antiparallel    chains. The sharpness of thisreflection indicates that the peptides form a periodic array of    chains that run normal to the long axis of the elongated assem-bly. An oriented sample of PrP-(82 – 146) wt  after solubilizationand drying showed a low angle reflection at 5.9 nm spacing. If this arises from the first intensity maximum of a long cylindri-cal (fibrillar) structure having radius  r 0 , the reciprocal coordi-nate  R  and  r 0  are related by 2   r 0  R  5.152 for a solid cylinderand 3.770 for a tubular cylinder (32). Given  R  (5.9 nm)  1 , theradius  r 0  was calculated to be 4.8 nm for a solid cylinder and 3.5nm for a tubular cylinder. Thus, the fibril diameter is   7 – 10nm. This estimate based on diffraction measurements wassimilar to the 9.7-nm width measured from electron micro-graphs of the second population of fibrils for PrP-(82 – 146) wt .This size is larger than that of the protofilaments (4.0 nm) of asimilar peptide ( i.e.  PrP-(90 – 145)) (33), indicating that thePrP-(82 – 146) fibril in the current study is likely constituted of a multiple of protofilaments. Unlike PrP-(82 – 146) wt  orienta-tion after S/D treatment, PrP-(82 – 146) 106 – 126scr  showed a weakcircular reflection at   0.47 nm, indicating lack of orientationand weaker scattering compared with the wild type peptide.Moreover, the larger breadth of the 0.47 nm reflection com-pared with that in the wild type suggests that the number of H-bonded strands is less in the structure formed by the scram-bled sequence. PrP-(82 – 146) 127 – 146scr  and PrP-(82 – 146) scr  didnot show any distinct reflections, indicating that these peptidesdid not form a sufficient number of organized macromolecularassemblies that could be detected by x-ray scatter.  FTIR Spectra of PrP-(82  – 146) wt  Assemblies Suggest Parallel  -Sheet Organization of Fibrils  — FTIR analysis was carried outon PrP-(82 – 146) wt  and PrP-(82 – 146) 106 – 126scr  aggregates be-cause only these peptides formed significant amounts of sedi-mentableprecipitateenablingthepreparationofdiscretequan-tities of samples. Fig. 8  A  shows two diagnostic regions of theFTIR spectrum recorded for PrP-(82 – 146) wt  fibrils. The narrowpeak 1 that falls at 1630 cm  1 (amide I region) indicates thepresence of an extended    sheet structure that arises fromsymmetric carbonyl stretch (34). The absence of the diagnostic F IG . 7.  X-ray diffraction from PrP-(82 – 146) wt  dried after solu-bilization and drying.  The exposure time was 70 h. The sharp me-ridional reflection had a Bragg spacing of 0.477 nm ( arrows ), and thefirst intensity maximum of the broad, small angle reflection (obscuredsomewhat by central scatter) was at 5.9 nm spacing ( arrowheads ).T  ABLE  II  Summary of x-ray diffraction measurements Sample a 82-146 wt  82-146 106-126scr  82-146 127-146scr  82-146 scr Condition L VH S/D L VH S/D S/D S/DSpacing  b 0.484 C 0.477 C 0.477 M 0.469 C    0.473 C    0.278 c Forward scatter d    5.9       Exposure (h) 43 40 70 72 22 141 106 118 a  L , lyophilized;  VH  , vapor-hydrated;  S  /   D , solubilized and dried. b Bragg spacing in nm;  C ; circular reflection,  M  ; meridional reflection.  indicates that discrete reflections were not detected. c This crystalline, sharp reflection likely arises from NaCl. d Forward scatter refers to the central scattering observed near the beam stop, likely arising from the structure factor of the macromolecularassembly. The solubilized and dried sample of PrP-(82-146) wt  is the only sample that gave an oriented pattern. A broad and weak intensitymaximum was observed at 5.9 nm Bragg spacing.  Structural Properties of PrP-(82  – 146) 48150
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