mechanism of amyloidogenesis: nucleation-dependent

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541http://bmbreports.org BMB reports

*Corresponding author. Tel: 82-2-880-7402; Fax: 82-2-888-1604;E-mail: [email protected]

Received 31 August 2009

Keywords: Amyloidogenesis, Fibrillar polymorphism, Nucleation- dependent fibrillation, Protein self-assembly, Template-dependentand template-independent fibrillations

Mechanism of amyloidogenesis: nucleation-dependent fibrillation versus double-concerted fibrillationGhibom Bhak, Young-Jun Choe & Seung R. Paik*

School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-744, Korea

Amyloidogenesis defines a condition in which a soluble and innocuous protein turns to insoluble protein aggregates known as amyloid fibrils. This protein suprastructure derived via chemically specific molecular self-assembly process has been commonly observed in various neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Prion diseases. Although the major culprit for the cellular degeneration in the diseases remains unsettled, amyloidogenesis is considered to be etio-logically involved. Recent recognition of fibrillar polymorphism observed mostly from in vitro amyloidogeneses may indicate that multiple mechanisms for the amyloid fibril formation would be operated. Nucleation-dependent fibrillation is the prevalent model for assessing the self-assembly process. Following thermo-dynamically unfavorable seed formation, monomeric poly-peptides bind to the seeds by exerting structural adjustments to the template, which leads to accelerated amyloid fibril formation. In this review, we propose another in vitro model of amyloido-genesis named double-concerted fibrillation. Here, two con-secutive assembly processes of monomers and subsequent oligomeric species are responsible for the amyloid fibril formation of α-synuclein, a pathological component of Parkinson’s disease, following structural rearrangement within the oligomers which then act as a growing unit for the fibrillation. [BMB reports 2009; 42(9): 541-551]

Amyloidogenesis

Amyloidogenesis is a biochemical process or related conditions in which a soluble and innocuous protein converts into insoluble and fibrillar protein aggregates known as amyloid fibrils (1, 2). The term ‘amyloid’ has been spared to describe the proteina-ceous deposits found in various pathological conditions (3). In particular, amyloid fibril formation has been widely observed among various neurodegenerative disorders of which no suc-

cessful treatments are yet available, including Alzheimer’s di-sease (AD), Parkinson’s disease (PD), Huntington’s chorea, and Prion disease (2, 4-6). Amyloid fibril formation is considered to be a valid and reliable pathological signature of the neuro-degenerations although the exact mechanism behind the cel-lular degeneration remains unknown (5). Nevertheless, several mechanisms of toxicity have been proposed: (a) Amyloids in-vade the extracellular space of organs, and destroy the archi-tecture and function of normal tissue (7, 8); (b) Oligomers con-structed into final amyloid fibrils could exhibit toxic effects by destabilizing cellular membranes (9, 10). Therefore, the final amyloid fibrils have been hypothesized as a non-toxic detoxifi-cation product of the toxic intermediates; (c) The incorporation of redox metals into amyloids and the subsequent generation of reactive oxygen species could affect cell viability (11-15); (d) Alternatively, the protein aggregates could trap and thus re-move some trophic proteins essential for cell survival. Natural amyloid fibrils exert their biological activities in var-ious living systems (16). In microorganisms, amyloid fibrils are utilized to form biofilm which is made up of curli amyloid fi-bers that shield enterobacteria from various antimicrobial agents (17-19). The amyloid fibrils of class I hydrophobins pro-teins in fungi and chaplin in filamentous bacteria form the functional coat to facilitate the branching process of hyphae (the threads composing the fungus body) by decreasing the water surface tension at the substrate-air interface (20, 21). Furthermore, chorion proteins found in insects and fish self-as-semble into the amyloid fibril layer that protects their eggs from physical and chemical stresses (22, 23). In the case of mammals, Pmel17 amyloid fibrils serve as templates to poly-merize reactive melanin precursors during melanin biosyn-thesis (24), and factor XII protein is activated by various amy-loids in human hemostasis (25). Amyloid fibrils have an average width of 10-20 nm, in which constituting proteins are aligned perpendicular to the fi-brillar axis by forming cross β-sheet conformation (26, 27) that affords the protein nanofibrils a mechanical strength com-parable to spider silk (28, 29). Amyloid fibrils are the products of a highly selective molecular self-assembly process that has been particularly emphasized in the area of nanotechnology (29, 30). In fact, amyloid fibrils have been recognized as bio-logically compatible protein nanofibrils. The fibrils actually serve as functional templates for the fabrication of novel

Mini Review

Mechanism of amyloidogenesis: nucleation-dependent fibrillation versus double-concerted fibrillationGhibom Bhak, et al.

542 BMB reports http://bmbreports.org

Protein Native structure Disease References

Amyloid βα-Synucleinβ-2 microglobulinHuntingtinImmunoglobulin VL domainIslet amyloid polypeptideLysozymePrion proteinTransthyretin

U (RC)U (RC)G (BS)Variable (polyGln)G (BS)U (RC)G (AH/BS)G (AH/BS)G (BS)

Alzheimer’s diseaseParkinson’s diseaseDialysis-related amyloidosisHuntington’s diseaseLight-chain amyloidosisType II diabetesHereditary systemic amyloidosisCreutzfeldt-Jakob diseaseSenile systemic amyloidosis

(54)(51)(64)(109)(62)(53)(65)(63)(61)

Representing each abbreviation in the part of native structure: U: unfolded structure, G: globular protein, RC: random coils, AH: α-helices, BS: β-sheets.

Table 1. Amyloidogenic proteins of amyloid fibril formation and their involvement in amyloid disease

nanomaterials. Amyloid fibrils of yeast prion Sup35(NM) dis-playing surface-accessible cysteine residues were utilized to align gold nanoparticles for the formation of solid metal nano-wires with conductive properties (31). Diphenylalanine nano-tubes were also demonstrated to be a suitable scaffold for plat-inum-peptide composites (32), silver nanowires (33), and co-axial metal nanocables (34). Au and Pd nanoparticles de-posited on the fibrils of a 12 residue synthetic peptide (T1) re-sulted in the formation of double helical and single-chain ar-rays of metal nanoparticles (35). In addition, amyloid fibril structures have been utilized for protein and enzyme immo-bilization. Diphenylalanine nanotubes were chemically modi-fied with biotin for the selective decoration of avidin-labeled species (36). Amyloid fibrils adorned with bioactive proteins were prepared with yeast prion Ure2 protein (a regulator of ni-trogen catabolism) decorated with proteins such as barnase protein, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein (37). The cytochrome b units suc-cessfully attached to amyloid fibrils of the SH3 domain al-lowed for efficient incorporation of heme moieties into func-tional amyloid fibrils exhibiting long-distance electron transfer (38). Additional effects have been also demonstrated as the fi-brils were transformed into liquid crystal and hydrogel states. Nematic liquid crystal phases were obtained from the pre-formed amyloid fibrils of hen lysozyme (39). For hydrogel for-mation, Lysβ-21 peptide (residues 41-61 of hen lysozyme pro-tein) forms a gel whose viscoelastic properties can be con-trolled chemically or physically (40). Several dipeptides has been also reported to assemble into a biocompatible and rigid hydrogel (41, 42). Hydrogels composed of amyloids or amy-loid-like fibrils can be used for enzyme entrapment, controlled drug release, three-dimensional cell culture, and eventual tis-sue engineering. Moreover, since current nanotechnology is being fashioned for biological and medical applications, these protein-based biomaterials should garner a great deal of atten-tion in the area of nano-bio fusion technology. Therefore, elucidating the molecular mechanism of amyloid fibril formation could benefit not only the development of neu-roprotective strategies (5), but also the application of biological nanomaterials to various areas of promising future nano-

