Báo cáo khoa học: Branched chain mechanism of polymerization and ultrastructure of prion protein amyloid fibrils ppt

MINIREVIEW Branched chain mechanism of polymerization and ultrastructure of prion protein amyloid fibrils Ilia V. Baskakov 1,2 1 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD, USA 2 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 that can also be infectious [1]. Despite enormous investments over the last 30 years in searching for a prion virus or virion [2–5], no prion-specific nucleic acids associated with infectious prion particles have ever been identified [6]. A notable shift has occurred in the last few years from debating the question of whe- ther a protein can be infectious to what makes a pro- tein infectious and how many proteins are infectious [7–9]. Elucidating the polymerization mechanisms and structure of misfolded and aggregated isoforms of the prion protein (PrP) will help solving these long-stand- ing research problems. Prion polymerization is a branched- chain reaction To model prion conversion, two kinetic models has been exploited: the nucleation-polymerization [10] and the template assisted [11]. These models have been previously discussed in numerous review articles [12–14] and therefore will not be presented here. Although these two models have played an important role in the evolution of our ideas regarding the mechanism of prion conversion, neither of them emphasize the importance of multiplication of the active centers of prion conversion, a key step in prion replication. When studying the kinetics of the in vitro fibril formation, we were surprised to discover that fibrillization of recombinant PrP (rPrP) displays several kinetic features that can not be explained by the nucleation-polymerization or the template assisted models [15,16]. These ‘atypical’ features include: (a) the dramatic effect of reaction volume on the length of the lag-phase; (b) a volume-dependent threshold effect; and (c) the highly cooperative sigmoidal kine- tics of polymerization [15,16]. Although these features could not be rationalized within nucleation-polymer- ization or the template assisted models, they are Keywords amyloid fibrils; branched-chain mechanism; in vitro conversion; polymerization kinetics; prion diseases; prion protein Correspondence I. V. Baskakov, 725 West Lombard Street, Baltimore, MD 21201, USA Fax: +1 410 706 8184 Tel: +1 410 706 4562 E-mail: baskakov@umbi.umd.edu (Received 9 March 2007, accepted 31 May 2007) doi:10.1111/j.1742-4658.2007.05916.x The discovery of prion disease and the establishment of the protein only hypothesis of prion propagation raised substantial interest in the class of maladies referred to as conformational diseases. Although significant pro- gress has been made in elucidating the mechanisms of polymerization for several amyloidogenic proteins and peptides linked to conformational dis- orders and solving their fibrillar 3D structures, studies of prion protein amyloid fibrils and their polymerization mechanism have proven to be very difficult. The present minireview introduces the mechanism of branched- chain reaction for describing the peculiar kinetics of prion polymerization and summarizes our current knowledge about the substructure of prion protein amyloid fibrils. Abbreviations AFM, atomic force microscopy; GdnHCl, guanidine hydrochloride; PK, proteinase K; PrP, prion protein; rPrP, recombinant prion protein. 3756 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS consistent with the mechanism of branched-chain reactions. Employing the theory of branched-chain reactions will greatly benefit our understanding of the prion rep- lication mechanism. The first branched-chain processes were described at the beginning of twentieth century and the branched-chain theory was developed shortly afterward in the 1920s by Nikolay Semenov [17]. Although this theory had enormous impact on the developing chemical industry and nuclear sources of energy, the Nobel Prize for this amazing discovery was not awarded until 1956, almost half a century later [18]. A number of odd features including a strong dependence of the reaction rate on the volume or the shape of reaction vessel, the presence of a lag-phase, threshold effects and a strong dependence of the reac- tion rate on microimpurities observed for this type of reactions raised serious cautions and even jokes among conventional chemists. It took more than 30 years for the chemical community to be convinced that this theory was not heretical. Certainly, the history of developing the branched-chain mechanism and the ‘protein-only’ hypothesis of prion replication share many things in common. What is more surprising, the