Báo cáo Y học: Amyloid-fibril formation Proposed mechanisms and relevance to conformational disease docx

REVIEW ARTICLE Amyloid-fibril formation Proposed mechanisms and relevance to conformational disease Eva Z ˇ erovnik Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Ljubljana, Slovenia The phenomenon of the transformation of proteins into amyloid-fibrils is of interest, firstly, because it is closely connected to the so-called conformational diseases, many of which are hitherto incurable, and secondly, because it remains to be explained in physical terms (energetically and structurally). The process leads to fibrous aggregates in the form of extracellular amyloid plaques, neuro-fibrillary tangles and other intracytoplasmic or intranuclear inclu- sions. In this review, basic principles common to the field of amyloid fibril formation and conformational disease are underlined. Existing models for the mechanism need to be tested by experiment. The kinetic and energetic bases of the process are reviewed. The main controversial issue remains the coexistence of more than one protein conformation. The possible role of oligomeric intermediates, and of domain- swapping is also discussed. Mechanisms for cellular defence and novel therapies are considered. Keywords: amyloid fibrils; conformational disease; domain swapping; kinetics; mechanism of fibrillogenesis. Protein folding is important for cellular events ranging from transport, accepting and transmitting signals, regulation at the gene and RNA levels, cell adhesion, changes in cytoskeleton, metabolic reactions involving various enzymes, etc. An active protein conformation is needed for successful cell functioning, and therefore important in maintaining health. Several types of disease have been found where protein misfolding and conformational change are the main causes of the appearance and progression of disease [1]. A list of conformational diseases, together with their associated protein component(s), is shown in Table 1 [2]. In some cases, more than one protein is involved with a disorder, coexisting in a plaque or making its formation easier. Often, proteolytically degraded fragments are more prone to forming fibrils, e.g. amyloid precursor protein (APP) where a, b and c secretases [3,4] are responsible for the initial processing, huntingtin and possibly also a-synuc- lein [5]. In Alzheimer’s disease, which represents a major problem in the Western world’s ageing population, the main protein component is APP, a transmembrane protein of approxi- mately 700 amino-acid residues [3,4,6,7]. In its normal processing Ab (1–40) peptide is produced which circulates extracellularly and usually does not deposit as plaques. It has been proposed that the peptide may exert an antioxi- dative function [8]. In sporadic cases, especially when allele 4 of apolipoprotein E is present, the peptide starts to form amyloid plaques. In the familial, more severe early- onset cases, prevalence of the hydrophobic Ab (1–42) peptide leads to extensive amyloid plaque formation. This has been linked to mutations in the APP and presenilins 1 and 2 [7], which all increase the production of the more fibrillogenic Ab (1–42) peptide. Fibrillary tangles of another protein, sau, are observed in the cell. sau is a microtubule- associated protein involved in stabilizing axonal microtubules. Other functions include a role in signal transduction, and anchoring various kinases and phospha- tases [9]. Importantly, an anti-amyloidogenic protein, gelsolin, has been found in plasma and central system fluid (CSF). This secretory protein is able, by making complexes with Ab, to inhibit fibril formation and even to break down already formed fibrils [10]. Recently, it has been found that the endopeptidase ÔneprilysinÕ degrades Ab peptide. In neprilysin gene-disrupted mice Ab was found to accumu- late, with the highest levels in the hippocampus [11]. In Parkinson’s disease, which is the second most common neurodegenerative disease, several proteins are implicated, a-synuclein, synphilin (an a-synuclein inteacting protein) andparkin[12].a-Synuclein is a small (140 amino acid) acidic protein. It is a naturally unfolded, intracellular and presynaptic polypeptide that becomes partly helical on binding to synaptic vesicles [13]. Its function may be, among others, regulation of synaptic vesicles and neurotransmitter release [13]. It