biotechnology (29, 30). The molecular self-assembly process that underlies amyloid fibrillation, however, is not clearly understood. To shed light, therefore, we discuss mechanisms of amyloidogenesis in terms of template-dependent and tem-plate-independent processes by focusing on the one prevailing model of nucleation-dependent fibrillation and the other bur-geoning hypothesis of double-concerted fibrillation model, which could explain recently recognized phenomenon of amyloid fibrillar polymorphism.

Various amyloidogenic proteins

Amyloid fibrils are derived from various proteins that are closely linked to the onset of diverse degenerative disorders, although their cause-or-effect relationship remains elusive (Table 1). In other words, amyloid fibrils could be either a culprit for the cel-lular degeneration or just a mere side product of the cellular dis-ruption process. Amyloidogenic proteins initially exist in either a ‘natively unfolded’ or ‘naturally folded’ state. For example, α- synuclein (43, 44), an islet amyloid polypeptide (IAPP) (45), and amyloid β-protein (Aβ) (46, 47) belong to a group of polypeptides whose structures are predominantly random. It is perhaps this unfolded state that allows the proteins to be readily self-assembled into the fibrillar suprastructures. α-Synuclein is a pathological component of Parkinson’s disease in that it contributes to the formation of intracellular Lewy body and its radiating filamentous structures (48-52). IAPP, also known as amylin, is a glycemic controlling peptide hormone secreted from pancreatic β-cells and its fibrillation is associated with diabetes mellitus type 2 (53). Aβ40/42, derived from the coincidental limited proteolyses of a transmembrane protein of Aβ precursor protein (APP) by β- and γ-secretases, forms the extracellular fibrillar aggregates known as senile plaques as a hallmark of Alzheimer’s disease (54). On the contrary, some amyloidogenic proteins retain their native struc-tures before undergoing conformational transition and eventual fibril formation (55). This group of proteins include prion (56), β2-microglobulin (β2m) (57, 58), lysozyme (59, 60), transthyretin (TTR) (61), and the variable region of immunoglobulin light- chain (VL) (62), which are responsible for mad cow disease (63), dialysis-related amyloidosis (64), hereditary systemic amyloidosis



Fig. 1. Template-dependent fibrillation. (A) Fibrillar growth is illustrated with monomers acting as 'active' growing unit.The monomers experience structural tran-sition to the amyloidogenic conformationupon binding to template. (B) Amyloid fibril growth is modeled with monomersacting as 'passive' growing unit. Fibrils are elongated by template-mediated selec-tion of the amyloidogenic conformation from the pre-existing conformational equili-brium.

Fig. 2. Template-independent fibrillations.(A) Ligand-induced fibrillation, (B) Fibrillar polymorphisms induced by multiple li-gand interactions.

(65), senile systemic amyloidosis (61) and light-chain amyloidosis (62), respectively. In addition, apomyoglobin prepared by phase separation using the acid methylethylketone method (66) formed amyloid-like structures upon denaturation in 50 mM sodium borate, pH 9.0, at 65oC, suggesting that amyloid fibril formation could be considered a generic phenomena of proteins as they experience misfolded structures (67, 68). As a matter of fact, the amyloid fibrils obtained with the various amyloidogenic proteins/peptides end up with the common cross β-sheet conformation (26, 27) despite the proteins/peptides sharing little amino acid sequence similarity. It has been, therefore, perceived that a common mechanism of amyloidogenesis might be operated based on the generic molecular feature of the resulting fibrillar end products derived from seemingly unrelated proteins/peptides. Moreover, there has been even an optimistic suggestion that revelation of a molecular mechanism of amyloidogenesis for one particular protein in relation to its corresponding disease could be universally applied to other amyloid-related diseases, since the amyloid fibrils ought to be produced via the common mechanism. Recently, however, it starts to recognize that amyloid fibrils exist in multi-

ple fibrillar forms by exhibiting so-called fibrillar polymorphism (69-71). A single amyloidogenic protein results in multiple forms of amyloid fibrils depending on their induction conditions. This may indicate that amyloidogenesis occurs via multiple mecha-nisms (72, 73). In this review, we introduce our amyloidogenesis model named double-concerted fibrillation as one of the mecha-nisms in comparison with the prevalent model of nucleation- dependent fibrillation. However, template-dependent and template-independent fibrillations need to be clarified first.

Template-dependent and template-independent fibrillations

Template-dependent fibrillation requires a pre-existing template to which amyloidogenic proteins accrete by undergoing con-formational adjustment in order to fit on the template with sub-sequent exposure of the interactive domains for molecular self-assembly (74). The role of template could be carried out by various states of amyloidogenic proteins such as conforma-tionally altered monomeric forms, partially assembled oligo-



meric intermediates, immature fibrils, and fibrillar fragments. The conformational transition of the accreting monomeric proteins must therefore be scrutinized in order to understand the template- dependent mechanism. Furthermore, the structural flexibility of amyloidogenic proteins may play a significant role in the template-dependent assembly process. This process could con-ceivably contain two consecutive steps of protein anchoring followed by induced-fit type structural adjustment on the site of template (Fig. 1A). As proteins exist in ‘natively unfolded’ state or turn to partially unfolded state from their native con-formation, the template interactive regions are likely to be exposed, which wait to be thermodynamically stabilized by binding to their partners of template. An alternative view to this rather simple induced-fit type explanation can also be suggested. The unstructured or partially unfolded state of amy-loidogenic proteins would increase their conformational entropy as they may exist in a kinetically trapped state with relatively high free energy content. Various conformations, therefore, should occur at equilibrium for a single amyloidogenic protein (Fig. 1B). As one of the conformers binds to the template and consequently is depleted, the conformational equilibrium shifts to replenish the depleted conformer, causing the protein bind the template in its complementary structure. This entire process could be regarded as disorder-to-order transition of the amyloidogenic proteins exerting structural flexibility. This type of template-dependent process, therefore, could be considered as template-mediated selection process toward a certain amy-loidogenic conformation to form the amyloid fibrils. Regardless of whatever the role the amyloidogenic proteins play between the active and the passive fibrillar growing units during the template-dependent elongation (Fig. 1A, B), the fibrillar growth always depends on the presence of template. Template-dependent fibrillation is most appropriate when considering the infectivity of prion molecules. Prion protein (PrPC) is localized on the cellular surface via glycosylphosphati-dylinositol linkage, and its conformational change into another structure (PrPSc) is related to a group of prion diseases known as transmissible spongiform encephalopathies (TSEs). Trans-missibility defines the unique nature of prion diseases. After in-gestion, PrPSc resists harsh conditions, such as low pH and di-gestion by proteases, and reaches the gut-associated lymphoid tissue where amplification of PrPSc takes place (75). Although the precise mechanism is not understood, PrPSc once amplified is believed to invade the central nervous system through the peripheral nerves. The exogenous PrPSc directs the conversion of PrPC into PrPSc conformation by acting a template and thus the protein-only infection can occur. In template-independent fibrillation, the amyloidogenic con-formations should be induced in the absence of template prior to the molecular assembly process. The conformational entropy of unstructured or partially unfolded proteins should be also be taken into consideration. By assuming a self-interactive con-former present in the conformational equilibrium, the emer-gence of that particular conformer favors self-assembly process

