Báo cáo khoa học: The reconstitution of mammalian prion infectivity de novo pot

MINIREVIEW The reconstitution of mammalian prion infectivity de novo Ilia V. Baskakov Medical Biotechnology Center, University of Maryland Biotechnology Institute, and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA Prion diseases are a group of fatal neurodegenerative maladies that can arise spontaneously or be inherited, and can also be infectious. The prion hypothesis postu- lates that the infectious agent of the diseases is an abnormally folded isoform of the prion protein (PrP Sc ) that propagates its abnormal conformation in an auto- catalytic manner by recruiting the normal isoform of the same protein (PrP C ) [1,2]. Potential infectivity of prion diseases raised major public concern owing mainly to the spread of mad cow disease in Europe, as well as to chronic wasting disease of deer and elk in North America and the possibility of transmission of prion diseases through blood and surgical instruments. This article summarizes our current knowledge about the biochemical nature of the prion infectious agent, and describes potential strategies and challenges rela- ted to the de novo generation of prion infectivity in vitro. Biochemical nature of the prion infectious agent While formal proof of the protein-only hypothesis [i.e. generation of synthetic prions in vitro using prion pro- tein (PrP) produced in Escherichia coli or PrP C purified from normal brains and achieving an infectivity titer high enough to cause disease in wild-type animals] has not yet been presented, substantial progress has been made in the last couple of years towards reaching this goal [3,4]. On the other hand, the opponents of the Keywords amyloid fibrils; conformational adaptation; in vitro conversion; prion diseases; prion protein; synthetic prions Correspondence I. Baskakov, 725 W. Lombard St., Baltimore, MD 21201, USA Fax: +1 410 706 8184 Tel: +1 410 706 4562 E-mail: Baskakov@umbi.umd.edu (Received 2 August 2006, revised 30 November 2006, accepted 1 December 2006) doi:10.1111/j.1742-4658.2007.05630.x The discovery of prion disease transmission in mammals, as well as a non- Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postu- lates that an abnormal protein conformation propagates itself in an auto- catalytic manner using the normal isoform of the same protein as a substrate and thereby acts either as a transmissible agent of disease (in mammals), or as a heritable determinant of phenotype (in yeast and fun- gus). While the prion biology of yeast and fungus supports this idea strongly, the direct proof of the prion hypothesis in mammals, specifically the reconstitution of the disease-associated isoform of the prion protein (PrP Sc ) in vitro de novo from noninfectious prion protein, has been difficult to achieve despite many years of effort. The present review summarizes our current knowledge about the biochemical nature of the prion infectious agent and structure of PrP Sc , describes potential strategies for generating prion infectivity de novo and provides some insight on why the reconstitu- tion of infectivity has been difficult to achieve in vitro. Several hypotheses are proposed to explain the apparently low infectivity of the first genera- tion of recently reported synthetic mammalian prions. Abbreviations CJD, Creutzfeldt–Jakob disease; PrP, prion protein; PrP*, an intermediate form of the prion protein formed on the pathway toward PrP Sc ; PrP C , normal cellular isoform of the prion protein; PrP Sc , disease-associated isoform of the prion protein; rPrP, recombinant prion protein; a-rPrP, a-helical isoform of rPrP; PK, proteinase K; PrP res , PK-resistant form of PrP; Tg, transgenic; TSE, transmissible spongiform encephalopathy. 576 FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS prion hypothesis have not put forward any strong experimental evidence to invalidate this hypothesis, despite many years of trials. To date, most prion researchers agree that the PrP is the major and essen- tial, if not the only, component of the prion infectious agent. The ongoing discussions no longer dispute the key role of PrP, but rather revolve around the ques- tions of whether additional cellular components are involved in prion replication and what role they may play. These cellular components could be divided into two large categories, namely (a) cofactors that are important for stabilizing PrP Sc structure and therefore are a constituent part of PrP Sc and ⁄ or (b) cofactors that are required for conversion from PrP C to PrP Sc , but are not necessarily incorporated into PrP Sc aggre- gates. With respect to the first group, polysaccharides that consist of an a-linked polyglucose