is interesting that a-synuclein is a target of serine/threonine [14] as well as tyrosine [15,16] kinases. A hallmark of Parkinson’s disease is the presence of Lewy bodies, which are found in sporadic cases of Parkinson’s disease, in dementia with Lewy bodies and in the Lewy body variant of Alzheimer’s disease [17]. a-Synuclein is the main component of the Lewy bodies [18]. Both a-synuclein and synphilin are required for formation of the Lewy bodies where ubiquitination of synphilin probably takes place [12,17]. Parkin is a 465-amino-acid ubiquitin-protein ligase [17,19]. Mutations in parkin and a-synuclein, in familial cases of Parkinson’s disease, prevent proper ubiquitination, Correspondence to E. Z ˇ erovnik, Department of Biochemistry and Molecular Biology, Jozˇ ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. E-mail: eva.zerovnik@ijs.si Abbreviations: APP, amyloid precursor protein; Ab, amyloid b pep- tide; CSF, central system fluid; AFM, atomic force microscopy; EM, electron microscopy. (Received 28 January 2002, revised 1 May 2002, accepted 27 May 2002) Eur. J. Biochem. 269, 3362–3371 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03024.x so that proteins are not sequestered in the inclusion bodies, leading to greater toxicity [12]. Even the prion diseases, a range of transmissible spongi- form encephalophaties (kuru, Creuzfeldt–Jacob disease and fatal familial insomnia in humans, bovine spongiform encelopathy in cattle, scrapie in sheep, and chronic wasting disease in deer) have many features in common with amyloidoses and most likely are ÔconformationalÕ [20]. No other agent accompanying the prion protein like virus (DNA–protein) or bare DNA has convincingly been shown in the infected tissue [21]. The transmission could be explained solely by inducing a wrong, irreversible conform- ational change, resistant to proteolysis, leading to accumu- lation of harmful protein aggregates. This hypothesis has recently been confirmed by inducing disease in transgenic mice inoculated by b rich conformation of mutant P101L (89–143) peptide of the human prion protein [21], in contrast to the ones inoculated by non-b-form of the peptide. The term ÔamyloidÕ was introduced in 1854 by the German physician R. Virchow, who named it in the belief that the iodine-staining component was starch-like [22,23]. The first criterion for detecting amyloid ex vivo was birefringence of the histological dye Congo Red, observed under polarized light. As the second criterion, electron microscopy showed that all amyloid deposits exhibited a similar fibrillar, submicroscopic structure, bundles of straight, rigid fibrils ranging in width from 60 to 130 A ˚ and in length from 1000 to 16000 A ˚ [23]. In addition to the fibrillar component of amyloid, nonfibrillar components were always found, including serum amyloid protein, heparan sulfate proteoglycans and apolipoprotein E [23]. The importance of the nonprotein and nonfibrillar compo- nents of amyloid as observed in vivo remains to be determined. In vitro studies of the disease related proteins, as well as other amyloidogenic proteins, have been concerned mostly with the morphology and kinetics of fibrillogenesis. It was concluded by Soto [20] that the pathogenesis of all the conformational diseases, including prion disease, involves conformational changes leading to aberrantly folded proteins, rich in b secondary structure that have a high tendency to form aggregates and are quite resistant to proteolysis [20,24]. The field is characterized by several scientific findings that challenge some of the commonly held dogmas in biology [24]. These findings are that a protein can exist in more than one conformation with distinct biological properties, and that biological function is mediated through changes in protein conformation. Some of the basic principles underlying protein fibril formation are described in the following sections. FIBRIL FORMATION, A GENERAL PROPERTY OF PROTEINS AND POLYPEPTIDES? Several authors have found that proteins that have not been associated with any disease can form amyloid-like fibrils [25–31]. Especially surprising was the finding that even a helical proteins, such as myoglobin [32] or apo-cyto- chrome c [33] can form fibrils under certain conditions. These observations led Dobson and coauthors to propose that amyloid-fibril formation is a generic property of proteins [27,32,34]. A common observation is that fibrilli- zation starts from an intermediate state, either partially unfolded or partially folded, molten globule or native-like intermediate [35]. In the case of globular proteins such as phosphoglycerate kinase [25], cystatin C [36], acylphospha- tase [29] and transthyretin [37], partial unfolding needs to occur to enable fibril formation and, in the case of unfolded polypeptides such as a-synuclein [38,39] and islet amyloid polypeptide, these must partially fold. The parts with the a helical structure must undergo an a to b transition and the b strands then associate into a regular fibrillar structure. An a to b transformation is well characterized with peptides, like poly( L -lysine). It has more recently been observed with proteins which are initially unfolded or predominantly b sheet [40–42] and which fold through an a helical intermediate [43–46]. In vitro, variation of solvent conditions by changing pH or adding organic solvents [47] can lead to partial unfolding Table 1. Protein fibrillar inclusions in neurodegenerative and other types of diseases. Data from [2,100]. TSE, transmissible spongiform encephalopathies. Disease Protein component Cellular inclusion Neurodegenerative Alzheimer’s sau, A42b peptide Neurofibrillary tangles Pick’s sau Pick bodies/cytoplasmic Progressive supranuclear palsy (PSP) sau, heat shock proteins Neurofibrillary tangles Dementia with Lewy bodies a-Synuclein Lewy bodies/cytoplasmic Parkinson’s a-Synuclein, crystallins Neurofilaments/cytoplasmic Huntington’s Expanded Glu repeats of Intranuclear inclusion huntingtin Spinocerebellar ataxias (SCA) Expanded Glu repeats of Intranuclear inclusion ataxins 1,3,7 TSE Prion protein, cathepsin B Endosome-like organelles System amyloidosis Diabetes type 2 Amylin Haemodialysis related A b-2 Microglobulin Reactive amyloidosis Amyloid A Cystic fibrosis CFTR protein Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. Biochem. 269) 3363 and subsequent protein fibril formation [29,48]. With unfolded polypeptides, partial folding can be obtained by lowering pH or by heating [39]. In vivo, partial unfolding may happen as a consequence of lowered protein stability due to mutation, local change in pH at membranes, oxidative and heat stress, whereas partial folding may happen on exposure to environmental hydrophobic sub- stances, such as pesticides [39]. AMYLOIDOGENIC CONFORMATION ANDCOMMONSTRUCTURAL TRAITS OF THE FIBRILS Amyloid fibrils cannnot be observed in solution whereas the preamyloidogenic conformation can be trapped in crystal- line or soluble form. NMR data exist on the native-like acid intermediate of transthyretin [49] where the authors have used hydrogen exchange in conjunction with NMR to trace structural features of the preamyloidogenic conformation. Similarly, by using hydrogen exchange in the native state, the labile parts of the prion peptide have been determined [50]. NMR has been used in combination with electrospray ionization mass spectrometry (ESI MS) to enable the population of the intermediate to be seen [51]. Recently, crystal structures of the domain-swapped dimers of human cystatin C [52] and prion peptide [53] have been determined. A solution structure of human stefin A (type I cystatin) dimer is also available [54] and confirms the main features observedincrystalstructureofcystatinC. Ordered fibrillar aggregates and the amyloid-fibrils themselves can be studied at lower resolution by trans- mission electron microscopy, atomic force microscopy (AFM) [55,56], cryo-electron mycroscopy [57], X-ray diffraction [58] and solid state NMR [59]. The fibrils appear long, of indefinite length, unbranched, with repeats that reflect the twisting of the component ÔfilamentsÕ around one another [60–62]. Common features of the fibrils are [58], b strands (separated by 4.7 A ˚ ) running perpendicular to the long axis of the fibrils and b sheets extending parallel to this axis. The bstrands form a b helical twist with the usual repeat at every 115 or 250 A ˚ [56,58]. There are two main types of the fibrils, type 2 fibrils are built from two intertwined filaments, with a diameter from 80 to 130 A ˚ . Type 1 fibrils are thinner and are formed from one filament only. There are other types of fibrils [62]; for example, a fibril and untwisted filaments of human stefin B [31] (type I cystatin) are illustrated in Fig. 1. ENERGETIC