leading to the final amyloid fibril formation by shifting the equilibrium to replenish that depleted conformer. If the formation of the self-associative conformer is facilitated by a specific li-gand interaction, then fibrillation can be further accelerated as observed in various examples of the chemical ligand-mediated accelerated fibrillation of amyloidogenic proteins (76-82) (Fig. 2A). Such ligands could include not only small chemicals but also physiological or pathological intra/extra-cellular compo-nents, which permits the template-independent fibrillation to be physiologically or pathologically relevant process. Apart from the ligands, the enrichment of an amyloidogenic con-former without encountering any template is critical for the template-independent fibrillation to occur. In addition, if there are multiple conformers, designed to develop into various amyloid fibrils, present in the conformational equilibrium and their enrichment is also favored by a specific ligand inter-action, then a single amyloidogenic protein could end up with a diverse set of amyloid fibrils with different morphologies (Fig. 2B). The high energy state of the natively or partially unfolded amyloidogenic proteins with increased conformational entropy could allow the self-interactive conformers to be stabilized via multiple pathways into various types of amyloid fibrils espe-cially in the presence of specific ligands. Polymorphism thus has been achieved. Template-dependent and template-independent fibrillations could act in combination to achieve amyloid fibril formation. The initial template, often referred to as the nucleation center or seed (see below), is produced by the template-independent fibrillation process. Once the template becomes available, the fibrillar growth with sequential addition of monomers through conformational transition is directed by the template-depend-ent fibrillation. Now, molecular dynamics of an amyloidogenic protein experiencing either the pre-structured self-associative conformer or the template anchoring and subsequent con-formational transition determines the on-going fibrillar growth patterns. Since the structure of the template determines the fi-nal morphology of the amyloid fibrils, a variety of the tem-plates would guarantee the fibrillar polymorphism.

Nucleation-dependent fibrillation model

Nucleation-dependent and double-concerted fibrillation models are two mechanisms of amyloidogenesis that represent template- dependent and template-independent fibrillation, respectively. Nucleation-dependent fibrillation is the prevailing notion re-flecting the template-dependent fibrillation. As traced with thi-oflavin-T binding fluorescence or turbidity of the incubation mixture of amyloidogenic proteins, normal fibrillation kinetics is divided into three phases - lag, exponential growth, and sta-tionary phases - under supersaturated condition (83) (Fig. 3). During the lag phase, thermodynamically unfavorable nucleus formation takes place, which requires prolonged incubation of the amyloidogenic proteins (Fig. 4A). As soon as the nucleus (seed) is generated, monomeric proteins accrete to the tem-



Fig. 4. Models of amyloidogenesis. (A) Nucleation- dependent fibrillation, (B) Double- concerted fibrillation.

Fig. 3. Kinetics of amyloidogenesis.

plate via undergoing conformational transition, which gives rise to the abruptly accelerated fibrillar growth phase (84-86) (Fig. 3). As monomeric proteins are consumed to a certain lev-el, the growth phase is decelerated to a stationary phase, where no additional fibrillar growth occurs. It is then pre-sumed that the mature fibrils are in assembly/disassembly equilibrium between the releasing and accreting monomers on the fibrillar growth ends. The nucleus formation is a rate-de-termining step for the entire fibrillation process as evidenced by the addition of mature fibrils to an incubation mixture of amyloidogenic proteins, which abolished the lag phase. Although the nucleation-dependent model generally supports the observed in vitro fibrillation kinetics, there are still many unanswered questions which remain to be explored. First of all, nature of the seed formation wants to be clarified and thus it has been a target of intensive investigations. It has been sug-gested that an amyloidogenic conformer of a protein present in

equilibrium with other conformations acts as the stable nu-cleus, which converts non-amyloidogenic conformations of the protein into self-assembled product of amyloid fibrils. In fact, conformational equilibrium of monomeric α-synuclein was investigated using single molecule force spectroscopy by stretching each molecule of α-synuclein and recording their mechanical properties indicative of its molecular folding states (87). Three different conformational categories were observed, and one of which resembled the fibrillar state of α-synuclein. The fibrillar state-like monomeric conformer was enriched in conditions which promote the fibrillation. It remains, however, to be seen that the amyloidogenic conformer actually accel-erates the fibrillation kinetics. Otherwise, the self-assembly of pre-structured amyloidogenic protein can not be discriminated from the template-independent fibrillation model. Stable dimers of α-synuclein obtained at low pH of the fi-brillation promoting condition were also suggested to partic-ipate in the fibrillation as a nucleation site (88). Spatial ar-rangement of monomers within the dimeric species must be carefully evaluated whether the resulting dimeric structure could provide interactive domains for the accreting monomers. The 3D domain swapping model has been suggested to ex-plain the development of protein oligomer assembly (89). Provided there are two interacting domains separated by a hinge region within the monomeric form, there may be two ways for the protein to be self-associated, one of which is a dead-end dimer and the other is polymers derived via domain swapping. In this respect, the stable dimer supposed to act as a seed would be a simple end product without participating in the fibrillar elongation procedure. If the dimer participates in the fibrillation via the 3D domain swapping process, the accel-eration phenomenon of amyloidogenesis must be demonstrated.



Protein Size Cytotoxicity On-/off- pathway

AlbebetinAβα-Synuclein

Barstarβ2MIAPPLENκ-CaseinPGKPI3-SH3PrionTTR

Height = 2 nm (110)RS = ∼10 nm (100)RS = ∼10 nm (112)Height = 2-6 nm (97)Diameter = ∼20 nm (106)Height = ∼2 nm (113)Variable (102)Diameter = ∼5 nm (114)UnknownDiameter = ∼20 nm (104)Diameter = ∼10 nm (103)Variable (116)RH = ~13 nmDiameter = ∼10 nm (117)

UnknownNeurotoxic (111)Neurotoxic (111)Neurotoxic (10)

UnknownUnknownNeurotoxic (111)UnknownUnknownUnknownUnknownUnknownUnknown

On (110)On (100)On/off (99, 112)

On (113)On (102)UnknownOff (115)On (104)On (103)On (116)On (101)On (117)