were shown to be permanent components of prion rods purified from scrapie brains [5,6]. Approximately 5–15% of PrP Sc is composed of polysaccharides, which are believed to form a structural scaffold. This scaffold should have a significant impact on the accessibility of PrP to proteo- lytic digestion and stability in a cell [5]; it might also impact strain diversity. In addition to polysaccharides, small amounts of sphingolipids and cholesterol were found in purified preparations of prion rods, which is not surprising considering that lipids represent the cel- lular environment for prion conversion [7]. After nearly two decades of searching, the attempts to identify nucleic acids as constituent components that are either essential for prion infectivity or specify strain-dependent properties, have not been successful [8,9]. While not essential for prion infectivity, nucleic acids might play some role in a complex process of pri- on conversion. Recent studies showed that antibody to DNA immunocaptured PrP Sc in brain homogenates, suggesting that PrP Sc might have high affinity for bind- ing to nucleic acids [10]. Another study that exploited an in vitro conversion assay suggested that RNA mole- cules might be involved in facilitating prion replication [11]. However, it is more likely that the stimulating effects of RNA are related to its polyanionic nature, rather than being dependent on specific sequences or types of RNAs [12]. Sulfated polysaccharides are another class of polyanions that have been found to stimulate prion conversion in a cell-free system [13]. In vitro, polyanions may provide a frame for aligning PrP C and PrP Sc molecules in a mutually favorable ori- entation via interacting with the positively charged N-terminal region. It is well known that the N-ter- minal region is not directly involved in the formation of the proteinase K (PK)-resistant b-sheet rich core in PrP Sc , but remains solvent-exposed in both PrP Sc and PrP C . There are six positively charged and no negat- ively charged residues within a short region spanning residues 23–48, which will probably bind to polyanions [14]. The functional roles of the DNA–PrP Sc complex, the a-linked polyglucose–PrP Sc complex, or other com- plexes that PrP Sc may form, need to be clarified in future studies, as it remains uncertain whether these complexes are formed in vivo or are only present in homogenized tissues. Structural models of PrP Sc In contrast to PrP C , little is known about the structure of PrP Sc . Several structural models of PrP Sc have been proposed recently [15–19]. Based on electron microsco- py (EM) imaging of 2D crystals produced from highly infectious brain fractions, Govaerts and co-authors proposed a model in which residues 89–175, including helix A, adopt a b-helical conformation in PrP Sc form [15,16]. The rest of the C terminus maintained a-helical structures, which formed a fibrillar interface in mature PrP Sc . Molecular dynamics simulations by DeMarco & Daggett [17] were in overall agreement with this model, which, however, was inconsistent with several experimental observations. First, incubation of PrP Sc with 2–3 m GndHCl was shown to destabilize the cen- tral region (residues  90–140), which acquired a PK-sensitive conformation, whereas the C-terminal region (residues  143–230) remained PK resistant [20]. Second, upon conversion from PrP C to PrP Sc , the epi- topes between residues  90–110 were found to become partially secluded [21]. However, this region still remained exposed to the solvent to such an extent that antibodies specific to this region immunoprecipitated native PrP Sc [22]. Taken together, these data argue that the central region (residues  90–140) may not be a constitutive part of the b -sheet-rich core in PrP Sc , but rather accounts for fibrillar interfaces. Contrary to this model of Govaerts et al., a simula- tion by Dima & Thirumalai suggests that residues 172–224, which correspond to helices B and C in the native PrP C form, have a high propensity to adopt a b-sheet-rich conformation in PrP* form, a possible intermediate produced on the pathway towards forma- tion of PrP Sc [18]. The result of these simulations were indirectly supported by crystallographic studies of the a-helical PrP monomer, which indicated that region 188–204 is able to undergo a partial a-helix- to b-sheet-switch within the monomeric state, presumably giving rise to early intermediate species [23]. These simulations were also consistent with the remarkably high intrinsic helix propensity of residues within the I. V. Baskakov Reconstitution of prion infectivity de novo FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS 577 helix A (residues 144–154), which are