AND KINETIC BASIS OF FIBRILLOGENESIS The molecular and energetic basis of protein misfolding and amyloid fibrillogenesis is still largely unknown [20,63]. In the conclusion to their review, Rochet & Lansbury [35] propose that future research should be directed towards understand- ing the mechanism of amyloid-fibril formation, including environmental factors, such as temperature, ionic strength, pH and oxidation potential. Proteins have been treated as an ensemble of rapidly interconverting conformational substates. In contrast, recent studies have shown that interconversion between different conformations may be slow (taking hours to days). For certain proteins the folding appears to be determined by kinetic rather than thermody- namic factors [64]. The free-energy barriers can be quite high [64–66], leading to persistence of parallel states, which possibly exhibit different biological functions. The forces involved are nonspecific, e.g. hydrophobic and repulsive electrostatic, and specific, e.g. hydrogen bonding and salt- bridges. As cooling causes reversible disaggregation of Ab fibrils, a significant contribution to stability must come from entropy-driven hydrophobic interactions. This led to trials of various hydrophobic compounds that should be effective in destabilizing and disaggregating amyloid fibrils. Fig. 1. Transmission electron micrographs. (A) Amyloid fibrils of human recombinant stefin B (cystatin B) prepared in vitro at pH 4.8, showing a b helical repeat. (B) Porous fibrillar aggregate and fine structure of a fibril (made from four filaments) resulting from the addition of trifluoroethanol. 3364 E. Z ˇ erovnik (Eur. J. Biochem. 269) Ó FEBS 2002 Despite enormous efforts, description of the process of fibrillogenesis is only qualitative at the moment. Various morphologic species are described in the literature on protein fibril formation. Fibrillogenesis often starts with dimers as initial building blocks [3,56]. These further oligomerize to tetramers, octamers, etc. The oligomeric species constitute Ôprefibrillar aggregatesÕ composed of fluid (micelle-like) nuclei [67]. From these, the ÔprotofibrilsÕ grow up to 200 nm in length and are slightly curved [67,68]. All these species accumulate in the so-called Ôlag-phaseÕ char- acteristic for the kinetics of fibril growth. The lag-phase ends with an exponential growth when proto-fibrils merge into ÔfilamentsÕ. Fully grown fibrils are then made from one or more filaments added laterally or, end by end [69]. The events in the lag-phase are especially important and some results have been obtained by real time AFM [70,71]. Presence of prefibrillar (oligomeric) intermediates is an emerging theme [68,72]. The kinetics of fibrillogenesis have been studied by light scattering [67,72]. Teplow and coauthors [67] have detec- ted the following steps: (a) peptide micelles form above a certain critical concentration, (b) fibrils nucleate within these micelles or on heterogenous nuclei (seeds), and (c) fibrils grow by irreversible binding of monomers to the fibril ends. Simpler, colorimetric methods exist for detect- ing amyloid fibrils. Use of histological dyes Congo Red [73] and Thioflavin T [74] is widespread. In fact, both dyes may actually label the filaments better than the fibrils (E. Z ˇ erovnik, unpublished observation). Thioflavin T fluorescence is a suitable method to follow the kinetics of fibril formation in an interrupted manner, whereas interference with the process on longer standing would be expected. Whether Congo Red is fibril specific has been questioned [75]. Substances based on Congo Red dye structure have been used to inhibit fibril formation in vivo [76] and others based on Thioflavin dye structure to label the amyloid plaques in brain imaging [77]. Teplow and coworkers [40] have recently reported that an intermediate with additional a helix structure was shown to be a key step in Ab fibrillogenesis. The a helical content (as revealed by CD) was observed immediately prior to the appearance of b structure, suggesting a precursor role for the intermediate. It was not until a helix formation had begun that fibrils were detected by electron microscopy. The occurrence of an a helical intermediate that associates into oligomers is not limited to Ab peptide. It has also been observed in insulin [41] and helix-turn-helix peptide [42]. The a helical intermediate is