Table 2. Spheroidal oligomer characteristics of various amyloidogenic proteins

Oligomeric species of amyloidogenic proteins have been hypothesized to play the nucleus of amyloid fibril formation (90). Soluble oligomeric species generally appear during the lag phase and subsequently disappear just before the fibrillar elongation starts to be accelerated (52). The fibrillation of α-synuclein became more efficient at high temperatures, in which the level of oligomeric species increases proportionally with temperature (91). The C-terminally-truncated Sup35, a yeast prion, showed only a ＜10-fold decrease in lag phase de-spite a ＞500-fold increase in monomer concentration during fibrillation. This indicates that the lag phase is not directly de-pendent on the monomeric association, and that oligomeric conversion might be the rate-limiting step (90). However, it re-mains controversial whether the oligomeric species are real on-pathway intermediates of the fibrillation pathway. We hy-pothesize that the initial stable seed formation appears to be thermodynamically unfavorable since the presumable oligo-meric nucleus production is an entropically expensive process which needs to be overcome by an enthalpic advantage. Among those oligomers, a few would survive to accommodate accreting monomers by exposing adequate interaction do-mains on the template. Those oligomers failed to be matured into the fibrils would be either disintegrated back to mono-mers or eliminated from the fibrillation process as an off-path-way product. For the nucleation-dependent fibrillation to be thoroughly understood, the accelerated fibrillar growth needs to be clari-fied, which accompanies conformational transition of mono-mers and subsequent attachment to the templates. Since this attachment between monomers and the growing fibrils should have thermodynamic advantage, the approaching monomers must exist in a relatively high-energy state as the ‘partially’ or ‘natively’ unfolded proteins are demonstrated to express their enhanced amyloidogenic propensity. For these structures to be available even transiently, they should exist in a kineti-cally-trapped state which waits for the seed formation to be stabilized into the final fibrillar structures. Partially unfolded

intermediates are suggested to be immediate precursors for the self-assembly process. Formation of partially unfolded inter-mediates depends on various factors and conditions. The parti-ally unfolded structures of α-synuclein were observed with low pH, high temperature (92), various pesticides such as rote-none, dieldrin, and paraquat (93), and polyvalent cations (94) by using circular dichroism spectroscopy and Fourier trans-form infrared spectroscopy. Under these conditions, α-synu-clein showed more highly accelerated fibrillation, indicating that the partially unfolded structures are precursors of fibrils. At the stationary phase, however, the mature fibrils do not ap-pear to grow any more, indicating that there would be a dy-namic equilibrium for the monomeric units associating and dissociating from the ends of existing fibrils. This would result in the molecular recycling within amyloid fibrils (95). Although the characteristic three subjects of nucleation-dependent fi-brillation - seed formation, accelerated fibrillar growth, and stationary phase - have been separately examined, additional studies are required for unambiguous revelation of the mecha-nism of amyloidogenesis.

Oligomeric species of the amyloidogenic proteins

Oligomeric structures of various amyloidogenic proteins have been identified and characterized in terms of their involvement in cytotoxicity and participation in the fibrillation process as an on- or off-pathway species (Table 2). Observations of various amyloidosis-related neurodegenerative disorders, including AD, PD, and Huntington’s chorea, have found that clinical manifestations precede actual formation of the fibrillar protein aggregates. Patients with the amyloid deposits sometimes do not show any neurodegenerative symptoms, indicating that amyloid fibrils do not cause diseases onset. Therefore, it has been suggested that the oligomeric intermediates are the toxic species while the amyloid fibrils are just detoxification products (9, 10). Spheroidal oligomeric species have been morphologically defined in terms of α-synuclein since they are thought to be



responsible for the cytotoxicity of neuronal cells observed in PD (96, 97). It was shown that oligomeric species of α-synuclein formed so-called amyloid pores, which significantly affect the cell viability by influencing membrane stability (9, 10). Oligomers were also laterally associated to form circularized chains which could intercalate into membranes by forming a different type of pores (98). Oligomeric spheroid formation preceding the appearance of amyloid fibrils was augmented under the condition for accelerated fibrillation, indicating that the oligomers act as on-pathway intermediates during amyloidogenesis (99). For Aβ42, large oligomer and protofibril formation was shown to follow the initial assembly of pentamer/hexamer units during initial oligomerization (100). Mouse prion protein potentially undergoes stepwise amyloid fibril formation, in which only β-rich oligomers not the α-rich monomers are transformed into worm-like amyloid fibrils (101). Mediated by Cu2+, dimeric β2-microglobulin intermediates assemble into tetra- and hexameric forms, which in turn gives rise to amyloid fibril formation (102). Yeast phos-phoglycerate kinase (PGK) forms the critical oligomers which assemble into protofibrils via a two-step process (103). Similar oligomeric species are also found for other amyloidogenic proteins such as albebetin protein exhibiting human interferon-like function in vitro, barstar protein inhibiting ribonuclease activity in Bacillus amyloliquefaciens, islet amyloid polypeptide (IAPP) contributing to glycemic control, κIV immunoglobulin light chain variable domain (LEN) discovered in the urine of a patient suffering multiple myeloma, SH3 domain of the α-subunit of bovine phosphatidyl-3’-kinase (PI3-SH3), and transthyretin (TTR) transporting thyroxine and retinol. Hence, critical role of the oligomeric species isolated in the middle of fibrillation process should be acknowledged to evaluate the mechanism of amyloid fibril formation by acting as the seeds or the growing unit for the fibrillar assembly, in addition to their suggested pathological activity of causing cytotoxicity (104, 105).

Double-concerted fibrillation model

Recently, we have proposed another mechanism for the amy-loidogenesis of α-synuclein, named double-concerted fibrillation (106), to parallel the prevailing nucleation-dependent fibrillation model (86). Based on this double-concerted fibrillation model, the amyloid fibril formation is achieved via two consecutive, concerted associations of monomers and subsequently formed oligomeric granular species, in which the oligomeric species act as a growing unit for the fibril formation in the absence of template (106, 107) (Fig. 4B). Structural rearrangement within the oligomeric species could be a major driving force for the suprastructures formation. Since the oligomers already contain interactive regions between constituting monomers, those in-teractions could be shifted from intra-oligomeric to inter-oligo-meric interactions, which results in the final fibril formation. In fact, we have been successful to observe the dramatically ac-celerated fibril formation of the oligomeric granular species of

