Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m413185200
ISSN1083-351X
AutoresAlexis Nazabal, Marie‐Lise Maddelein, Marc Bonneu, Sven J. Saupe, Jean‐Marie Schmitter,
Tópico(s)Infectious Encephalopathies and Encephalitis
ResumoThe HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218–289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218–289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218–289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218–289) sequence. The HET-s-(218–289) fibrils are thus likely to be organized as a succession of β-sheet segments interrupted by short turns or short loops. The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218–289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218–289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218–289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218–289) sequence. The HET-s-(218–289) fibrils are thus likely to be organized as a succession of β-sheet segments interrupted by short turns or short loops. Amyloids are fibrillar protein aggregates composed of a “cross-β” structure in which β-strands are oriented perpendicular to the fiber axis. This type of protein aggregate is associated not only with prion diseases but with a variety of protein deposition diseases including Alzheimer disease and Parkinson disease (1.Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2475) Google Scholar). Resolution of the structure of amyloid assemblies is of foremost importance from both a fundamental and a biomedical point of view. As a consequence, much effort has been devoted to the acquisition of structural information on a number of amyloid proteins using a variety of methods including proline scanning mutagenesis (2.Williams A.D. Portelius E. Kheterpal I. Guo J.T. Cook K.D. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (344) Google Scholar, 3.Wood S.J. Wetzel R. Martin J.D. Hurle M.R. Biochemistry. 1995; 34: 724-730Crossref PubMed Scopus (318) Google Scholar), hydrogen exchange (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 5.Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar, 6.Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. 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Among the amyloidogenic proteins, prion proteins possess the unique ability to replicate the amyloid conformation and thus display an infectious character. In mammals, prions are infectious proteinaceous particles that cause fatal neurodegenerative diseases termed spongiform encephalopathies (12.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). Mammalian prions correspond to an altered form of a cellular protein termed PrP that is converted to a protease-resistant aggregated form in diseased individuals. Proteins capable of propagating an altered conformational state have also been identified in eukaryotic microorganisms (13.Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1086) Google Scholar). The best characterized prion models are the yeast Ure2p and Sup35p proteins and the HET-s protein from the fungus Podospora anserina (14.Wickner R.B. Edskes H.K. Roberts B.T. Baxa U. Pierce M.M. Ross E.D. Brachmann A. Genes Dev. 2004; 18: 470-485Crossref PubMed Scopus (64) Google Scholar). All of these proteins form amyloid fibrils in vitro (15.Glover J.R. Kowal A.S. Schirmer E.C. Patino M.M. Liu J.J. Lindquist S. Cell. 1997; 89: 811-819Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 16.Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1343Crossref PubMed Scopus (264) Google Scholar, 17.King C.Y. Tittmann P. Gross H. Gebert R. Aebi M. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6618-6622Crossref PubMed Scopus (293) Google Scholar, 18.Dos Reis S. Coulary-Salin B. Forge V. Lascu I. Begueret J. Saupe S.J. J. Biol. Chem. 2002; 277: 5703-5706Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). These fungal prion proteins represent valuable models for exploring the mechanism of prion propagation. It is essential to gain insight into the structure of prion proteins in their infectious conformation to elucidate the details of prion replication and also to determine whether specific structural features are associated with the infectious character. The HET-s prion protein of P. anserina is involved in a genetically programmed cell death reaction termed heterokaryon incompatibility. This cell death reaction is triggered when the prion form of HET-s interacts with a natural variant of HET-s, designated HET-S, which differs from HET-s by 13 residues and is devoid of prion behavior (19.Coustou V. Deleu C. Saupe S. Begueret J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9773-9778Crossref PubMed Scopus (407) Google Scholar). HET-s aggregates specifically in vivo upon transition to the prion state (20.Coustou-Linares V. Maddelein M.L. Begueret J. Saupe S.J. Mol. Microbiol. 