highly stable against environmental changes and therefore less likely to initiate transition into PrP Sc [24]. Future studies on the PrP Sc ultrastructure should resolve the controversy regarding PrP regions that acquire the b-structure in mature PrP Sc form. Two strategies for de novo generation of prion infectivity in vitro In an attempt to generate the infectious form of PrP, two different strategies have been pursued, namely (a) amplification of PrP Sc in PrP Sc -dependent conversion reactions, and (b) conversion of PrP C or recombinant PrP (rPrP) in the absence of a PrP Sc template. There are many technical challenges related to the generation of prion infectivity in vitro. Despite sub- stantial conformational differences between PrP C and PrP Sc (PrP C is a PK-sensitive a-helical monomer, whereas PrP Sc is an assembled multimer characterized by enhanced resistance towards PK digestion and an increased amount of b-structure [25,26]), it is still unclear which physical property can be used as a valid probe for monitoring the formation of PrP Sc de novo in a cell-free system. While the PK-digestion assay remains the common method for detecting PrP Sc , numerous studies have demonstrated that PK resist- ance, in fact, does not correlate well with the prion infectious titer [27–30]. It is well known that acquiring a b-sheet-rich conformation by PrP molecules, or aggregation into insoluble fibrillar states, are not exclu- sively associated with the generation of prion infectiv- ity. The fact that PrP Sc is intrinsically polymorphic and heterogeneous with respect to conformation, PK resistance and aggregation state creates additional con- fusion and challenges for generating prion infectivity de novo. It will be beneficial if future studies can deter- mine whether any specific physical property, such as a unique pattern of surface-exposed epitopes, distingui- shes the infectious subpopulation of PrP from the broad range of abnormally folded noninfectious PrP species [31]. PrP Sc -specific antibodies might be useful in this respect. In the past, three different strategies have been exploited successfully by different laborator- ies to produce antibodies that specifically recognize PrP Sc , but not PrP C [32–34]. Amplification of PrP Sc Several in vitro amplification protocols have been developed, in which PrP Sc was used for seeding the conversion of PrP C into nascent PrP Sc [11,35,36]. As differences in PK resistance have been used historically to distinguish PrP C from PrP Sc isoforms, all previously established amplification protocols exploited an increase in the PK resistance of PrP to track the con- version reactions. In 1995, Caughey and coworkers demonstrated that PrP C can be converted into the PK-resistant form, referred to as PrP res , in the presence of PrP Sc in a cell- free system [37,38]. Conversion of PrP C into PrP res was found to display two peculiar features of prion replica- tion: species barrier and strain-specificity [35,39,40]. However, in these studies, only small amounts ( 20%) of PrP C supplied in the reaction mixtures were converted into the PrP res form, despite a 50-fold molar excess of PrP Sc used as a seed. A detailed review of these studies has been published [41]. In the studies that followed, Soto and coworkers developed a cyclic amplification system, where  30-fold amplification of PrP res was obtained upon repetitive cycles of sonica- tion of brain homogenate containing PrP Sc and PrP C at molar ratios less than 1 : 100 [36]. In the most recent studies, unlimited amplification of PrP res was achieved upon subsequent serial dilution-amplification cycles of the miniscule amounts of initial PrP Sc seeds [4,42]. These elegant approaches demonstrated that PrP res molecules generated in vitro were able to cata- lyze the formation of new PrP res molecules, confirming the autocatalytic properties of de novo-generated PrP res . Amplification of PrP res was accompanied by amplification of prion infectivity. Without sonication, substantially lower levels of PrP res amplification were achieved, suggesting that sonication generates active replication centers [11,43]. The mechanisms for frag- mentation of PrP Sc aggregates and generation of active replication centers in vivo are currently unknown. Amplification of PrP res was also found to be less effi- cient in mixtures of purified PrP C and PrP Sc than in crude brain homogenates, suggesting that additional cellular cofactors may be necessary for prion conver- sion in vivo [12]. Future experiments on the amplifica- tion of PrP res in mixtures of purified components may eventually reveal all the molecular factors required for the efficient replication of the prion infectious agent. The