α-synuclein by selectively distorting the oligomeic structures with shear stress and organic solvents (106, 107). When such stresses were applied, the fibrillation starts to occur almost in-stantaneously, in which the monomers, on the other hand, no longer participate for the fibrillar growth process. The partic-ulate state and pseudo-stability of the oligomeric species sensi-tizes those structures to the physical and chemical influences. Involvement of the oligomeric units for the amyloid fibril formation has been also suggested for other amyloidogenic proteins based mostly on kinetic studies. The C-terminally trun-cated yeast prion protein Sup35(NM) was demonstrated to form the oligomers in a fluid-like state, which might act either a growing unit to the pre-existing fibrils or the nucleation cen-ter to accommodate structurally malleable oligomers or mono-mers (90). PGK was shown to become protofibrils through a two-step mechanism in which monomers and subsequent crit-ical oligomers associated among themselves (103). Although no structural transition has been described within the oligo-meric species, this two-step mechanism basically shares the common notion with our double-concerted fibrillation model in terms of the amyloid fibril formation. In addition, the amy-loid fibril formation of insulin revealed helical oligomers as a fibrillar elongation unit by performing a kinetic x-ray solution scattering study (108). Bovine κ-casein was also hypothesized to form amyloid fibrils via structural rearrangement of pre-formed oligomeric micellar aggregates into more organized fi-brillar species (104). As confirmed with the various amyloido-genic proteins, the double-concerted fibrillation mechanism is indeed operated as one of multiple mechanisms responsible for amyloidogenesis. One might argue whether this mechanism is feasible under physiological or pathological conditions. Since the major driv-ing force for the oligomers to be assembled is the subtle struc-tural rearrangement within the pseudo-stable oligomeric spe-cies, many intra-/extra-cellular components including not only soluble entities but also insoluble structures could affect the oligomeric structures. If those two fibrillation processes of the double-concerted and the nucleation-dependent mechanisms work in cooperation in vivo, amyloid fibril formation would be more likely. The multiple pathways of amyloidogenesis and mutual cooperations also guarantee the fibrillar polymorphism in vivo. Since the real toxic causes in relation to the amyloido-genesis are still elusive at the moment, all the components found in the fibrillation process need to be carefully examined to identify therapeutic targets, which includes the oligomers acting as either the seeds or the growing units and the multiple forms of amyloid fibrils. The targets are certainly diversified.

Conclusions

As witnessed with the fibrillar polymorphism, multiple mecha-nisms for amyloidogenesis are expected to operate. Template- dependent and template-independent fibrillations would di-versify the amyloidogenic conformers of self-associated mono-



meric proteins and the subsequent formation of nucleation centers which determine the final morphology of mature amy-loid fibrils. In combination, the fibrillar polymorphism could be generated. Nucleation-dependent and double-concerted fi-brillation are two specific fibrillation models differentiated on the basis of the seed formation and differences on the fibrillar growing unit between monomers and oligomeric species. These mechanisms could operate individually or/and coopera-tively in vivo, which leads to the formation of diversified oligomers and amyloid fibrils. If cytotoxicity lies on the diversi-fied components of the amyloidogenesis, the possible toxic causes would vary accordingly. Similarly, since those amyloid fibrils could be employed as protein nanofibrils, the fibrillar polymorphism would provide an opportunity to have novel materials to be used for future nanobiotechnology.

Acknowledgements This study has been supported by the Korea Healthcare Technology R&D Project (A08-4217-A21723-08N1-00010A) of Ministry for Health, Welfare, and Family Affairs.

REFERENCES

1. Sipe, J. D. (1992) Amyloidosis. Annu. Rev. Biochem. 61, 947-975.

2. Chiti, F. and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333-366.

3. Virchow, R. (1854) Zur celluslose-frage. Virchows Arch. 6, 415-426.

4. Selkoe, D. J. (2003) Folding proteins in fatal ways. Nature 426, 900-904.

5. Sacchettini, J. C. and Kelly, J. W. (2002) Therapeutic strategies for human amyloid diseases. Nat. Rev. Drug. Discov. 1, 267-275.

6. Hardy, J. and Selkoe, D. J. (2002) The amyloid hypoth-esis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356.

7. Tan, S. Y. and Pepys, M. B. (1994) Amyloidosis. Histo-pathology 25, 403-414.

8. Merlini, G. and Bellotti, V. (2003) Molecular mecha-nisms of amyloidosis. N. Engl. J. Med. 349, 583-596.

9. Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T. and Lansbury, P. T. Jr. (2002) Amyloid pores from pathogenic mutations. Nature 418, 291.

10. Caughey, B. and Lansbury, P. T. Jr. (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the re-sponsible protein aggregates from the innocent bystan-ders. Annu. Rev. Neurosci. 26, 267-298.

11. Huang, X., Cuajungco, M. P., Atwood, C. S., Hartshorn, M. A., Tyndall, J. D. A., Hanson, G. R., Stokes, K. C., Leopold, M., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Saunders, A. J., Lim, J., Moir, R. D., Glabe, C., Bowden, E. F., Masters, C. L., Fairlie, D. P., Tanzi, R. E. and Bush, A. I. (1999) Cu(II) potentiation of Alzheimer Ab neurotoxicity. J. Biol. Chem. 274, 37111-37116.

12. Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup, G., Goldstein, L. E., Scarpa, R. C., Cuajungco, M. P.,

Gray, D. N., Lim, J., Moir, R. D., Tanzi, R. E. and Bush, A. I. (1999) The Ab peptide of Alzheimer's disease di-rectly produces hydrogen peroxide through metal ion reduction. Biochemistry 38, 7609-7616.

13. Cuajungco, M. P., Goldstein, L. E., Nunomura, A., Smith, M. A., Lim, J. T., Atwood, C. S., Huang, X., Farrag, Y. W., Perry, G. and Bush, A. I. (2000) Evidence that the b-amyloid plaques of Alzhermer's disease represent the redox-silencing and entombment of Ab by zinc. J. Biol. Chem. 275, 19439-19442.

14. Opazo, C., Huang, X., Cherny, R. A., Moir, R. D., Roher, A. E., White, A. R., Cappai, R., Masters, C. L., Tanzi, R. E., Inestrosa, N. C. and Bush, A. I. (2002) Metalloen-zyme-like activity of Alzheimer's disease β-amyloid. J. Biol. Chem. 277, 40302-40308.

15. Barnham, K. J., Ciccotosto, G. D., Tickler, A. K., Ali, F. E., Smith, D. G., Williamson, N. A., Lam, Y. -H., Carrington, D., Tew, D., Kocak, G., Volitakis, I., Sepa-rovic, F., Barrow, C. J., Wade, J. D., Masters, C. L., Cherny, R. A., Curtain, C. C., Bush, A. I. and Cappai, R. (2003) Neurotoxic, redox-competent Alzheimer's b-amy-loid is released from lipid membrane by methionine oxidation. J. Biol. Chem. 278, 42959-42965.

16. Fowler, D. M., Koulov, A. V., Balch, W. E. and Kelly, J. W. (2007) Functional amyloid - from bacteria to humans. Trends Biochem. Sci. 32, 217-224.

17. Barnhart, M. M. and Chapman, M. R. (2006) Curli bio-genesis and function. Annu. Rev. Microbiol. 60, 131-147.

18. Costerton, J. W., Stewart, P. S. and Greenberg, E. P. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284, 1318-1322.

19. Kikuchi, T., Mizunoe, Y., Takade, A., Naito, S. and Yoshida, S. (2005) Curli fibers are required for develop-ment of biofilm architecture in Escherichia coli K-12 and enhance bacterial adhearence to human uroepithelial cells. Microbiol. Immunol. 49, 875-884.

20. Gebbink, M. F. B. G., Claessen, D., Bouma, B., Dijkhui-zen, L. and Wösten, H. A. B. (2005) Amyloids - a func-tional coat for microorganisms. Nat. Rev. Microbiol. 3, 333-341.

