[Frontiers in Bioscience 15, 373-396, January 1, 2010]
Methods and models in neurodegenerative and systemic protein aggregation diseases
Ann-Christin Brorsson1, Janet R. Kumita2, Ian MacLeod3,4, Benedetta Bolognesi2, Elena Speretta4, Leila M. Luheshi2,4, Tuomas P. J. Knowles5 David A. Lomas3, Christopher M. Dobson2, Damian C. Crowther3,4
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TABLE OF CONTENTS
1. ABSTRACT
Protein misfolding and aggregation are implicated in a wide range of increasingly prevalent human diseases ranging from dementia to diabetes. In this review we discuss the current experimental strategies that are being employed in the investigation of the pathogenesis of three important protein misfolding disorders. The first, Alzheimer's disease (AD), is the most prevalent neurodegenerative disease and is thought to be initiated by the aggregation of a natively unstructured peptide called amyloid beta (Abeta). We discuss methods for the characterization of the aggregation properties of Abeta in vitro and how the results of such experiments can be correlated with data from animal models of disease. We then consider another form of amyloidosis, where a systemic distribution of amyloid deposit is caused by aggregation and deposition of mutational variants of lysozyme. We describe how experiments in vitro, and more recently in vivo, have provided insights into the origins of this disease. Finally we outline the varied paradigms that have been employed in the study of the serpinopathies, and in particular, a dementia caused by neuroserpin polymerization.
2. INTRODUCTION
In the most highly developed countries the major challenges to human health over the coming decades will be disorders of affluence and old age, such as diabetes, Parkinson's disease and Alzheimer's disease. Indeed, as the population progressively ages we can reasonably expect a doubling in the prevalence of these common diseases by the middle of the century (1). While these diseases characteristically appear in the elderly, it now seems that they are not the inevitable consequence of "wear and tear", but instead they are the result of specific changes in the cellular metabolism of folded and unfolded proteins. To understand how best to intervene in disorders of protein misfolding and aggregation, it is vital that we investigate the molecular and cellular processes that result in disease, both at the molecular and at the organism level. In this chapter, we discuss some of the in vitro techniques and the cellular and animal modeling approaches that can be used to study the aggregation of a range of pathogenic proteins (reviewed in reference (2)). Some of these in vitro and in vivo assays, including their advantages and limitations, are listed in Tables 1a and 1b respectively. The specific proteins that we mention here are members of three important classes of disease-related proteins; first we will discuss the amyloid-beta peptide as a representative of a group of proteins and peptides that are natively unfolded, while in disease they form microscopic, cytotoxic amyloid deposits within tissues, often in the nervous system. Secondly, we will discuss lysozyme as a representative of a group of natively folded proteins, that when misfolded form macroscopic amyloid deposits throughout the body. Thirdly we review neuroserpin, a protein that retains a near-native fold even after forming aggregates in the endoplasmic reticulum of neurons.
3. THE BETA-AMYLOID PEPTIDE AND ALZHEIMER'S DISEASE
In Alzheimer's disease, as in the majority of protein misfolding disorders, the characteristic protein deposits are composed of amyloid fibrils (2). It is remarkable that a wide range of polypeptides are able to adopt this well-defined fibrillar structure, characterized by a cross beta-structure in which beta-strands are oriented perpendicular to the direction of the fibril axis, and by the ability to display red-green birefringence when stained with Congo Red (3-7). There are two types of deposits that are characteristic of AD, the first is extracellular neuritic plaques composed of Abeta peptides and the second is intracellular tangles that are comprised of the protein tau. The plaques are composed of a number of isoforms of the Abeta peptide, each of which has distinct C-termini resulting from variations in the site of the endoproteolytic cleavage that generates the peptide from the amyloid precursor protein (8). The two major Abeta isoforms present in the human brain are Abeta40 and Abeta42 and the ratio of these peptides is clinically important; specifically an increased ratio of the longer Abeta42 to Abeta40 correlates with an increased risk of subsequently developing AD. Despite being secreted at much lower concentrations than Abeta40, it is Abeta42 that is the predominant component of plaques and is believed to be critically important in AD (9, 10).