2001; 42: 1325-1335Crossref PubMed Scopus (49) Google Scholar). Recombinant HET-s forms typical amyloid fibrils in vitro (18.Dos Reis S. Coulary-Salin B. Forge V. Lascu I. Begueret J. Saupe S.J. J. Biol. Chem. 2002; 277: 5703-5706Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We were able to show that introduction of amyloid aggregates of recombinant HET-s into P. anserina cells induces the [Het-s] prion with a very high efficiency (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). Hence, the amyloids generated in vitro represent infectious material that can propagate the [Het-s] prion. The HET-s protein is 289 amino acids in length and is composed of two distinct domains: an N-terminal globular domain spanning approximately residues 1 to 230 and a flexible domain spanning approximately residues 230 to 289 (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). In the amyloid form of HET-s, the C-terminal domain of HET-s forms the protease-resistant amyloid core of the fibril, whereas the N-terminal domain remains accessible to proteolysis. The C-terminal domain of HET-s (residues 218–289) is sufficient for [Het-s] propagation in vivo and amyloid formation in vitro (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). Hydrogen/deuterium exchange combined with mass spectrometry (HXMS) 1The abbreviations used are: HXMS, hydrogen/deuterium exchange combined with mass spectrometry; H/D, hydrogen/deuterium; PrP, prion protein; H2O MQ, Ultrapure milli-Q H2O; MS, mass spectroscopy; LC-MS/MS, liquid chromatography-tandem mass spectroscopy. has become a powerful tool for the study of protein structures and dynamics (3.Wood S.J. Wetzel R. Martin J.D. Hurle M.R. Biochemistry. 1995; 34: 724-730Crossref PubMed Scopus (318) Google Scholar, 23.Smith D.L. Deng Y. Zhang Z. J. Mass Spectrom. 1997; 32: 135-146Crossref PubMed Scopus (385) Google Scholar, 24.Wagner D.S. Melton L.G. Yan Y. Erickson B.W. Anderegg R.J. Protein Sci. 1994; 3: 1305-1314Crossref PubMed Scopus (57) Google Scholar, 25.Engen J.R. Smith D.L. Anal. Chem. 2001; 73: 256A-265ACrossref PubMed Google Scholar, 26.Maier C.S. Schimerlik M.I. Deinzer M.L. Biochemistry. 1999; 38: 1136-1143Crossref PubMed Scopus (52) Google Scholar, 27.Tito P. Nettleton E.J. Robinson C.V. J. Mol. Biol. 2000; 303: 267-278Crossref PubMed Scopus (40) Google Scholar). This technique exploits the ability of mass spectrometry to determine the incorporation of deuterium atoms in proteins following a reaction of exchange with labile hydrogens located at backbone amide positions. Isotope exchange rates for amide hydrogens depend primarily on intramolecular hydrogen bonding and access to the solvent. This exchange rate depends also on the pH and the temperature of the exchange reaction. Protein dynamics can be studied by hydrogen exchange and depend on the unfolding and refolding rate constants of the protein (28.Englander S.W. Mayne L. Bai Y. Sosnick T.R. Protein Sci. 1997; 6: 1101-1109Crossref PubMed Scopus (269) Google Scholar, 29.Raschke T.M. Marqusee S. Curr. Opin. Biotechnol. 1998; 9: 80-86Crossref PubMed Scopus (88) Google Scholar). For a detailed characterization of solvent accessibility in specific regions, the protein can be submitted to proteolysis after the exchange reaction (30.Zhang Z. Smith D.L. Protein Sci. 1993; 2: 522-531Crossref PubMed Scopus (902) Google Scholar). More details concerning HXMS methodology are presented in a review by Engen and Smith (25.Engen J.R. Smith D.L. Anal. Chem. 2001; 73: 256A-265ACrossref PubMed Google Scholar). Hydrogen/deuterium exchange combined with mass spectrometry or NMR has been used to analyze the structural properties of several amyloid peptides or proteins (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 5.Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar, 31.Kuwata K. Matumoto T. Cheng H. Nagayama K. James T.L. Roder H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14790-14795Crossref PubMed Scopus (121) Google Scholar, 32.Yamaguchi K. Katou H. Hoshino M. Hasegawa K. Naiki H. Goto Y. J. Mol. Biol. 2004; 338: 559-571Crossref PubMed Scopus (99) Google Scholar, 33.Kheterpal I. Lashuel H.A. Hartley D.M. Walz T. Lansbury Jr., P.T. Wetzel R. Biochemistry. 2003; 42: 14092-14098Crossref PubMed Scopus (114) Google Scholar, 35.Nazabal A. Dos Reis S. Bonneu M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: 8852-8861Crossref PubMed Scopus (41) Google Scholar). It has been shown that amide protons in core regions of amyloid aggregates are highly resistant to hydrogen exchange (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 6.Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (166) Google Scholar, 7.Kraus M. Bienert M. Krause E. Rapid Commun. Mass Spectrom. 2003; 17: 222-228Crossref PubMed Scopus (28) Google Scholar). H/D exchange can thus be used to estimate the number of backbone amides involved in a highly hydrogen-bonded state. Kheterpal et al. (36.Kheterpal I. Wetzel R. Cook K.D. Protein Sci. 2003; 12: 635-643Crossref PubMed Scopus (36) Google Scholar) have reported H/D exchange data for Aβ1–40 fibrils, showing that 48–55% of backbone amide protons are highly protected from exchange. H/D exchange can also be used to distinguish highly hydrogen-bonded peptide segments from regions that are not involved in stable secondary structure elements. Highly protected regions are generally interpreted as representing β-sheet core elements, whereas regions of higher exchange are interpreted as disordered or loop segments. Our previous analysis by hydrogen exchange/mass spectrometry has revealed that the prion-forming domain of full-length HET-s protein is highly protected from hydrogen exchange in the amyloid form (35.Nazabal A. Dos Reis S. Bonneu M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: 8852-8861Crossref PubMed Scopus (41) Google Scholar). Herein we have verified by means of a biolistic procedure that the amyloid form of the recombinant HET-s-(218–289) peptide can induce the [Het-s] prion when introduced in vivo. We then used HXMS at high resolution to gain structural information on the infectious amyloid form of this peptide. HET-s-(218–289) Purification and Amyloid Formation—The histidine-tagged HET-s-(218–289) peptide was expressed and purified from inclusion bodies under denaturing conditions as described previously (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). To eliminate urea, the peptide was then submitted to gel filtration on a Sephadex G-25 column using 175 mm acetic acid as an eluent. Protein concentration was adjusted to 125 μm. To initiate the spontaneous amyloid aggregation of HET-s-(218–289), pH was brought to 8 by the addition of Tris base. Amyloid formation was monitored by thioflavin T binding and by electron microscopy as described previously (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). After amyloid aggregation, aggregates were recovered by centrifugation (20 min at 12,000 × g), and aggregates were resuspended in H2O. Biological Ballistic Infectivity Assay—The biolistic infectivity assay was performed as described previously (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). Strains of the het-s genotype expressing the HET-s protein in its non-prion form (designated [Het-s*]) were grown for 2 days at 26 °C on solid medium. Four strains were grown per Petri dish. 10 μg of HET-S-(218–289) or full-length HET-s protein was overlaid onto each [Het-s*] mycelium in a final volume of 100 μl of 180 mm Tris base, 175 mm acetic acid, pH 8. After evaporation of the buffer, plates were bombarded with 2 mg of tungsten particles (0.4 μm) in a Bio-Rad PDS-1000/helium system using 1100 p.s.i. rupture disks. Plates were incubated for 2 days at 26 °C, and two mycelial fragments were sampled from each strain confronted with a het-S tester strain. In this assay, strains that retained the prion-free [Het-s*] phenotype present