21. Talbot, N. J. (2003) Aerial morphogenesis: enter the chaplins. Curr. Biol. 13, R696-R698.

22. Iconomidou, V. A., Vriend, G. and Hamodrakas, S. J. (2000) Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145.

23. Podrabsky, J. E., Carpenter, J. F. and Hand, S. C. (2001) Survival of water stress in annual fish embryos: dehy-dration avoidance and egg envelope amyloid fibers. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R123-R131.

24. Fowler, D. M., Koulov, A. V., Alory-Jost, C., Marks, M. S., Balch, W. E. and Kelly, J. W. (2006) Functional amyloid formation within mammalian tissue. PLoS Biol. 4, e6.

25. Shibayama, Y., Joseph, K., Nakazawa, Y., Ghebreihiwet, B., Peerschke, E. I. B. and Kaplan, A. P. (1999) Zinc-de-pendent activation of the plasma kinin-forming cascade by aggregated β amyloid protein. Clin. Immunol. 90, 89-99.

26. Sunde, M. and Blake, C. C. F. (1998) From the globular to the fibrous state: protein structure and structural con-version in amyloid formation. Q. Rev. Biophys. 31, 1-39.

27. Sunde, M., Serpella, L. C., Bartlama, M., Frasera, P. E.,



Pepysa, M. B. and Blake, C. C. F. (1997) Common core structure of amyloid fibrils by synchrotron X-ray diff-raction. J. Mol. Biol. 273, 729-739.

28. Knowles, T. P., Fitzpatrick, A. W., Meehan, S., Mott, H. R., Vendruscolo, M., Dobson, C. M. and Welland, M. E. (2007) Role of intermolecular forces in defining material properties of protein nanofibrils. Science 318, 1900-1903.

29. Cherny, I. and Gazit, E. (2008) Amyloids: Not only pathological agents but also ordered nanomaterials. Angew. Chem. Int. Ed. 47, 4062-4069.

30. Zhang, S. (2003) Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21, 1171-1178.

31. Scheibel, T., Parthasarathy, R., Sawicki, G., Lin, X. -M., Jaeger, H. and Lindquist, S. L. (2003) Conducting nano-wires built by controlled self-assembly of amyloid fibers and selective metal deposition. Proc. Natl. Acad. Sci. U.S.A. 100, 4527-4532.

32. Song, Y., Challa, S. R., Medforth, C. J., Qiu, Y., Watt, R. K., Peῇa, D., Miller, J. E., Swol, F. and Shelnutt, J. A. (2004) Synthesis of peptide-nanotube platinum-nano-particle composites. Chem. Commun. 1044-1045.

33. Reches, M. and Gazit, E. (2003) Casting metal nanowires within discrete self-assembled peptide nanotubes. Science 300, 625-627.

34. Carny, O., Shalev, D. E. and Gazit, E. (2006) Fabrication of coaxial metal nanocables using a self-assembled pep-tide nanotube scaffold. Nano Lett. 6, 1594-1597.

35. Fu, X., Wang, Y., Huang, L., Sha, Y., Gui, L., Lai, L. and Tang, Y. (2003) Asseblies of metal nanoparticles and self-assembled peptide fibrils - formation of double heli-cal and single-chain arrays of metal nanoparticles. Adv. Mater. 15, 902-906.

36. Reches, M. and Gazit, E. (2007) Biological and chemical decoration of peptide nanostructures via biotin-avidin interactions. J. Nanosci. Nanotechnol. 7, 2239-2245.

37. Baxa, U., Speransky, V., Steven, A. C. and Wickner, R. B. (2002) Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein. Proc. Natl. Acad. Sci. U.S.A. 99, 5253-5260.

38. Baldwin, A. J., Bader, R., Christodoulou, J., MacPhee, C. E., Dobson, C. M. and Barker, P. D. (2006) Cytochrome display on amyloid fibrils. J. Am. Chem. Soc. 128, 2162-2163.

39. Corrigan, A. M., Müller, C. and Krebs, M. R. H. (2006) The formation of nematic liquid crystal phases by hen ly-sozyme amyloid fibrils. J. Am. Chem. Soc. 128, 14740- 14741.

40. Aggeli, A., Bell, M., Boden, N., Keen, J. N., Knowles, P. F., McLeish, T. C. B., Pitkeathly, M. and Radford, S. E. (1997) Responsive gels formed by the spontaneous self-assembly of peptides into polymeric β-sheet tapes. Nature 386, 259-262.

41. Zhang, Y., Gu, H., Yang, Z. and Xu, B. (2003) Supramo-lecular hydrogels respond to lignad-receptor interaction. J. Am. Chem. Soc. 125, 13680-13681.

42. Yang, Z., Liang, G., Wang, L. and Xu, B. (2006) Using a kinase/phosphatase switch to regulate a supramolecular hydrogel and forming the supramolecular hydrogel in vivo. J. Am. Chem. Soc. 128, 3038-3043.

43. Conway, K. A., Harper, J. D. and Lansbury, P. T. Jr. (1998) Accelerated in vitro fibril formation by a mutant

a-synuclein linked to early-onset Parkinson disease. Nat. Med. 4, 1318-1320.

44. Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. and Lansbury, P. T. Jr. (1996) NACP, a protein im-plicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry 35, 13709-13715.

45. Kayed, R., Bernhagen, J., Greenfield, N., Sweimeh, K., Brunner, H., Voelter, W. and Kapurniotu, A. (1999) Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vitro. J. Mol. Biol. 287, 781-796.

46. Teplow, D. B. (1998) Structural and kinetic features of amyloid b-protein fibrillogenesis. Amyloid: Int. J. Exp. Clin. Invest. 5, 121-142.

47. Walsh, D. M., Hartley, D. M., Kusumoto, Y., Fezoui, Y., Condron, M. M., Lomakin, A., Benedek, G. B., Selkoe, D. J. and Teplow, D. B. (1999) Amyloid β-protein fibrillogenesis. Structure and biological activity of proto-fibrillar intermediates. J. Biol. Chem. 274, 25945-25952.

48. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. and Goedert, M. (1998) α-Synuclein in filamentous inclusions of Lewy bodies form Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. U.S.A. 95, 6469-6473.

49. Spillantini, M. G., Schmidt, M. L., Lee, V. M. -Y., Troja-nowski, J. Q., Jakes, R. and Goedert, M. (1997) α- Synuclein in Lewy bodies. Nature 388, 839-840.

50. Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D. and Giusti, P. (2004) a-Synuclein and Parkinson's disease. FASEB 18, 617-626.

51. Goedert, M. (2001) Alpha-synuclein and neurodege-nerative diseases. Nat. Rev. Neurosci. 2, 492-501.

52. Fink, A. L. (2006) The aggregation and fibrillation of α-synuclein. Acc. Chem. Res. 39, 628-634.

53. Jaikaran, E. T. A. S. and Clark, A. (2001) Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology. Biochim. Biophys. Acta-Mol. Basis Dis. 1537, 179-203.