The clinical importance of AD has encouraged a great deal of interest in elucidating the mechanism of Abeta aggregation and neurotoxicity. Although the mature amyloid fibrils, which comprise the neuritic plaques in AD, have historically been considered the pathogenic species, more recent research has focused interest on the prefibrillar oligomeric Abeta assemblies that have the potential to cause neuronal failure in AD (11, 12). Abeta amyloid fibrils formed in vitro have been shown to exhibit the same morphological properties as ex vivo amyloid fibrils and are also toxic to neurons (13-16). Therefore, synthetic Abeta peptides are widely used for characterization of the Abeta aggregation mechanism and its pathological consequences.
3.1. Preparation and handling of the Abeta peptide
The Abeta peptide has proven difficult to handle due to its high propensity to aggregate and therefore, many technical barriers must be overcome in preparing solutions of the peptide to allow well defined and reproducible biophysical and biological behaviors. Abeta peptides can be produced either by solid-phase synthesis, a technique in which amino acids are successively coupled to a growing chain attached to a polymer support, or by recombinant protein expression methods (17-20). As the aggregation propensity differs between Abeta variants, each variant may require specific modifications to the production process, consequently leading to problems in comparing the behavior of different variants or isoforms of a particular peptide. Depending on the batch of peptide or the method of preparation, there are likely to be variation in the aggregation state of the initial material, and the aim is to achieve protocols that can minimize this source of experimental variability.
A wide range of methods have been employed to prepare monomeric peptide stock solutions, ranging from the use of high and low pH, a variety of organic solvents and different biophysical techniques such as centrifugation, sonication, size exclusion chromatography and filtration (21). It is important to use a preparation method that will give a reproducibly homogenous initial state of the peptide, and that is free from pre-formed aggregates but also maintains a high yield of material. Notably, even in an apparently aggregate-free Abeta peptide solution, prepared at micromolar concentration, there is an equilibrium between monomeric and oligomeric structures that is not well understood. These species are described in the literature as low molecular weight Abeta aggregates (21, 22). In our experience, no single method of peptide preparation yields material that is optimal for every biophysical technique; however, we describe here a range of preparation methods and their uses and limitations (summarized in Table 2).
Using high pH is advantageous for solubilizing Abeta peptides as the transition from basic to neutral conditions avoids passing through the isoelectric point of Abeta (pI ≈ 5.5) (23). However, high pH might not be sufficient to disaggregate fibrillar forms of Abeta and thus a second step, such as filtration or size exclusion chromatography, is often needed to produce high quality monomeric Abeta (21). Such an additional step will reduce the yield of peptide and may result in greater variability in the final concentration of the peptide solution, but is essential to obtain reproducible aggregation kinetics.
The use of hexafluoroisopropanol (HFIP) at low pH has also been shown to be successful in disrupting pre-formed Abeta fibrils (24). However, in our experience, synthetic Abeta peptides can not be efficiently solubilized using HFIP alone. Instead, we find the addition of trifluoroacetic acid followed by sonication and lyophilization is a good starting point for preparing synthetic Abeta peptides. After this step the peptides can readily be dissolved in HFIP, divided into appropriate aliquots and dried by rotary evaporation. The amount of peptide in each aliquot can then be determined by performing quantitative amino acid analysis at least on the first and the last aliquots to ensure that the peptide concentration remains constant in each sample. Using this preparation method, the resulting Abeta40 peptide in solution gives rise to a typical "random coil" CD spectrum with a strong negative ellipticity reading at 200 nm (Figure 1A) (25, 26). Furthermore, only three bands, corresponding to monomer, dimer and trimer are visible in western blots (Figure 1B). This finding is consistent with the observation of SDS-stable Abeta dimers in solutions of Abeta40 at nanomolar concentrations (27). Using this preparation method, we have been able to reproducibly characterize the aggregation behavior of a range of different Abeta peptide variants on a routine basis (28).