invention of a cyclic amplification system opened new opportunities for the detection of ultra- low levels of prion infectivity in blood and for rapid testing of the prion transmission barrier between dif- ferent mammalian species [44]. Utilizing rPrP, instead of PrP C , as a substrate for cyclic amplification would simplify this assay and expand the range of potential applications. However, to date, attempts to use rPrP have not been successful [45,46] (C Soto, University of Texas Medical Branch at Galveston, TX; personal communication). The inability of rPrP to replace PrP C Reconstitution of prion infectivity de novo I. V. Baskakov 578 FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS in the cyclic amplification reaction is quite puzzling as it appears that this deficiency is unlikely to be caused by the lack of glycosylation or a GPI anchor [47–51]. In its a-helical conformation, rPrP resembles PrP C ,as both proteins were shown to have very similar, if not identical, secondary and tertiary structures [52]. The PrP conformation does not change if the protein is placed in the vicinity of the membrane. Recombinant a-PrP that was attached to the lipid membrane via a GPI anchor mimetic was shown to have similar struc- ture to that of PrP C [53]. Subtle differences in configuration of the proline peptide backbone between rPrP and PrP C might account for their markedly different efficiency in amplifying PrP Sc in vitro. Because rPrP is expressed in E. coli and purified from inclusion bodies, this mole- cule could differ from PrP C in the amount of cis- ver- sus trans-isomers of proline. In proteins synthesized in mammalian cells, proline is found predominantly in trans-conformation, and the balance between cis- and trans-isomers is strictly controlled by prolyl isomerases [54]. It is unclear whether recombinant proteins, expressed as inclusion bodies in E. coli, display the same proportion of trans- versus cis-isomers of proline. Backbone isomerization of a conserved proline was recently found to induce a conformational change that initiated amyloid fibril formation of b2-microglobulin [55]. There are several conservative prolines in PrP, including those located at positions 102 or 105, replacement of which with a leucine was found to cause inherited forms of prion disease [56]. It remains to be determined why rPrP is deficient, and whether rPrP can eventually be used in cyclic amplification. In vitro conversion of recombinant PrP or PrP C in the absence of PrP Sc An alternative strategy for the de novo generation of PrP Sc in vitro involves the conversion of PrP C or rPrP in the absence of PrP Sc . This approach has more paral- lels with the sporadic formation of prions, rather than with prion diseases acquired through transmission. In contrast to the amplification approach, where the physical properties of PrP res are expected to mimic that of PrP Sc seeds, conversions in the absence of PrP Sc pose uncertainty regarding the conformation of the final products of the reaction. To monitor conversion, most researchers have followed an increase in the PK resistance or b-sheet-rich structures. It is not sur- prising, that in the absence of a PrP Sc template, the conversion of rPrP or PrP C produced a large diversity of abnormal b-sheet-rich isoforms, which acquired some, but not all, characteristics of the PrP Sc [57–65]. Together, these studies illustrate the intrinsic propen- sity of the PrP molecule to adopt a diverse range of conformations depending on solvent conditions and cofactors supplemented in the conversion reactions. Effect of cofactors on PrP conversion The conversion of PrP C to PrP Sc in vivo is believed to occur through at least partial unfolding of PrP C . Therefore, the cofactors that either destabilize the native state or stabilize the transition state may facili- tate the conversion process in vitro. Several classes of bioorganic molecules were found to be capable of assisting in the transition of the a-helical isoform of rPrP (a-rPrP) into b-sheet-rich states. In 2001, Silva and co-authors demonstrated that double-stranded DNA facilitates the conversion of a-rPrP23-231 into a soluble b-sheet-rich isoform [64]. Interestingly, a small molecule, 4,4¢-dianilino-1,1¢-binaphthyl-5,5¢-sulfonate (bis-ANS), had a similar effect on the conversion of a-rPrP23-231 to the b-isoform [66]. In addition to DNA, rPrP was shown to interact with other polyani- ons, including sulfated glycans [14,67] and RNA [68,69]. RNA was found to stimulate the conversion of rPrP into an aggregated, partially PK resistant, form [69]. Using hamster rPrP90-231, Pinheiro and co- authors showed that PrP has a high affinity for the negatively charged lipid membranes of