theory of branched- chain reactions explains equally well such diverse pro- cesses as an atomic explosion or prion replication. Among key characteristics of the branched-chain mechanism is the multiplication of active or catalytic centers in the time course of the reactions, a feature that makes these processes similar to the autocatalytic reactions (Fig. 1). In a simplified expression, the reac- tion rate is determined by the multiplication coefficient (r), which is proportional to the probability of gener- ating new active ⁄ catalytic centers divided by the prob- ability of their loss or quenching. Depending upon the rate of multiplication versus quenching, the reactions may switch between auto-acceleration and decay modes. When multiplication exceeds quenching (r > 1), the reaction proceeds with self-acceleration. If the rate of quenching is higher than the rate of multiplication (r < 1), the reaction decays. When r is equal to 1.00, the number of active centers remains constant during the reaction time; therefore, the kinetics of such reac- tions follow the formal mechanism of enzyme catalysis (Fig. 1). However, apparently negligible changes in experimental parameters, such as the presence of microimpurities or a change in the shape of the reac- tion vessel, may alter the r-value and switch the reac- tion to decay mode or to auto-acceleration mode. The branched-chain reactions have been known to be unusually ‘sensitive’ to slight changes in experimental parameters that might be seen as stochastic behavior, in which the reaction follows the ‘all or nothing’ rule. It is important to indicate that the branched-chain mechanism is consistent with the sigmoidal kinetics of fibrillation, which has been previously referred to as ‘nucleation-elongation’ kinetics (Fig. 2). According to the nucleation-polymerization model, the lag-phase in the fibrillation process corresponds to the nucleation step, a stage when mature fibrils are not yet formed (Fig. 2A). By contrast to this prediction, we found that mature fibril were present at the lag-phase of rPrP fibrillation [16,19]. This observation is consistent with the branched-chain mechanism that attributes the lack of an observable signal during the second part of the lag-phase to the limitations in detecting small amounts of the final reaction products (i.e. in this case, fibrils) (Fig. 2B). As soon as the final reaction products are formed even in miniscule amounts, the reaction rate is accelerated due to the branched-chain mechanism of multiplication of active centers. Therefore, in a Branched chain reactions are similar to autocatalytic processes (multiplication coefficient) probability of formation of new active centers probability of loss of active centers r ~ reaction time r = 1 r > 1 r >> 1 fibril elongation The kinetics is similar to catalytic processes Fig. 1. Schematic representation of the branched-chain mechanism. If no fibril frag- mentation occurs, the fibril elongation reac- tion follows the formal kinetics of enzyme catalysis. Branched chain reactions are accompanied by multiplication of active centers (r >> 1). In prion polymerization, multiplication of active centers occurs, pre- sumably, as a result of fibril fragmentation. Quenching or clearance of active centers could partially counteract the process of their multiplication (r > 1). I. V. Baskakov Branched chain mechanism of polymerization FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS 3757 branched-chain mechanism, the length of the lag-phase is regulated by the rate of multiplication of active cen- ters. The higher the rate of multiplication, the shorter is the lag-phase (Fig. 2C). The branched-chain mech- anism predicts that the rate of fibril fragmentation controls the length of lag-phase and the cooperativity of sigmoidal kinetics (Fig. 2C). In our yet unpublished studies, we, indeed, observed substantial differences in the length of the lag-phase and polymerization rate of PrP fibrillation reactions that were carried out under identical solvent conditions, but subjected to different fragmentation intensities (O. V. Bocharova & I. V. Baskakov, unpublished results). The mechanism of the branched-chain reaction pre- dicts three potential outcomes for prion disease. Depending on the dynamic balance between the rate of multiplication versus clearance, prion disease could: (a) progress very quickly to the clinical form (if >>1, the kinetics of PrP Sc (Sc-scrapie) accumulation follow the formal mechanism of branched-chain reactions); (b) develop very slowly and exist at subclinical level for a long period of time (r ¼ 1, the kinetics of PrP Sc formation follow the formal mechanism of enzyme cata- lysis), or (c) never progress (r < 1, PrP Sc is cleared, the rate of clearance follow apparent first order kinetics). It has been shown that the concentration of PrP Sc in the brain of experimental animals drops substantially in the first week after intracerebral inoculation [20,21], indica- ting that the rate of clearance may exceed the rate of multiplication during the initial stage of prion transmis- sion. Despite substantial resistance to proteolytic diges- tion, the life-time of PrP Sc was found to be relatively short (only 28 h) [22,23]. Therefore, for the disease to progress to the clinical stage, the rate of PrP Sc multipli- cation should eventually exceed the rate of clearance. If the process of multiplication of the active PrP Sc form is slower than the degradation, PrP Sc will be cleared throughout an animal’s lifetime. The critical role of the multiplication of active cen- ters is reflected by the history of the development of an experimental procedure for cell-free prion repli- cation. Successful amplification of prion infectivity in vitro was not achieved until the repetitive steps of fibril fragmentation were introduced as a part of the experimental protocol. In 1995, Caughey and coworkers demonstrated that PrP C (C-cellular) can be converted into the proteinase K (PK)-resistant form, referred to as PrP-res, in the presence of PrP Sc in a cell-free sys- tem [24,25]. In these studies, however, only small amounts (approximately 20%) of PrP C supplied to the reaction mixtures were converted into the PrP-res form despite a 50-fold molar excess of PrP Sc used as a seed. In subsequent studies, unlimited amplification of PrP Sc was achieved in the conversion reactions referred to as misfolding cyclic amplification by introducing repetitive cycles of elongation and fragmentation, ThT lluorescence The branched chain mechanism nucle -ation elongation and fragmentation Time Time A B C ThT lluorescence nucleation elongation The nucleation-polymerization model Time r >>>1 r >>1 r >1 r = 1 ThT fluorescence Fig. 2. Sigmoidal kinetics of rPrP polymerization. (A) The nuclea- tion-polymerization model postulates that fibrillation consists of two consecutive stages: nucleation that accounts for a lag-phase and elongation. (B) The branched-chain mechanism predicts that the for- mation of mature fibrils has already taken place during so-called ‘lag-phase’. However, only a small fraction of the rPrP monomer converts into fibrils. Two parallel processes of fibril elongation and fragmentation occur during the second part of a lag-phase and a subsequent stage that has been referred to as ‘elongation’. Arrows indicate the time point where the mature fibrils could be detected according to the branched-chain mechanisms. (C) The branched- chain mechanism predicts that the length of the lag-phase and the polymerization rate are controlled by the r-value. Schematic repre- sentation of four polymerization reactions that are carried out under identical solvent conditions, but showed different lag-phase and polymerization rates as a result of differences in fragmentation con- ditions (I. V. Baskakov, unpublished data). Branched chain mechanism of polymerization I. V. Baskakov 3758 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS where fragmentation was induced by short pulses of sonication [26–28]. Without sonication, substantially lower levels of PrP Sc amplification were reported, illus- trating that sonication is critical for multiplication of active replication centers [29,30]. What factors regulate the clearance and multiplica- tion of active PrP Sc centers? Multiple effects may contribute to the clearance of PrP Sc : strain-specific intrinsic stability of PrP Sc [31,32]; species and tissue- specific variations in proteolytic activity [33,34]; interactions of PrP Sc with cellular cofactors such as glycosaminoglycans [35–37] or polysaccharides [38] that stabilize PrP Sc . Removal of active PrP Sc centers could also occur due to aggregation of PrP Sc into large plaques or oxidative modification of amino acid resi- dues on the PrP Sc surface that are involved in prion replication. Our previous studies revealed that sorption of self-propagating amyloid fibrils to walls of reaction vessels may account for deactivation of active seeds in vitro, resulting in dramatic volume-dependent threshold effects [15,16]. For the majority of branched- chain reactions, the multiplication coefficient r depends on the ratio of surface to volume of the reaction vessel [18]. Vessel surfaces may either catalyze or deactivate active centers, thus having a significant impact on the lag-phase and final yield of the reactions. The volume- dependent threshold is consistent with the scenario