3.2. In vitro aggregation of the Abeta peptide
A number of spectroscopic methods including thioflavin T fluorescence (29-31), light scattering (32), circular dichroism (CD) spectroscopy (33), size exclusion chromatography (SEC) (34) and transmission electron microscopy (TEM) have been used, alone or in combination, to study fibril formation of Abeta peptides. Perhaps the most commonly used method is to follow the spectroscopic signal from thioflavin T, where a substantial increase in the fluorescence emission at 482 nm can be detected upon binding to fibrillar structures (35, 36). A typical thioflavin T fluorescence profile of Abeta aggregation is presented in Figure 2, in which Abeta40 displays a sigmoidal, characteristically bipartite aggregation profile consisting of a lag and growth phase (37). From this curve the rate of aggregation (k), which is a measure of the efficiency of the aggregation reaction, can be extracted from the maximal slope of the sigmoidal curve, or equivalently by fitting the growth phase to an exponential form: y = q + Ae (-kt) (38, 39). The length of the lag time (tl) can also be determined by fitting the initial data to a straight line a and a tangent b to the steepest region of the growth curve; tl is defined as the time point where the lines a and b intersect (Figure 2). The lag time is often considered to be the time required to form growth nuclei, and it has been assumed that its duration is determined only by the rate of nucleation events. However, recent measurements show that lag phases can occur even under conditions where nucleation is negligible and that signs of amyloid fibril growth can be detected during the lag phase (40). Therefore, it is likely that the lag time contains contributions both from the growth of aggregates, as well as from the de novo formation of new aggregates from soluble protein through nucleation.
Much interest has focused on identifying the elementary steps which underlie amyloid growth, by extracting information from bulk kinetic measurements and observing how changes in the microscopic rate constants affect the overall characteristics of the sigmoidal growth kinetics. It has become apparent that in addition to the processes of nucleation and elongation, fragmentation of existing fibrils is an essential factor for understanding amyloid growth (41-43). Indeed, as spontaneous nucleation is inherently a slow higher order reaction, the formation of new fibrils through fragmentation of existing structures could, in many cases, be a more effective source of new fibrils, the combination of elongation and fragmentation leading to exponential growth of the fibril population (43). For prions, it has been established that the most aggressive strains are those composed of fibrils with the greatest propensity to fragment and proliferate (44, 45); the finding that the Abeta peptide can also exist in different strain-analogous configurations in vitro and in vivo raises the question of whether fragmentation could have a similarly central role for the understanding of the aggregation of Abeta peptide and the different activities of the structures resulting from this process (46, 47). In this context, it is interesting to speculate that the less robust and less rigid nature of protofibrillar species, that precede the formation of fully formed amyloid fibrils, may allow them to fragment, proliferate and interact with cellular components more effectively than fully formed fibrils, thereby leading to increased pathogenicity (48).
The characteristics of a typical Abeta aggregation profile, probed by thioflavin T fluorescence, are strongly dependent on the design of the experiment and on the handling of the Abeta sample. In situ measurement of thioflavin T fluorescence has the advantage of leaving the Abeta sample undisturbed in the fluorometer throughout each experiment. But, the presence of thioflavin T in the reaction mixture may interfere with the aggregation process. In contrast, ex situ assays measure thioflavin T binding to aggregates in aliquots that are taken from a stock solution of polypeptide. Although ex situ assays avoid dye artifacts, the repeated sampling may in turn disturb the aggregation process. Another consideration when interpreting thioflavin T fluorescence data is that the intensity of the signal at the end of the aggregation process varies greatly between different Abeta aggregates, likely reflecting variations in the binding of thioflavin T to aggregates with different morphologies. For example, despite differing by only two C-terminal residues, the Abeta40 and Abeta42 peptides display profound differences in their aggregation behavior. The Abeta42 peptide has a higher propensity to form protofibrils than Abeta40 and while the end stage aggregation products of Abeta40 are usually long well-ordered fibrils (Figure 3A), the Abeta42 fibrils are commonly accompanied by smaller aggregated species (Figure 3B). In our experience, the aggregation products of Abeta40 give a higher thioflavin T fluorescence signal than aggregates of Abeta42, highlighting the differences in the respective aggregated structures and probably reflecting an enhanced ability of Abeta40 aggregates to bind to thioflavin T as compared to Abeta42.