54. Hardy, J. A. and Higgins, G. A. (1992) Alzheimer's disease: The amyloid cascade hypothesis. Science 256, 184-185.

55. Chiti, F. and Dobson, C. M. (2009) Amyloid formation by globular proteins under native conditions. Nat. Chem. Biol. 5, 15-22.

56. Harris, D. A. and True, H. L. (2006) New insights into prion structure and toxicity. Neuron 50, 353-357.

57. Bernier, G. M. (1980) b2-Microglobulin: structure, func-tion and significance. Vox Sanguinis. 38, 323-327.

58. Trinh, C. H., Smith, D. P., Kalverda, A. P., Phillips, S. E. V. and Radford, S. E. (2002) Crystal structure of mono-meric human β-2-microglobulin reveals clues to its amy-loidogenic properties. Proc. Natl. Acad. Sci. U.S.A. 99, 9771-9776.

59. Artymiuk, P. J. and Blake, C. C. F. (1981) Refinement of human lysozyme at 1.5 Å resoution analysis of non- bonded and hydrogen-bond interactions. J. Mol. Biol. 152, 737-762.

60. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. and Sarma, V. R. (1965) Structure of hen egg-white lysozyme: a three-dimensional fourier syn-thesis at 2 Å resolution. Nature 206, 757-761.



61. Kelly, J. W., Colon, W., Lai, Z., Lashuel, H. A., McCulloch, J., McCutchen, S. L., Miroy, G. J. and Peterson, S. A. (1997) Transthyretin quaternary and ter-tiary structural changes facilitate misassembly into amyloid. Adv. Protein. Chem. 50, 161-181.

62. Bellotti, V., Mangione, P. and Merlini, G. (2000) Review: Immunoglobulin light chain amyloidosis - The archetype of structural and pathogenic variability. J. Struct. Biol. 130, 280-289.

63. Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383.

64. Koch, K. M. (1992) Dialysis-related amyloidosis. Kidney Int. 41, 1416-1429.

65. Pepys, M. B., Hawkins, P. N., Booth, D. R., Vigushin, D. M., Tennent, G. A., Soutar, A. K., Totty, N., Nguyen, O., Blake, C. C. F., Terry, C. J., Feest, T. G., Zalin, A. M. and Hsuan, J. J. (1993) Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 362, 553-557.

66. Teale, F. W. J. (1959) Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta. 35, 543.

67. Fändrich, M., Fletcher, M. A. and Dobson, C. M. (2001) Amyloid fibrils from muscle myoglobin. Nature 410, 165-166.

68. Fändrich, M., Forge, V., Buder, K., Kittler, M., Dobson, C. M. and Diekmann, S. (2003) Myoglobin forms amy-loid fibrils by association of unfolded polypeptide segments. Proc. Natl. Acad. Sci. U.S.A. 100, 15463- 15468.

69. Kodali, R. and Wetzel, R. (2007) Polymorphism in the in-termediates and products of amyloid assembly. Curr. Opin. Struct. Biol. 17, 48-57.

70. Petkova, A. T., Leapman, R. D., Guo, Z., Yau, W. -M., Mattson, M. P. and Tycko, R. (2005) Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science 307, 262-265.

71. Wetzel, R., Shivaprasad, S. and Williams, A. D. (2007) Plasticity of amyloid fibrils. Biochemistry 46, 1-10.

72. Goldsbury, C., Frey, P., Olivieri, V., Aebi, U. and Müller, S. A. (2005) Multiple assembly pathways underlie amy-loid-b fibril polymorphisms. J. Mol. Biol. 352, 282-298.

73. Gosal, W. S., Morten, I. J., Hewitt, E. W., Smith, D. A., Thomson, N. H. and Radford, S. E. (2005) Competing pathways determine fibril morphology in the self-assem-bly of b2-microglobulin into amyloid. J. Mol. Biol. 351, 850-864.

74. Griffith, J. S. (1967) Self-replication and scrapie. Nature 215, 1043-1044.

75. Nuvolone, M., Aguzzi, A. and Heikenwalder, M. (2009) Cells and prions: a license to replicate. FEBS Lett. 583, 2674-2684.

76. Park, J. -W., Ahn, J. S., Lee, J. -H., Bhak, G., Jung, S. and Paik, S. R. (2008) Amyloid fibrillar meshwork formation of ion-induced oligomeric species of Aβ40 with phthalo-cyanine tetrasulfonate and its toxic consequences. Chem. Bio. Chem. 9, 2602-2605.

77. Kim, Y. -S., Lee, D., Lee, E. -K., Sung, J. Y., Chung, K. C., Kim, J. and Paik, S. R. (2001) Multiple ligand interaction of a-synuclein produced various forms of protein ag-gregates in the presence of Aβ25-35, copper, and eosin.

Brain Res. 908, 93-98.78. Paik, S. R., Lee, D., Cho, H. -J., Lee, E. -N. and Chang, C.

-S. (2003) Oxidized glutathione stimulated the amyloid formation of a-synuclein. FEBS Lett. 537, 63-67.

79. Lee, D., Lee, E. -K., Lee, J. -H., Chang, C. -S. and Paik, S. R. (2001) Self-oligomerization and protein aggregation of a-synuclein in the presence of coomassie brilliant blue. Eur. J. Biochem. 268, 295-301.

80. McLaurin, J., Yang, D. -S., Yip, C. M. and Fraser, P. E. (2000) Review: modulating factors in amyloi-b fibril for-mation. J. Struct. Biol. 130, 259-270.

81. Dong, J., Canfield, J. M., Mehta, A. K., Shokes, J. E., Tian, B., Childers, W. S., Simmons, J. A., Mao, Z., Scott, R. A., Warncke, K. and Lynn, D. G. (2007) Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity. Proc. Natl. Acad. Sci. U.S.A. 104, 13313-13318.

82. Koppaka, V. and Axelsen, P. H. (2000) Accelerated accu-mulation of amyloid b proteins on oxidatively damaged lipid membranes. Biochemistry 39, 10011-10016.

83. Naiki, H. and Gejyo, F. (1999) Kinetic analysis of amy-loid fibril formation. Methods Enzymol. 309, 305-318.

84. Jarrett, J. T. and Lansbury, P. T. Jr. (1993) Seeding ''one- dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73, 1055-1058.

85. Harper, J. D. and Lansbury, P. T. Jr. (1997) Models of amyloid seeding in Alzheimer's disease and scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem. 66, 385-407.

86. Wood, S. J., Wypych, J., Steavenson, S., Louis, J. -C., Citron, M. and Biere, A. L. (1999) α-Synuclein fibrillo-genesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease. J. Biol. Chem. 274, 509-512.

87. Sandal, M., Valle, F., Tessari, I., Mammi, S., Bergantino, E., Musiani, F., Brucale, M., Bubacco, L. and Samori, B. (2008) Conformational equilibria in monomeric α-synu-clein at the single-molecule level. PLoS Biol. 6, e6.

88. Uversky, V. N., Lee, H. -J., Li, J., Fink, A. L. and Lee, S. -J. (2001) Stabilization of partially folded conformation dur-ing a-synuclein oligomerization in both purified and cy-tosolic preparations. J. Biol. Chem. 276, 43495-43498.