that self-propagating forms of rPrP are adsorbed and deactivated by the vessel surface. As the reaction volume decreases, the surface-to-volume ratio grows. Therefore, the threshold may be reached when the rate of surface-dependent deactivation exceeds the rate of multiplication of self-propagating forms. Indeed, we found that amyloid fibrils have high propensity to adsorb to walls of the reaction tubes made from differ- ent materials [16]. Binding of fibrillar rPrP to surfaces is reminiscent of that of PrP Sc . It is known that prion diseases can be efficiently transmitted through wires and surgical instruments contaminated with PrP Sc [39–42]. Although sorption of the active amyloid seeds seems to be a peculiar property of in vitro fibrillization, it may, in fact, mimic the clearance of the PrP Sc in vivo, and therefore provide mechanistic insight into prion replication mechanisms. With regards to the multiplication of active centers, both external cofactors and the intrinsic fragility of PrP Sc fibrils should control the rate of multiplication. It is important to note that the fibril elongation does not result in multiplication of the active or catalytic centers, unless fibril fragmentation occurs (Fig. 1). Cel- lular chaperones were found to be involved in frag- mentation of yeast prion fibrils [43]. Cellular cofactors participating in fragmentation of mammalian prion fibrils have yet to be identified. The intrinsic fragility (i.e. the ability of fibrils to fragment into shorter pieces) seems to be controlled by the conformational stability of amyloid fibrils and, specifically, by the stability of the cross-b-fibrillar structure [8] (Y. Sun & I. V. Baskakov, unpublished data). Recent studies have revealed a strong link between conformational stability and the intrinsic infectivity of fibrils formed by the yeast prion protein Sup35 [44]. The amyloid fibrils that displayed low conformational stability exhibited a high efficiency of infection with the large majority of colon- ies showing a strong phenotype. Vice versa, fibrils that had high conformational stability displayed low infec- tivity and produced ‘weak’ strains that disappeared fast or that could be easily cured. A similar correlation between conformational stability and infectivity was observed for synthetic mammalian prions [45,46]. Both yeast and mammalian prion studies indicated that the intrinsic infectivity of fibrils might be controlled, at least in part, by the conformational stability of the cross-b-sheet core, an unexpected lesson that we have learned [8]. If the intrinsic fragility of PrP Sc aggregates does dictate the rate of prion propagation, this prop- erty could account for substantial differences in the incubation times produces by different strains of PrP Sc . Future studies will determine whether conformational stability proves to be the missing link in our search for the physical determinants of prion fibril infectivity. Elucidating the relationship between conformational stability and infectivity may help us to answer the intriguing questions as to why are some but not all amyloidogenic proteins capable of forming infectious fibrils, and why are some but not all types of amyloid fibrils made of the same protein infectious. Ulstrastructure of PrP amyloid fibrils In recent years, there has been considerable debate as to whether small nonfibrilar oligomeric particles are more pathogenic or infectious than amyloid fibrils [47,48]. A discussion regarding a plausible role for fibrillar or nonfibrillar PrP aggregates in the pathologi- cal process is meaningless unless the physical proper- ties of b-structures and their origin are specified. The key criterion in our classification of variable b-sheet rich forms should be their substructure, and not size. Our judgment as to whether PrP aggregates are fibril- lar or nonfibrillar is often made solely base on tech- niques with poor spatial resolution such as light microscopy. Light microscopy has been utilized histor- ically for neuropathological studies and used often for classification of prion aggregates. Using light micro- scopy only, it is easy to confuse nonfibrillar oligomers I. V. Baskakov Branched chain mechanism of polymerization FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS 3759 with small fibrillar fragments (Fig. 3). In fact, the size distribution of fibrils is very broad and, at any given time, includes very small or short fibrillar fragments. Short fibrils or their fragments can be generated at the initial stages of fibril elongation, but also produced as a result of fibril fragmentation. In addition to small C A B Fig. 3. Fluorescence and electron microscopy of rPrP amyloid fibrils. Amyloid