Circular dichroism spectroscopy has been used in several studies to define the conformational states of the Abeta peptide and to monitor transitions in secondary structure from random coil to beta-sheet (49-52). A typical spectrum recorded for a beta-rich amyloid structure is shown in Figure 3C, displaying a minimum at 219 nm and a maximum at 196 nm. CD spectroscopy has the advantage that measurements of secondary structure during the aggregation process can be made without the need for reporter dyes such as thioflavin T. In general agreement with the thioflavin T measurements, kinetics of Abeta aggregation recorded by CD spectroscopy typically show a characteristic three-step sigmoid profile comprised of a lag phase, an exponential growth phase and a plateau phase (53-55). The CD approach has also been found to be useful for measuring the effects of inhibitors of Abeta aggregation (52).
Size exclusion chromatography is a technique that allows the separation of aggregates of different size and shape and has been used to isolate and characterize specific aggregated species such as in vitro generated Abeta-derived diffusible ligands (soluble oligomeric forms of Abeta, abbreviated ADDLs) which have been shown to be neurotoxic (56-57). This technique has also be used to monitor the transition of monomeric peptide into protofibrils and then subsequently into amyloid fibrils (58). However, during the chromatography step, which can take up to an hour to complete, the equilibrium between aggregated species will be disturbed and thus the final chromatogram might not reflect the true composition of aggregates in the reaction mixture.
Dynamic light scattering can provide information about the size distribution profile of smaller oligomeric assemblies. For example, in a detailed study using a range of biophysical techniques including photo-induced cross-linking of unmodified proteins and size exclusion chromatography in addition to dynamic light scattering, it was possible to show that while the Abeta40 peptide exists in an equilibrium between monomeric, dimeric, trimeric and tetrameric forms, the Abeta42 peptide preferentially forms pentameric or hexameric units (paranuclei) which seem to be the initial and minimal structural elements from which assembly of Abeta into fibrils takes place (32).
The increasing evidence that it is oligomeric, rather than fibrillar, aggregates of Abeta that cause toxicity in AD has prompted much interest in the visualization of these smaller aggregates. We have found that a combination of negative stained TEM imaging and atomic force microscopy (AFM) is optimal for this purpose; whereas AFM gives three-dimensional information about morphology at the nanometer scale and can be performed under physiological conditions (48), the electron microscope provides convenient data regarding the length and the width of the Abeta aggregates. Indeed, using such an approach, it has been found that the mutation E22G of Abeta42, which causes familial, early onset AD, not only enhances the process of fibril formation but also accelerates the formation of oligomeric intermediate species (58, 59). Despite the transient nature of these neurotoxic aggregates (reviewed in reference (60)), these techniques show them to have a range of appearances including spheres, "beads on a string" and annular oligomers, where the latter have been suggested to have a potential role as specific membrane disrupting pores. A general scheme for Abeta aggregation is presented in Figure 4 where soluble Abeta monomers are converted to mature fibrils.
Structural studies of Abeta40 and Abeta42 fibrils, based on nuclear magnetic resonance and electron microscopy techniques, have revealed a protofilament, defined as the minimal fibrillar unit that is observed in the experiments, which is characterized by two stacked, in-register parallel beta-sheets that propagate along the fibril axis resulting in two-fold symmetry around the fibril growth axis (13, 14). The fibrillar core of Abeta40 involves residues 12-24 (beta1) and residues 30-40 (beta2). In comparison, the fibrillar core of Abeta42 was found to be slightly shifted towards the C-terminus, with beta1 and beta2 composed of residues 18-26 and residues 31-42 respectively. However, fibril morphology of the Abeta peptide is by no means homogeneous in nature. Recently, structural studies of Abeta40 fibrils, with a characteristic periodically-twisted appearance observed by TEM, revealed a protofilament constructed of three cross-beta units that result in three-fold symmetry around the fibril growth axis (61). This implies that differences in the molecular structures of the fibrils underlie the diverse morphological nature of amyloid fibrils.