palmitoyloleoyl- phosphatidylglycerol [1-palmitoyl-2-oleoyl-sn-glycero-3- phospho-rac-(1-glycerol), and that the binding of rPrP to palmitoyloleoylphosphatidylglycero membranes pro- motes the conversion into b-sheet structures [70,71]. Unexpectedly, salt was found to decrease the thermo- dynamic stability of a-rPrP23-230 [72]. This destabil- izing effect may explain, in part, the observations that transition into b-sheet-rich forms was stimulated in the presence of salt [58,73], as well as by polyanions [74]. The effect of Cu 2+ and other bivalent metal ions on the conformation of PrP is not discussed here, as it has been summarized in a review article by Millhauser [75] and in our recent study [76]. Generation of amyloid fibrils By analogy to the studies where amyloid structure was shown to be equivalent to a prion state for several yeast prion proteins [77–79], converting rPrP into amy- loid fibrils seemed to be one possible mechanism for the de novo generation of mammalian prion infectivity in vitro. Over the last few years, several protocols for converting rPrP or PrP C into a fibrillar shape have been developed by different groups. In contrast to yeast prion proteins, in which the amyloidogenic I. V. Baskakov Reconstitution of prion infectivity de novo FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS 579 regions are natively unfolded, the region associated with mammalian prion infectivity is structured and thermodynamically stable. Because chemical denatu- rants and elevated temperatures represent the most common ways of manipulating the dynamic balance between different unfolding intermediate states, the first experimental protocol for generating amyloid fibrils from the structured C-terminal domain of a-rPrP (rPrP90-231) utilized partially denaturing con- ditions using either chemical denaturants [57,58,65,80] or combinations of elevated temperature and high pressure [63]. Under partially denaturing conditions, a- myloid fibrils were produced from both oxidized [80] and reduced [57,60,81] forms of rPrP90-231. Further- more, Riesner and coworkers established an alternative refolding procedure, where amyloid fibrils were pro- duced by incubating rPrP90-231 or PrP C in the pres- ence of low concentrations of sodium dodecyl sulfate or in the presence of lipids [82,83]. For a mechanistic understanding of prion conversion it is important for us to dissect the intermediate steps of amyloid formation. In recent studies, Caughey and co-authors showed that maximal converting activity and infectivity belongs to a small oligomeric species, rather than large fibrils [84]. Whether these oligomers were produced via fragmentation of larger fibrils and therefore possess amyloid substructure, or appeared through an alternative amyloid-independent pathway, remains to be determined. Fibrillar fragmentation might occur as a result of the intensive sonication used in aforementioned work. To our surprise, our early studies of polymerization pathways in vitro revealed that small soluble b-sheet-rich oligomeric species are off the kinetic pathways to amyloid fibrils [80,85,86]. Synthetic mammalian prions The most stringent criteria for judging whether prion infectivity is generated in vitro de novo is a bioassay in animals. There have been no reports showing the pro- duction of infectious prions in vitro with an infectivity titer high enough to cause disease directly in wild-type animals. After many years of effort, this experiment remains ‘the most wanted’ for providing formal proof of the prion hypothesis. In 2000, Kaneko and co-authors reported that a chemically synthesized 55-residue peptide (residue nos 90–145) harboring a P101L mutation and refolded in vitro into a b-sheet-rich state, induced disease sim- ilar to Gerstmann)Straussler)Scheinker disease in transgenic mice that express PrP (P101L mice) [87]. The disease-associated PrP conformer identified in these mice, however, was conformationally indistin- guishable from those found in P101L mice that devel- oped disease spontaneously [28]. This finding raised a concern as to whether the synthetic peptide caused the disease de novo or just accelerated the spontaneous dis- ease [88]. In subsequent studies, we found that amyloid fibrils generated in vitro from wild-type mouse rPrP89– 230 induced prion disease in transgenic (Tg) mice over- expressing PrP89–231 (Tg 9949) (Fig. 1) [3,80]. This disease could be efficiently transmitted to the same line of Tg mice, to Tg mice expressing full-length PrP23– 231 at an eight-fold higher level, or to wild-type mice (Fig. 1). Unique biochemical and neuropathological features indicated that amyloid fibrils induced a novel strain of transmissible spongiform encephalopathy (TSE) in