a normal contact line with the het-S tester, whereas strains that acquired the [Het-s] prion phenotype present an abnormal contact line designated “barrage” and resulting from the incompatibility cell death reaction. If at least one of the two subcultures produced a barrage reaction with the het-S tester, the strain was scored as positive for the [Het-s] prion phenotype. Kinetics Study of Deuterium Incorporation in HET-s-(218–289)—A 1-ml suspension of aggregated HET-s-(218–289) at 90 μm, in Ultrapure milli-Q H2O (H2O MQ) was centrifuged for 30 min at 10,000 × g. The pellet was resuspended with 1 ml of H2O MQ. A 5-μl aliquot (90 μm) of the aggregated solution was diluted at 1:20 in H2O. After an incubation time from 5 min to 12 h, 5 μl of the labeled protein was submitted to a C4 ZipTip. The elution fraction (3 μl, 5:5:1 of H2O/acetonitrile/0.1% trifluoroacetic acid) was loaded into a nanospray needle (PicoTip emitters, standard coating, New Objective, Woburn, MA) prior to ion trap mass analysis. Peptide Mass Fingerprinting of Aggregated HET-s-(218–289) Prion Protein—Aggregated HET-s-(218–289) (1 ml, 90 μm, H2O MQ) was centrifuged for 30 min at 10,000 × g. The pellet was resuspended with 1 ml of H2O MQ. A 5-μl aliquot (90 μm) of the aggregated solution was diluted 1:20 in H2O. A 5-μl aliquot of the solution was treated under strong agitation in 4 m urea with immobilized pepsin (10 μl, 45 units) at pH 2.2 and 0 °C for 5 min. Digestion was followed by a short centrifugation (10,000 × g, 30s), and a 10-μl aliquot of the supernatant containing the HET-s digest was submitted to nanospray LC-MS/MS analysis. HET-s-(218–289) peptides were analyzed with an ion trap mass spectrometer (LCQ DECA XP, Thermo Finnigan, San Jose, CA) interfaced to a Dionex-LC Packing chromatographic system (C18 column, 75 μm ID, 150 mm long). After analysis, the TurboSequest program was used to assign peptide sequences from their fragment ions. Hydrogen/Deuterium Exchange on HET-s-(218–289) Amyloid Fibrils—Aggregated HET-s-(218–289) (1 ml, 90 μm, H2O MQ) was centrifuged for 30 min at 10,000 × g. The pellet was resuspended with 1 ml of H2O MQ. A 5-μl aliquot (90 μm) of the aggregated solution was diluted 1:20 in D2O and vortexed before incubation at 25 °C, pH 7, for variable times (between 5 min and 12 h). After incubation, 5 μl of the labeled solution was treated under strong agitation in 4 m urea with immobilized pepsin (5 min, 10 μl, 45 units) at pH 2.2 and 0 °C (exchange quenching conditions). Digestion was followed by a short centrifugation (10,000 × g, 30 s), and a 10-μl aliquot of the supernatant containing the HET-s digest was separated into three fractions by stepwise gradient elution from a C18 ZipTip. The three fractions were eluted with 2 μl of a solution containing 9:1:1; 8:2:1, and 6:4:1 of H2O/acetonitrile/0.1% trifluoroacetic acid, respectively. Each elution fraction was loaded into a nanospray needle (PicoTip emitters, standard coating) prior to mass analysis. The time from sample loading to data collection was kept constant for each sample analysis. Mass value given in the text for exchange experiments correspond to m/z of the centroid of the envelope of a pseudo-molecular ion cluster. In- and Back-exchange Controls—For back-exchange control, a pepsin digest of HET-s-(218–289) was diluted 19:1 in D2O and incubated for 12 h at 25 °C, pH 7, to achieve complete exchange of backbone amide protons for deuterium atoms. After incubation, the exchange was quenched at 0 °C by the addition of 0.1% trifluoroacetic acid (Pierce). After quenching, the peptide mixture was loaded on a C18 ZipTip (Millipore). The peptide fraction eluted from the ZipTip (the eluent was a solution containing 6:4:1 acetonitrile/H2O MQ with 0.1% trifluoroacetic acid kept at 0 °C) was directly loaded into a nanospray needle (PicoTip) before mass analysis. The relative back-exchange (%) was determined for each peptide by comparing the maximal number of exchanged amide hydrogen atoms and the corresponding experimental value. For in-exchange control, a pepsin digest was diluted 19:1 in the quenched solution (H2O MQ, 0.1% trifluoroacetic acid, 0 °C). The peptide mixture was loaded on a C18 ZipTip and eluted with a solution containing 6:4:1 