reminiscent of several cases reported in the field of protein folding [43–46]. The same authors [40] have studied the effect of various substitutions ontherateofa helical appearance. To test the hypothesis that aspartic acid and histidine residues control the kinetics of a helix formation, mutations were made in Ab peptide where Asp and His were replaced by neutral residues. Specific influence of Asp23 and His13 was observed. Substitution of His13 by Ala dramatically inhibited fibril formation and altered fibril morphology. Similarly, substi- tution of Asp23 by Asn delayed a helix formation and fibril formation. This was explained with salt-bridges, which form in pH range from 4 to 5.5, where Asp is negatively and His positively charged. A mechanism for amyloid fibril formation was proposed by Massi & Straub [78] based on the energy landscape description. The authors predict that temperature and denaturants would initially increase the rate of fibril elongation with a turnover at higher temperatures or denaturant concentrations. In his study, Friedhoff [9] has shown that polyanions stimulate filament growth whereas phosphorylation retards growth. In a study based on statistical mechanics by Aggeli et al. [69], the kinetics of fibril-growth of two rationally designed peptides have been compared. One peptide was made more hydrophobic by replacing Glu by Phe and Trp residues. At 100 l M concentration this peptide formed b sheet ribbons and at a concentration of > 600 l M the ribbons were transformed into rigid fibrils. Due to the balance of weak forces, fibril and fibre formation is characterized by slow kinetics. In the particular case [69], fibril formation takes up to several weeks to complete, as monitored by CD and TEM. Serio et al. [79] have studied the yeast prion, sup 35. Detailed kinetics showed that seeding accelerated the fibril growth while, with no seeds present, a lag phase was observed. During this phase, smaller fibrils (seeds) form that allow rapid assembly. The lag time should decrease exponentially with increasing soluble protein concentration if the nucleated polymerization model were applicable, which was not the case. They have therefore proposed a new model, termed the nucleated conformational conversion (NCC) model, which states that oligomers lacking a conformation leading to fibril formation accumulate and associate with the nuclei where conformational conversion takes place as a rate-determining step. Several other mechanistic models, in addition to the NCC model, have been proposed: the monomer-derived conver- sion (MDC) model [60], which is similar to the template assisted (TA) model [24,60], the nucleated polymerization (NP) model proposed by Teplow and coauthors [67,72], and, lately, a mathematical model by Pallito & Murphy [80], which is termed here the off-pathway folding (OFF) model. It is difficult to judge which of the models best describes a Ôgeneral processÕ of amyloid fibril formation. It may even be, similarly to protein folding, that several mechanisms apply to different specific cases. More studies of the influence of protein concentration, temperature and seeding on the rate of amyloid fibril formation are needed. A description of the two most recent models follows. Nucleated conformational conversion (NCC) model This model states that oligomers lacking a fibril-competent conformation accumulate and associate into a ÔnucleusÕ where conformational conversion takes place as a rate- determining step. Fluid oligomeric complexes appear to be crucial intermediates in forming the amyloid nucleus. When these complexes undergo a conformational change on association with the nuclei, rapid assembly follows [79]. Off-pathway folding (OFF) model In the initial refolding step, an amyloidogenic intermediate, I, forms (A-state) in a parallel reaction [80]. The step is practically irreversible, in contrast to the normal folding phase where monomer (M) and dimer (D) are in equilib- rium (equivalent to S-state). Nucleus formation follows the initial partitioning of Ôfibril competentÕ and noncompetent Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. Biochem. 