experimental animals [89]. The incubation times observed upon inoculation of fibrillar rPrP89– 230, however, were much longer than those exhibited by most known PrP Sc strains. Why, despite a high expression level of PrP89–231 in Tg 9949, does a long incubation time precede the progression of prion dis- ease? The simplest explanation of the apparently lim- ited infectivity titer is that only a tiny subfraction of the fibrils generated in vitro were infectious (Fig. 2A). While searching for an explanation for the long incubation time, it is important to acknowledge that the natural strains of PrP Sc evolved through natural selection and evolution [90]. It is quite possible that TSE strains are the result of selection and evolution of different conformational subtypes of PrP Sc of sporadic origin. Only those strains that show a very fast rate of replication and, correspondingly, shorter incubation times, have a chance to ‘survive’ in the natural envi- ronment and are also preferred by many laboratory investigators. Moreover, most strains that are currently used in laboratories were passaged numerous times and adapt well to a particular host. Therefore, the long incubation time observed in testing the in vitro-gener- ated fibrils would not be that surprising if one consid- ers that no selective pressure was applied to the synthetic prions produced in a plastic tube. For the above reasons, the first synthetic prions may simply fall into a category of strains propagating very slowly. One can argue, however, why then did the incubation time become much shorter in subsequent passages of synthetic prions? Here, we propose two hypotheses, which reconcile this paradox, and will guide the future studies on production of the next generation of syn- thetic prions. ‘Maturation’ hypothesis The idea that only a small subfraction of the amyloid fibrils is infectious (referred to as ‘small-subfraction’ Reconstitution of prion infectivity de novo I. V. Baskakov 580 FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS hypothesis) does not explain substantial differences in the incubation times observed in the first and second passages of the synthetic prions (Fig. 1). Alternatively to the ‘small-subfraction’ hypothesis, the ‘maturation’ hypothesis speculates that the amyloid fibrils generated in vitro are not yet mature prions, but rather corres- pond to a semi-infectious metabolic intermediate referred to as PrP*, which is produced on the pathway towards production of PrP Sc (Fig. 2B). Our previous studies revealed that the amyloid fibrils are more PK-sensitive than ‘classical’ PrP Sc and therefore further conversion steps may be required for acquiring a proper PK-resistant conformation [91]. Specifically, we identified three major PK-resistant fragments in the amyloid form. These fragments encompassed residues 138 ⁄ 141–230, 152 ⁄ 153–230 and 162–230 (Fig. 3), and these fragments remained assembled in fibrillar struc- tures after PK treatment and maintained high b-sheet content with high seeding activity, as determined in a cell-free conversion assay [91]. Similar PK-resistant C-terminal fragments (starting at positions Y166 and F174) were first identified in mice infected with the ME7 strain of scrapie as minor products of digestion of PrP Sc [92]. In recent studies, the PK-resistant C-terminal fragments were also found in patients with sporadic Creutzfeldt–Jakob disease (CJD) [93]. These fragments encompassed residues 152 ⁄ 154–231 and 162 ⁄ 167–231 and accounted for up to 24% of all PK-resistant PrP polypeptides, including ‘classical’ PrP Sc in brains from patients with sporadic CJD (Fig. 3). To date, the relationship between the short C-ter- minal PK-resistant fragments and ‘classical’ PrP Sc remains speculative, but the results of proteolytic digestion of PrP Sc carried out under partially denatur- ing conditions may provide an important link between the two forms. Caughey and co-authors showed that PK treatment of hamster PrP Sc , in the presence of 2.5 m guanidine hydrochloride, resulted in digestion of the N-terminal regions encompassing residues 90–115 Incubation time: 516+27 (days) Tg PrP 89-230 154+4 First passage Second passage 258+25 Tg PrP 23-230 90+1 Tg PrP 89-230 16x 16x8x 1x rPrP 89-230 Wild type Fig. 1. Transmission of synthetic prions. Amyloid fibrils were generated in vitro from rPrP89–230 and inoculated into transgenic (Tg) mice expressing PrP89–230 (Tg 9949). The animals inoculated with the fibrils devel- oped neurologic, clinical signs of prion dis- ease and died between days 380 and 620 following inoculation. The brain tissues of these mice were injected into wild-type mice and two groups of Tg mice expressing PrP89–230 and PrP23-231. All