fibrils were produced as described by Bocharova et al. [55] and (A) stored in Na-acetate buffer, pH 5.5; (B) stored in Na-acetate buffer, pH 5.5, and sonicated for 1 min prior to imaging; and (C) stored in Tris ⁄ HCl buffer, pH 7.4. All three samples were analyzed in parallel by thioflavine T-fluorescence microscopy (left panels) and by electron microscopy (right panels). When observed by fluorescence microscopy, the fibrils subjected to 1 min of sonication (B) appeared as small nonfibrillar oligomers. (A,B) Scale bars ¼ 1 lm; (C) scale bar ¼ 10 lm. Branched chain mechanism of polymerization I. V. Baskakov 3760 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS fragments, fibrils might form aggregates of various shapes and densities (Fig. 3). Although fibrillar aggre- gates or plaques are believed to be less pathogenic, they might serve as repositories of more pathogenic small fibrillar fragments and therefore are equally important. Regardless of the fibril size and shape, the key feature of fibrils is cross- b-sheet structure, which is essential for the prion self-propagating activity. More- over, the cross- b-sheet structure of amyloid fibrils is substantially more stable kinetically and thermody- namically than the structure of nonfibrillar oligomeric species, ensuring that fibrils remain assembled and pre- serving their physical properties even at low biologi- cally relevant concentrations of PrP. Because the infectious form of PrP has been often referred to as nonfibrillar in nature, it is important to evaluate the validity of such claims. First, if infectious prions are indeed nonfibrillar, the question of how could oligomeric nonfibrillar species be infectious in the absence of the self-propagating cross-b structure needs to be answered. Second, the vast majority of experimental procedures used for extraction and purifi- cation of PrP Sc involved sonication, treatment with detergents and, sometimes, freezing and thawing [49–51]. All of these steps cause severe fragmentation of fibrils. In our experience, sonication for only 1 min is sufficient to fragment fibrils into small fibrillar frag- ments that could easily be confused with nonfibrillar particles (Fig. 3B). In our search for physical properties that are essen- tial for prion infectivity it is important to gain infor- mation about the substructure of PrP fibrils. What regions of PrP molecule adopt cross-b-sheet conforma- tion within amyloid fibrils? Can we control the con- formational stability of cross-b-sheet core? The large size of PrP molecules in combination with the highly aggregated, heterogeneous and insoluble nature of PrP fibrils precluded application of NMR and other high-resolution techniques. In the absence of methods to solve structure of PrP fibrils in the near future, we employed several alternative approaches for elucidating ultrastructure of fibrils. High resolution atomic force microscopy revealed that fibrils produced in vitro from the full-length rPrP consisted of several laterally assembled filaments [52]. In our recent studies, we found that the fibrils produced under single growth conditions varied with respect to the number of consti- tutive filaments and the manner in which the filaments were assembled. The high-order fibrils formed through a highly hierarchical mechanism of lateral assembly. At each step, filaments were found to associate in pairs in a pattern resembling dichotomous coalescence (Fig. 4) [19,52]. Because of alternative modes of lateral assembly, fibrils produced under a single growth condi- tion were heterogeneous with respect to the width, height and twisting morphology. How many PrP molecules are packed per 1 nm within an amyloid fibril? As judged from atomic force microscopy (AFM) measurements and atomic volume calculations, a single full-length rPrP polypeptide occu- pied a distance of approximately 1.2 nm within a single filament (Fig. 5A) [52]. The amyloid fibrils are Dichotomous mechanism of lateral assembly Width (nm) 20 40 60 80 Height, nm 0 5 10 15 AB Fig. 4. Hierarchical mechanism of lateral assembly. (A) Electron microscopy image of an amyloid fibril taken at the intermediate stage of lateral assembly. Several ‘coales- cent forks’ (marked by arrows) could be observed within an individual fibril. Sche- matic representation of the mechanism of dichotomous assembly is shown in inset. Based on data from [19]. (B) Height–width profiles of fibrils grown under single growth conditions illustrate polymorphism in fibril dimension that occurred as a result of the hierarchical mechanism of lateral assembly. Based on data from [52]. I. V. Baskakov Branched chain mechanism of polymerization FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS 3761 known to be build of b-strands oriented perpendicular to the fibrillar axis with the distance between two neighboring b-strands of approximately 4.8 A ˚ . There- fore, the axial distance occupied by one rPrP molecule should be equivalent to approximately 2.5 layers of b-strands. Our studies using PK digestion assay revealed that the PK resistant core of the amyloid fibrils consisted of residues 138 ⁄ 141–230, 152 ⁄ 153–230 and 162–230, where the fragment 162–230 was the most resistant to PK digestion (Fig. 5) [53,54]. Upon treatment with PK, the 152 ⁄ 153–230 and 162–230 PK-resistant fragments maintained fibrillar structure and preserved a high b-sheet context with strong inter- molecular hydrogen bonds. Remarkably, the b-sheet rich fibrillar cores encompassed by residues 152 ⁄ 153– 230 and 162–230 were found to maintain high seeding activity in vitro despite cleavage of the N-terminal and central regions [53,55]. Consistent with these studies, the rPrP regions 159–174 and 224–230 were observed to be buried in the fibril interior and were the most resistant to GdnHCl-induced denaturation as judged from the newly developed dual color immunofluores- Fig. 5. (A) Three-dimensional AFM image of amyloid fibril. The fibril consists of several filaments assembled laterally in horizontal and vertical dimensions as seen by a stepwise increase in fibrillar height. Although atomic volume calculations predicted that single PrP molecule occu- pies the distance of approximately 1.2 nm (52), the precise 3D structure of PrP within amyloid fibrils has yet to be determined. Despite changes in the shape of the PrP molecule upon conversion from the native a-helical form (inset) into the fibrillar form, the atomic volume occupied by a single PrP polypeptide chain does not change substantially. (B) Schematic diagram illustrating mapping of PrP regions within amyloid fibrils. The PK-resistant b-sheet rich core of amyloid fibrils composed of residues 152–230 and 162–230; PK-cleavage sites are indicated by red arrows. Based on data from [55]. The epitopes to antibodies AH6 and R1 were solvent unaccessible and were the most resistant to GdnHCl-induced denaturation (highlighted in red); the epitope to antibody D18 was found to be cryptic under native conditions and solvent exposed under partially denaturing conditions (highlighted in orange), whereas the epitopes to antibodies D13 and AG4 were solvent-accessible regardless of the solvent conditions (highlighted in green); based on data from [56]. Residues 98, 127, 144, 196 and 230 (blue) showed cooperative unfolding, whereas unfolding at residue 88 (green) was noncooperative; based on data from [58]. Branched chain mechanism of polymerization I. V. Baskakov 3762 FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS cence microscopy assay (Fig. 5) [56]. The 132–156 segment was cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas region 95–105 was solvent-accessible regard- less of the solvent conditions [56]. In fibrils formed from truncated rPrP 90–230, the residues 169–230 showed the slowest hydrogen exchange rate confirming that the C-terminal part is involved in the b-sheet structure [57]. Site-specific conformational studies revealed that the C-terminal region accounts for the high conformational stability of amyloid fibrils [58]. As judged from the C 1 ⁄ 2 values, the conformational stabil- ity of the residues within the region 127–230 were found to be similar to the global stability of the amy- loid structure, whereas the stability of residue 98 was substantially lower than the global stability, but approached that of natively folded proteins [58]. Taken together, the data accumulated to date have indicated that the C-terminal part of the rPrP molecule encompassing residues 152–230 and 162–230, and poss- ibly 169–230, acquires cross-b-sheet self-propagating core in amyloid fibrils [53,54,56–58]. These regions account for the high conformational stability and structural integrity of fibrils. The central regions encompassing residues 90–150 are likely to be involved in forming the fibrillar interface that participates in lateral interactions between filaments within mature fibrils. 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Baskakov Branched chain mechanism of polymerization FEBS Journal 274 (2007) 3756–3765 ª 2007 The Author Journal compilation ª 2007 FEBS 3765 . MINIREVIEW Branched chain mechanism of polymerization and ultrastructure of prion protein amyloid fibrils Ilia V. Baskakov 1,2 1 Medical. polymerization mechanisms and structure of misfolded and aggregated isoforms of the prion protein (PrP) will help solving these long-stand- ing research problems. Prion