269) 3365 conformations. Filament formation then takes place, followed by filament elongation by end-to-end addition of the intermediate I (equivalent to A-state). Fibrils form by lateral and end-to-end association of the filaments [80]. We believe that it would be possible to include irreversible domain-swapped dimers (A-state dimers) in the model, inplace of monomeric I. ROLE OF DOMAIN SWAPPING IN FIBRILLOGENESIS It is to be noted that several amyloidogenic proteins form domain swapped-dimers. Such is the case with prion protein [53], human cystatin C [36,52] and human stefin A [54,82], a type 1 cystatin. It remains to be seen if these irreversible transitions, due to high energetic barriers [81,82], have relevance to amyloidogenesis. Eisenberg and coworkers have proposed a method by whichdomain-swappeddimerscouldleadtohigher oligomerization and amyloid fibrillization [30,81]. If the exchange of secondary structure elements is not recipro- cated but propagated along multiple polypeptide chains, higher order assemblies may form. In principle, any protein is capable of oligomerization by 3D domain-swapping [83]. By designing an a helical structure that could domain swap, Eisenberg et al. [84] have shown that it was possible to design a sequence that permits a reciprocated swap and another that promotes a propagated swap. Indeed, domain- swapped dimer and fibrils resulted, as expected. An interesting observation was also made with ribonuclease where pair of domain-swapped structures involving N- and C-terminal parts can coexist. This suggests another possible mechanism for propagated domain swapping [30]. Staniforth et al. [54] discuss ways in which the domain- swapped dimer of cystatin could propagate into a fibrillar structure. It is assumed that open ends on the N- and C-termini would allow further interactions. The electronic density of a Ôgeneric fibrilÕ could be fitted by two rows of dimers, each row extending in both directions indefinitely (Fig. 2). Janowski et al. who determined the crystal struc- ture of human cystatin C domain-swapped dimer [52], believe that the dimers most probably represent a Ôdead endÕ to further amyloidogenesis or, at least, hinder the process. If domain-swapped dimers were rate-limiting for fibril formation, a high energetic barrier would be expected; this could be deduced from the influence of temperature on the process. In the case of plant monnelin, a structurally analogous protein to cystatins, the authors [85] did not look for existence of the dimer. It has been found that heating was needed for the prenucleus stage of fibril growth and that maturation of the nucleus proceeded at lower temperature. A similar observation has been made with human stefin A (cystatin A), which demonstrates a high activation energy of 99 kcalÆmol )1 for domain swapping [82] and forms dimers when heated to 85 °C for  1 h. A preheated sample can make fibrils at ambient temperature if the structure is additionally destabilized by lowering pH to 2.4 (E. Z ˇ erovnik, unpublished observa- tion). More importantly, the disease-causing variant of human cystatin C (L68Q) forms dimers under physiolo- gical conditions [36]. It has been suggested by Bergdoll et al.[86]and confirmed by Itzhaki and coauthors [87] that a proline in the linker region might facilitate domain swap. It could rigidify the hinge region and keep it extended [83]. Parallel reactions in folding have largely been attributed to the difference in peptide bond configuration at some critical proline [88] in the denatured state ensemble. This option, too, should be considered in searching for an explanation for slow formation of domain-swapped dimers and fibrils. The energy of activation determined for the lag and growth phases in a-synuclein fibrillization [39] was  20 kcalÆmol )1 , which would be consistent with a proline isomerization reaction. Of course, there may be other slow events with high activation energy. It has been found that a slow rate of unfolding (a high E barrier) prevents amyloid fibril forma- tion [89] and that fast unfolding leads to increased rate of fibrillization. CONNECTION OF PROTEIN FIBRIL FORMATION TO PATHOPHYSIOLOGY AND DISEASE So far, about 20 human proteins have been found in proteinaceous deposits in various conformational diseases. These do not demonstrate any sequence or structural homology. The common event is thought to be a conformational change, leading to lack of biological function or gain of toxic activity, and possibly, formation of amyloid fibrils. It is a matter of debate as to whether the fibrillar aggregates and amyloid plaques are the side-product of some other