89. Bennett, M. J., Schlunegger, M. P. and Eisenberg, D. (1995) 3D domain swapping: a mechanism for oligomer assembly. Protein Sci. 4, 2455-2468.

90. Serio, T. R., Cashikar, A. G., Kowal, A. S., Sawicki, G. J., Moslehi, J. J., Serpell, L., Arnsdorf, M. F. and Lindquist, S. L. (2000) Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289, 1317-1321.

91. Li, J., Uversky, V. N. and Fink, A. L. (2001) Effect of fami-lial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human α-synuclein. Biochemistry 40, 604-613.

92. Uversky, V. N., Li, J. and Fink, A. L. (2001) Evidence for a partially folded intermediate in α-synuclein fibril formation. J. Biol. Chem. 276, 10737-10744.

93. Uversky, V. N., Li, J. and Fink, A. L. (2001) Pesticides di-rectly accelerate the rate of α-synuclein fibril formation:



a possible factor in Parkinson's disease. FEBS Lett. 500, 105-108.

94. Uversky, V. N., Li, J. and Fink, A. L. (2001) Metal-trig-gered structural transformations, aggregation, and fi-brillation of human α-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure. J. Biol. Chem. 276, 44284-44296.

95. Carulla, N., Caddy, G. L., Hall, D. R., Zurdo, J., Gairi, M., Feliz, M., Giralt, E., Robinson, C. V. and Dobson, C. M. (2005) Molecular recycling within amyloid fibrils. Nature 436, 554-558.

96. Apetri, M. M., Maiti, N. C., Zagorski, M. G., Carey, P. R. and Anderson, V. E. (2006) Secondary structure of a-syn-uclein oligomers: characterization by Raman and atomic force microscopy. J. Mol. Biol. 355, 63-71.

97. Conway, K. A., Lee, S. -J., Rochet, J. -C., Ding, T. T., Williamson, R. E. and Lansbury, P. T. Jr. (2000) Acceler-ation of oligomerization, not fibrillization, is a shared property of both a-synuclein mutations linked to ear-ly-onset Parkinson's disease: implications for patho-genesis and therapy. Proc. Natl. Acad. Sci. U.S.A. 97, 571-576.

98. Ding, T. T., Lee, S. -J., Rochet, J. -C. and Lansbury, P. T. Jr. (2002) Annular a-synuclein protofibrils are produced when spherical protofibrils are incubated in solution of bound to brain-derived membranes. Biochemistry 41, 10209-10217.

99. Hoyer, W., Cherny, D., Subramaniam, V. and Jovin, T. M. (2004) Rapid self-assembly of a-synuclein observed by in situ atomic force microscopy. J. Mol. Biol. 340, 127-139.

100. Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B. and Teplow, D. B. (2003) Amyloid b-pro-tein (Ab) assembly: Ab40 and Ab42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. U.S.A. 100, 330-335.

101. Jain, S. and Udgaonkar, J. B. (2008) Evidence for step-wise formation of amyloid fibrils by the mouse prion protein. J. Mol. Biol. 382, 1228-1241.

102. Eakin, C. M., Attenello, F. J., Morgan, C. J. and Miranker, A. D. (2004) Oligomeric assembly of native-like pre-cursors precedes amyloid formation by β-2 microglo-bulin. Biochemistry 43, 7808-7815.

103. Modler, A. J., Gast, K., Lutsch, G. and Damaschun, G. (2003) Assembly of amyloid protofibrils via critical oligomers - A novel pathway of amyloid formation. J. Mol. Biol. 325, 135-148.

104. Leonil, J., Henry, G., Jouanneau, D., Delage, M. -M., Forge, V. and Putaux, J. -L. (2008) Kinetics of fibril for-mation of bovine k-casein indicate a conformational re-arrangement as a critical step in the process. J. Mol. Biol. 381, 1267-1280.

105. Cheon, M., Chang, I., Mohanty, S., Luheshi, L. M., Dobson, C. M., Vendruscolo, M. and Favrin, G. (2007) Structural reorganisation and potential toxicity of oligo-meric species formed during the assembly of amyloid

fibrils. PLoS Comput. Biol. 3, 1727-1738.106. Bhak, G., Lee, J. -H., Hahn, J. -S. and Paik, S. R. (2009)

Granular assembly of a-synuclein leading to the accel-erated amyloid fibril formation with shear stress. PLoS ONE 4, e4177.

107. Lee, J. -H., Bhak, G., Lee, S. -G. and Paik, S. R. (2008) Instantaneous amyloid fibril formation of a-synuclein from the oligomeric granular structures in the presence of hexane. Biophys. J. 95, L16-L18.

108. Vestergaard, B., Groenning, M., Roessle, M., Kastrup, J. S., Weert, M., Flink, J. M., Frokjaer, S., Gajhede, M. and Svergun, D. I. (2007) A helical structural nucleus is the primary elongating unit of insulin amyloid fibrils. PLoS Biol. 5, e134.

109. Bates, G. (2003) Huntingtin aggregation and toxicity in Huntington's disease. Lancet 361, 1642-1644.

110. Morozova-Roche, L. A., Zamotin, V., Malisauskas, M., Öhman, A., Chertkova, R., Lavrikova, M. A., Kostanyan, I. A., Dolgikh, D. A. and Kirpichnikov, M. P. (2004) Fibrillation of carrier protein albebetin and its bio-logically active constructs. Multiple oligomeric inter-mediates and pathways. Biochemistry 43, 9610-9619.

111. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W. and Glabe, C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486- 489.

112. Kaylor, J., Bodner, N., Edridge, S., Yamin, G., Hong, D. -P. and Fink, A. L. (2005) Characterization of oligomeric intermediates in a-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F α-synuclein. J. Mol. Biol. 353, 357- 372.

113. Kumar, S. and Udgaonkar, J. B. (2009) Conformational conversion may precede or follow aggregate elongation on alternative pathways of amyloid protofibril formation. J. Mol. Biol. 385, 1266-1276.

114. Kayed, R., Sokolov, Y., Edmonds, B., McIntire, T. M., Milton, S. C., Hall, J. E. and Glabe, C. G. (2004) Perme-abilization of lipid bilayers is a common conformation- dependent activity of soluble amyloid oligomers in pro-tein misfolding diseases. J. Biol. Chem. 279, 46363- 46366.

115. Souillac, P. O., Uversky, V. N. and Fink, A. L. (2003) Structural transformations of oligomeric intermediates in the fibrillation of the immunoglobulin light chain LEN. Biochemistry 42, 8094-8104.

116. Bader, R., Bamford, R., Zurdo, J., Luisi, B. F. and Do-bson, C. M. (2006) Probing the mechanism of amyloido-genesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fi-bril formation. J. Mol. Biol. 356, 189-208.

117. Quintas, A., Vaz, D. C., Cardoso, I., Saraiva, M. J. M. and Brito, R. M. M. (2001) Tetramer dissociation and monomer partial unfolding precedes protofibril for-mation in amyloidogenic transthyretin variants. J. Biol. Chem. 276, 27207-27213.

mechanism of amyloidogenesis: nucleation-dependent

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