three groups of animals developed scrapie after a much shorter incubation time, as indicated. I. V. Baskakov Reconstitution of prion infectivity de novo FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS 581 and 90–143, whereas the C-terminal regions remained intact (Fig. 3) [20]. The C-terminal fragments of PrP Sc generated under partially denaturing conditions [20] were similar to the C-terminal fragments associated with sporadic CJD [93]. Therefore, the C-terminal PK- resistant fragments found in sporadic CJD and pro- duced in vitro might be proteolytic products of PrP*, a metabolic intermediate or byproduct of the formation of ‘classical’ PrP Sc . Consistent with this hypothesis is our recent observation that in vitro-generated fibrils acquired the PK-resistant core (residues 97–230), sim- ilar to that of ‘classical’ PrP Sc upon brief heating or prolonged incubation with brain homogenate, a proce- dure referred to as ‘maturation’ [94]. If the fibrils gen- erated in vitro indeed represent PrP*, the long lag- phase observed in the first passage of amyloid fibrils simply reflects the additional incubation time required for the conversion of PrP* into mature PrP Sc . BCA Fig. 2. Three hypotheses explaining long incubation time observed for the first passage of synthetic prions. (A) The ‘small subfraction’ hypo- thesis proposes that only a small subfraction of amyloid fibrils generated in vitro are infectious. (B) The ‘maturation’ hypothesis postulates that the amyloid fibrils generated in vitro correspond to an intermediate form of prion protein (PrP*), which occurs on the pathway of conver- sion from the cellular isoform of the prion protein (PrP C ) to the disease-associated isoform of the prion protein (PrP Sc ). Yet-unidentified cellu- lar cofactors may be required for maturation. (C) The ‘adaptation’ hypothesis postulates that efficient propagation of synthetic prions in the first passage is precluded owing to an apparent transmission barrier. Transmission barriers arise because the subset of abnormal conformers that can be generated from recombinant prion proteins (rPrPs) in vitro does not overlap with the subset of PrP Sc strains produced in vivo. The differences in the incubation time in the first and second passages of the synthetic prions are attributed to adaptation. 89 230 138/141 152/153 162 90 231 154/156 162/167 amyloid fibrils of Mo rPrP 89-230 A B C novel Hu PrP Sc subpopulation Ha PrP Sc + GdnHCl 90 231 115 143 Fig. 3. Diagram illustrating proteinase K (PK)-resistant fragments. (A) PK-resistant core of the amyloid fibrils of mouse (Mo) rPrP89–230; sites of PK digestion were identified by epitope-mapping and LC ⁄ MS [91]. (B) PK-resistant core of the novel subpopulation of the disease-associ- ated isoform of the prion protein (PrP Sc ) found in sporadic CJD; sites of PK digestion were identified by N-terminal sequencing using Edman degradation [93]. (C) PK-resistant core of hamster (Ha) PrP Sc generated in the presence of 2.5 M guanidine hydrochloride. The approximate location of PK cleavage was found within residues 115–143 by epitope mapping, and is represented by the light gray area [20]. PK-resistant regions are represented by the dark gray area, and partial PK-resistant regions by the light gray area. Reconstitution of prion infectivity de novo I. V. Baskakov 582 FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS Are there any molecules that may assist the matur- ation and production of infectious prions? It is plaus- ible that yet-unidentified cellular cofactors are required for the maturation of PrP* and the generation of fully infectious PrP Sc . Such a cofactor might promote assembly by counteracting the electrostatic repulsion of positively charged N-terminal regions that remain exposed to the solvent in the amyloid form. Polyani- ons, such as sulfated glycans or RNA, may serve this function [95]. RNA and heparan sulfate have been shown to bind to the N-terminal region of PrP C [14,68,96]. In summary, the ‘maturation’ hypothesis assumes that the amyloid fibrils (or PrP*) and PrP Sc have a similar substructure, and that maturation may involve binding of yet-unknown cellular cofactors that can stabilize PK-sensitive regions of PrP*. ‘Adaptation’ hypothesis There is an alternative hypothesis which postulates that the shortening of the incubation times observed in the second passage of synthetic prions may be attrib- uted to adaptation to the host, suggesting that an apparent transmission barrier precludes efficient propa- gation of the synthetic prions in the first passage (Fig. 2C). A transmission barrier is typically observed when the sequence of PrP Sc in the inoculum does not match that of PrP C in the recipient animals [97–99]. Besides the differences in the sequences of PrPs between recipient and donor species, other factors, such as strain-specific conformational differences, were shown to account for the transmission barrier. Note- worthy, different TSE strains have notably different propensities to overcome the transmission barrier, pre- sumably as a result of different conformations of PrP Sc [100]. Furthermore, some, but not all, recipient species can propagate certain strains from donor species. It is clear that the species barrier and the strain phenomena are closely connected. Figure 4 illustrates a framework that provides some insight into the strain-specificity of the species barrier. This framework helps to describe, at a very simplistic level, why some strains from donor species can be transmitted to recipient species, whereas other strains cannot; and why some, but not all, recipi- ent species can propagate certain strains from donor species. Because the sequence of rPrP89–230 used to produce the amyloid fibrils was identical to that of endogenously expressed PrP C (mouse PrP89–230), it is probable that the apparent transmission barrier may arise owing to the unique conformational properties of the amyloid fibrils and, in particular, as a result of the proteolytic liability of residues 90–138 [91]. In addition, the lack of glycosylation in rPrP89–230 may affect the conforma- tional preferences of the PrP molecule for acquiring the fibrillar form of a specific conformation in vitro.Asa result, the subset of abnormal b-sheet-rich conformers that can be produced in vitro from recombinant PrPs may not overlap with those that are formed from PrP C in a brain under pathological conditions (Fig. 2C). If this is correct, an apparent transmission barrier occurs that may account for the slow propagation rate observed in the first passage. Remarkably, after primary passage was accomplished, substantially shorter incuba- tion times were observed in the second passage of the synthetic prions [3]. Several lines of experimental evidence are consistent with the adaptation hypothesis. Among them is the fact that the amyloid fibrils of rPrP89–230 produced a novel strain of TSE. This strain is characterized by distinct incubation time, a unique neuropathological lesion profile and an unusually high thermodynamic stability to chemical denaturation [89]. The C ½ value [C ½ is the GdnHCl (guanidine hydrochloride) concen- Species AB C PrPC PrPSc strains Conformational diversity m n Fig. 4. The new conceptual framework postulates that (a) the pri- mary structure of prion protein (PrP) of each individual species (A, B and C) determines, to a large extent, the conformational diversity of disease-associated isoforms of the prion protein (PrP Sc ) strains. (b) However, PrP Sc seed or template specifies a particular confor- mation within a given conformational space. (c) Subsets of con- formers (strains) formed by PrPs from different species may overlap, suggesting that some PrP Sc strains are more universal and can be shared by two or more species, whereas other strains are more species-specific. For instance, strain n is shared by species A, B, and C, while strain m is shared by species A and C. Only those strains that occupy overlapping areas can be propagated by more than one species. I. V. Baskakov Reconstitution of prion infectivity de novo FEBS Journal 274 (2007) 576–587 ª 2007 The Author Journal compilation ª 2007 FEBS 583 tration at the half-maximal denaturation] determined for the nascent PrP Sc produced in the brains of Tg9949 mice after inoculation with amyloid fibrils was found to be substantially higher than those displayed by nat- ural prion strains [89,101]. Remarkably, the C ½ value of seeds (i.e. amyloid fibrils generated in vitro) (4.2 m) [91] was identical to the C ½ value of PrP Sc formed in Tg9949 mice [89]. The ‘maturation’ and ‘adaptation’ hypotheses are different in one key aspect. The ‘maturation’ hypothe- sis postulates that amyloid fibrils or PrP* generated in vitro have a substructure very similar to that of PrP Sc , and that this substructure is largely preserved upon maturation of PrP* into PrP Sc . The ‘adaptation’ hypothesis, on the other hand, suggests that the amy- loid fibrils are primitive surrogates of PrP Sc and that the substructure of amyloid fibrils may change dramat- ically in the course of adaptation. The ‘maturation’ and ‘adaptation’ hypotheses are not mutually exclusive of each other. Future studies should determine whether either of the above hypotheses are correct. Acknowledgements IVB is supported by National Institute of Health grant NS045585. 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Yet-unidentified cellu- lar cofactors