pathology or whether they are the main cause of the disease. Co-localization of protein aggregates with degenerating tissue and association of their presence with disease symptoms are a strong indication of the involvement of amyloid deposition in the pathogenesis of conformation- al diseases [20]. In familial cases of some neurodegenerative diseases (Table 1), evidence has been obtained for a direct link between the ability of mutated protein to form fibrils and the appearance of signs of the pathology [2,90]. Studies with transgenic animals have also confirmed the contribu- tion of the mutation in the amyloidogenic protein and disease pathogenesis [20,91,92]. Whether the fibrils or the prefibrillar aggregates are the dangerous species for the cell metabolism is still disputed. In animal studies it has been shown that significant tissue damage and clinical symptoms appear before any protein aggregates are detected, implicating an intermediate on the amyloidogenic pathway, which could be the real cause of the pathogenesis [3,4,6,7]. It was proposed that protein aggregation into fibrils could even represent a protective event that depletes the cell of the toxic prefibrillar species [3]. Careful usage of fibril inhibitors is indicated as they may cause accumulation of the toxic precursor [68]. Evidence has been obtained in studies on Alzheimer’s disease that fibrils are not the most neurotoxic form of Ab [6]. The peptide also assembles into soluble proto-fibrils and smaller oligomers. The proto-fibril of Ab was shown by AFM to be a slightly curved, of 4–11 nm diameter and < 200 nm long [56]. Isolated protofibrils were found to be toxic, causing oxidative stress and, eventually, neural death [72,93]. The smaller oligomers can interfere with signal transduction, possibly binding a tyrosine kinase important for memory formation (long-term synaptic potentiation) and sau phosphorylation [6]. 3366 E. Z ˇ erovnik (Eur. J. Biochem. 269) Ó FEBS 2002 In prion diseases [20,24,94], no abundant amyloid deposition was found in the brain, even though PrP Sc (the disease-related conformer of the protein) has a strong tendency to aggregate in vitro. An interesting observation was made that PrP C (the normal, cellular protein) binds to survival factors and that the PrP C to PrP Sc transition might result in apoptotic cell death. In Huntington’s disease, activation of microglia following disruption of neuronal architecture may be the death trigger rather than the apoptotic pathway [91]. This is consistent with findings in a transgenic mouse model of Huntington’s disease, where cell death was neither apoptotic nor necrotic [92]. MEANS OF NATURAL DEFENCE AND REGULATION Cellular defence against unfolded and aberrantly folded proteins consists of several protective systems that prevent aggregation, refold unfolded proteins or, degrade them. If the rate of damage to cellular proteins is increased, for example on exposure to increased temperature, oxygen free radicals or other stress conditions, or when mutations occur, this can disturb normal cellular functions and trigger apoptosis [95]. In such harsh conditions, cells respond by the induction of heat shock proteins (Hsp) that comprise chaperones, antioxidant enzymes and ubiquitin–protea- some components. The largest group of heat shock proteins act as chaperones that bind to denatured or partially folded proteins [96–98]. Certain combinations of chaperones, in particular Hsp70, Hsp104 and Hsp40, can serve to dis- assemble intracellular protein aggregates [99]. Especially, Hsp104 was found to be of importance for disassembly– disaggregation [97,100]. The ubiquitin- and proteasome-mediated degradation of proteins plays an important role in cellular quality control by removing mutated, misfolded and post-translationally damaged proteins [20,100]. In many cellular inclusions ubiquinated proteins are found together with proteasome components [100]. If the cell is still overburdened by aggregated proteins, apoptosis programs are switched on. A novel finding is that heat shock proteins have a dual function. As well as a role in refolding aberrantly folded proteins and keeping them from aggregation, a second function involves regulation of apoptosis [95,101]. Among the heat shock proteins are anti-apoptotic and pro- apoptotic proteins [101]. The recently discovered BAG family of proteins operate as molecules that