acetonitrile/D2O with 0.1% trifluoroacetic acid kept at 0 °C. The incorporation of deuterium for in-exchange was determined in the same way as the back exchange control. MS Measurements—For MS experiments on the LCQ ion trap instrument, the samples were loaded in the source by mean of PicoTip needles (PicoTip Emitters, standard coating). The capillary temperature was set to 140 °C. The spray voltage was set to 0.90–1.40 kV. Microscopy—A fraction of the protein suspension (30 μm) used for exchange experiments on amyloid fibers was put onto a 400 mesh copper electron microscopy grid-coated with a plastic film (Formvar). To avoid rapid desiccation, sedimentation was allowed during 10 to 30 min in a moist Petri dish. Grids were then rinsed with 15–20 drops of freshly prepared 2% uranyl acetate in water (passed over 0.22-μm Millipore filters), dried with filter paper, and observed with a Phillips Tecnai 12 Biowin electron microscope at 80 kV. Data Analysis—Mass spectra were base line-corrected, and the number of deuterium atoms incorporated in a given peptide was determined from the centroid value of its isotopic peak cluster using the formula given in Equation 1 (37.Liu Y. Smith D.L. J. Am. Soc. Mass Spectrom. 1994; 5: 19-28Crossref PubMed Scopus (41) Google Scholar). D(t)=m(t)−m(0)m(100)−m(0)×N (Eq. 1) Where m(t) is the observed centroid mass of the peptide at incubation time point t, m(0) is the observed mass at time point 0 (for unlabeled peptides, see in-exchange control procedure), m(100) is the observed mass for a fully exchanged peptide (see back-exchange control procedure), and N is the total number of peptide exchangeable amide protons in the peptide. The percentage of deuterium incorporation is calculated in Equation 2 for each peptides of the peptide mass fingerprint. %Deuterium incorporation=D(t)/N (Eq. 2) Amyloids of HET-s-(218–289) Are Infectious—The proteinase K-resistant amyloid core of HET-s amyloids corresponds to the region spanning residue 218–289 (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar, 38.Balguerie A. Dos Reis S. Coulary-Salin B. Chaignepain S. Sabourin M. Schmitter J.M. Saupe S.J. J. Cell Sci. 2004; 117: 2599-2610Crossref PubMed Scopus (29) Google Scholar). We have previously shown that HET-s fibrils submitted to proteinase K digestion retain infectivity (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). Together these experiments suggest that HET-s-(218–289) amyloids generated in vitro should be infectious. It however remained possible that infectivity of the proteinase K digested HET-s amyloids could be due to an undigested contaminating fraction of full-length protein or else that the HET-s-(218–289) domain adopts a different structure in full-length HET-s amyloid as in amyloids formed from the HET-s-(218–289) peptide. To directly ascertain that amyloid aggregates of HET-s-(218–289) display an infectious character, we introduced such aggregates into living cells using the previously used biolistic assay. The HET-s-(218–289) peptide was incubated for 5 h at 125 μm at pH 8. Aggregates were recovered by centrifugation. As previously shown under these conditions, HET-s-(218–289) formed typical amyloid aggregates. By electron microscopy, HET-s-(218–289) amyloids appeared as unbranched fibrils of about 5 nm in width (Fig. 1). HET-s-(218–289) amyloids were then overlaid on the surface of a mycelium of a wild-type [Het-s*] (prion-free) strain grown on solid medium. Mycelia were then bombarded under vacuum with tungsten particles as described previously (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar) to introduce the recombinant peptide into the fungal cells. 48 h after bombardment, the strains were tested for the presence of the [Het-s] prion in incompatibility tests (Fig. 2). Of the 88 [Het-s*] mycelia that were bombarded in the presence of HET-s-(218–289) amyloids, 87 had acquired the [Het-s] prion. In the control experiment with full-length HET-s fibers, 64 of 64 bombarded [Het-s*] mycelia acquired [Het-s]. With buffer alone, all 32 tested mycelia maintained the [Het-s*] non-prion state. We conclude from this experiment that, as shown previously for the full-length HET-s protein, amyloid aggregates of the recombinant HET-s-(218–289) peptide are infectious.Fig. 2Biolistic introduction of the HET-s-(218–289) peptide in Podospora induces the [Het-s] prion. [Het-s*] strains have been overlaid with either buffer alone (control), full-length HET-s fibers, or HET-s-(218–289) fibers, bombarded with tungsten particles, and tested for the presence of the [Het-s] prion by confronting the strains with het-S tester strains. Formation of a dark line in the confrontation zone indicates that the strain has acquired the [Het-s] prion.