recruit chaperones to target proteins. Such diverse proteins as Bcl2, Raf1, various receptor, transcription factor mole- cules and Hsp70 compete for binding to members of the BAG-family of proteins [102]. This binding induces changes in protein conformation that may have a profound effect on protein function. Unfortunately, studying the conformational changes in proteins in vivo remainsratherelusive. NOVEL THERAPEUTIC APPROACHES Novel therapeutic approaches are being directed towards achieving one of the following goals: either to inhibit and/or reverse the conformational change, or to dissolve the smaller aggregates and disassemble the amyloid fibrils. Several successful attempts have been cited in the literature including the use of monoclonal antibodies that bind to the active conformation of the protein and thus inhibit conformational changes. In Alzheimer’s disease, vacci- nation is on the horizon, in this case targeting the smaller oligomers and prefibrillar aggregates [103]. Soto and coworkers have designed the so called Ômini-chaperonesÕ, also termed Ôb sheet breakersÕ [20,24], which are peptides that bind to the sequence of the protein region responsible for self association. In the prion disease, similarly to Alzheimer’s, trials are underway using monoclonal anti- bodies that prevent conformational change [104]. Some drugs already in use for other purposes have been screened and several were found that both retard or reverse neuro- degeneration if used for early intervention and also improve the disease state in quite desperate cases, as reported by the Prusiner’s group [105]. One of these drugs, quinacrine, is an anti-malarial agent and the other, chlorpromazine, is used to treat schizophrenia. Other blockers of amyloid fibril formation have been found, ranging from Congo Red derivatives, anti-cancer and antibiotic drugs to nicotine and melatonin [76]. CONCLUSIONS Understanding amyloid-fibril formation may contribute to resolving some of the today’s most devastating diseases and, at the very least, increase our general knowledge about protein structure, folding and stability. Many properties of amyloid fibrils have emerged: a common structure for filaments and fibrils [58], nucleation dependent kinetics [67], the role of oligomeric intermediates [68,72] and the existence of at least two protein conformations separated by a high energetic barrier, which behave as two macroscopic states [64,81]. The following are some of the challenges still facing us: (a) Can domain-swapping be a mechanism for fibrilliza- tion of globular proteins? (b) What is the role of a helical parts of proteins? Do they remain helical in the fibrils? (Periodicity characteristic for a helices has been observed in an X-ray diffraction study on the apolipoprotein A1 variant [106]). (c) What is the role of a helical intermediates observed in folding [107] and fibrillization studies [40] where temporarily non-native a helices appear? ACKNOWLEDGEMENTS For financial support the author thanks the Ministry of Education, Science and Sport of the Republic of Slovenia. Professor R. H. Pain (JSI, Ljubljana, Slovenia) is indebted for reading the manuscript, giving useful comments and editing English. I also thank T. Zavas ˇ nik-Bergant (JSI, Ljubljana, Slovenia) and K. Goldie (EMBL, Heidelberg, Germany) for taking the TEM picture reproduced in Fig. 1. I am thankful to M. Ravnikar and M. Pompe-Novak (both National Institute of Biology, Ljubljana) and I. Mus ˇ evic and M. S ˇ karabot (Department of Physics, JSI, Ljubljana) for continuous TEM and AFM work on human stefins. My gratitude goes to Professor V. Turk and his team: L. Kroon-Z ˇ itko and M. Kenig (at JSI, Ljubljana), for preparing the recombinant stefins. The author additionally thanks J. P. Waltho for the model of cystatin A–stefin A dimer reproduced in Fig. 2B, and to R. A Staniforth (Krebs Institute, University of Sheffield, UK) for reading the manuscript and giving useful sugges- tions. Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. 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ARTICLE Amyloid-fibril formation Proposed mechanisms and relevance to conformational disease Eva Z ˇ erovnik Department of Biochemistry and Molecular Biology,. transformation of proteins into amyloid-fibrils is of interest, firstly, because it is closely connected to the so-called conformational diseases, many of which