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Kinetics Study of Deuterium Incorporation in HET-s-(218–289) Amyloid Aggregates—H/D exchange was allowed to occur on HET-s-(218–289) amyloid aggregates for variable times from 5 min to 12 h. The deuterium content was found to increase rapidly during the first 60 min of incubation in the deuterium solvent (Fig. 3). Incorporation reached 38 deuterium atoms after 12 h, which corresponds to 48% of the total exchangeable hydrogens of the protein. Thus, after a 12-h incubation time in D2O, half of the exchangeable hydrogen atoms of HET-s-(218–289) are still protected in the amyloid aggregate. Because no significant difference was found for the incorporation of deuterium after 7 h of incubation time (36 deuterium atoms incorporated, 45% exchange rate), we chose to study the solvent accessibility of aggregated HET-s protein after this incubation time. This observation indicates that a majority of the residues are highly protected from exchange, thus confirming the results already obtained with the full-length HET-s protein (35.Nazabal A. Dos Reis S. Bonneu M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: 8852-8861Crossref PubMed Scopus (41) Google Scholar). These results correlate well with those obtained by Kheterpal et al. (36.Kheterpal I. Wetzel R. Cook K.D. Protein Sci. 2003; 12: 635-643Crossref PubMed Scopus (36) Google Scholar) for Aβ1–40 fibrils; in their study, after 100 h of incubation time in D2O, 48–55% of backbone amide protons were highly protected from exchange. Peptide Mass Fingerprinting of HET-s-(218–289)—The proteolysis of HET-s-(218–289) by immobilized pepsin generates a large number of peptides. After on-line LC-MS/MS analysis, 104 peptides could be assigned to their sequence. These peptides cover the sequence of the HET-s-(218–289) protein completely. However, to avoid a possible back-exchange caused by the chromatographic process, HXMS experiments were preferably conducted in the off-line nanospray mode. Thus, to find optimal analytical conditions, the pepsin digest was separated by micro-chromatography with a three-step gradient on a C18 ZipTip. In this way, 88 peptides of the 104 peptides assigned by on-line LC-MS/MS could be found in the three fractions (Fig. 4). Finally, 76 peptides yielded a separation of isotopic clusters sufficient for HXMS experiments, in order to determine the deuterium incorporation in HET-s-(218–289) aggregates. Hydrogen/Deuterium Exchange on the Amyloid Form of the HET-s-(218–289) Protein—Aggregates of HET-s-(218–289) were obtained as described previously, and amyloid formation was monitored by electron microscopy (Fig. 1). Aggregates were then incubated for 7 h in the presence of deuterium. Three different experiments have been performed to determine the incorporation of deuterium atoms in aggregated HET-s-(218–289) after this 7-h incubation. For in- and back-exchange experiments, the incorporation of deuterium atoms was found homogeneous for all peptides. In the back-exchange control, after overnight incubation of the pepsin digest, the level of deuterium incorporation for each peptide reached (84 ± 4)%. In the in-exchange control, the level of deuterium incorporation for each peptide under exchange quenching conditions reached (12 ± 3)%. The deuterium incorporation along the HET-s-(218–289) sequence was found to be very heterogeneous. Individual peptides exchange levels were found as low as 10% (HET-s-(275–282)) or as high as 52% (HET-s-(222–224)) (Fig. 5). Globally, deuterium incorporation was higher in peptides corresponding to the N-terminal region of HET-s-(218–289) and lower in C-terminal peptides. Use of Shared Boundaries between Pe
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