3.3. Abeta pathogenicity
To address the need to link the well-characterized biophysical processes of peptide aggregation to the biological pathways that mediate the neuronal dysfunction and death that characterize AD, a range of cell culture and animal models of AD have been established, each system having particular strengths and limitations.
Cell cultures are frequently used to examine the proteolytic processing of the amyloid precursor protein (APP), which generates the Abeta peptide (62-64). In particular, there has recently been great interest in determining the composition and activities of the proteolytic complexes that process APP. These activities named alpha, beta and gamma secretase, are of pharmaceutical interest because of their upstream location in the pathogenic cascade. Determining the proteolytic mechanisms of the secretases and their sub-cellular sites of action has been a particular achievement of the past five years. Building on this knowledge, Andersen and colleagues have shown that the subcellular trafficking of APP determines whether or not the protein comes into contact with the secretase machinery. In particular, a protein called SORL1 have been found to influence the risk of developing AD by determining whether APP is recycled to the plasma membrane after endocytosis or, alternatively, enters the amyloidogenic processing pathway (65-67). There are many other applications of cell biological models of AD, but crucially none give a robust model of Abeta toxicity. Although the generation of Abeta or Abeta-oligomers is taken as a surrogate endpoint for toxicity, specific data on Abeta toxicity can be obtained by the use of cell cultures, in which Abeta peptide is added, in relatively high concentrations, to the medium and then impairment in cellular function and viability is measured. An alternative and complimentary approach is to create whole-animal models of AD.
Traditionally, animal models of AD have been murine; indeed the best accepted models are exemplified by the triple transgenic mouse of LaFerla (68, 69). These mice express disease-associated variants of human APP, presenilin-1 and tau, and faithfully replicate many of the features of AD, including initial intracellular accumulation of Abeta, accompanying cognitive impairment and subsequent appearance of plaques and tau pathology. This model also exhibits neuronal death - a feature present in AD, but absent from many other mouse model systems.
Despite the dominance of murine models of AD, it is clear that the power and speed of the techniques that can be applied to invertebrate systems have provided an impetus to perform C. elegans and Drosophila studies. Link and colleagues have created a C. elegans model of AD based on the secretion of Abeta from cells as a transgenic peptide attached to a secretion signal peptide (70). While Abeta accumulates within the muscle cells of the worm in this model, rather than reaching the extracellular compartment, it remains very useful as it develops a clear phenotype characterized by progressive paralysis. This has been successfully exploited by Dillin and colleagues to dissect the complementary contributions of the heat shock and insulin growth factor-1 like signaling pathways to protection against toxicity and to observe how this balance changes with aging (71).
In many ways the use of worm and fly models is complementary; however Drosophila models of AD have some advantages, in particular the fly has a complex brain and hence the complexities of the phenotypes that may be assessed are correspondingly greater (Figure 5). For example the Abeta-expressing fly models allows disease-relevant phenotypes such as learning, memory (72), habituation (73, 74) and locomotor function to be explored in the context of progressive histological disruption (75, 76). It is likely that the quantitative analysis of complex behavioral phenotypes such as these will provide markers of pathological progression in the fly that resemble the clinical features of patients with AD. Furthermore, the short lifespan of the fly allows us to look at phenotype progression within days rather than months or years (life spans typical of rodent models) and this, combined with the potential sensitivity of behavioral phenotypes, will allow the rapid generation of data. In particular we find reductions in longevity and climbing ability to be strong indicators of Abeta toxicity. It is of particular interest that functional phenotypes, such as locomotor impairment, are seen early in the life span of the flies, when only intracellular accumulation of Abeta is apparent, and in the absence of mature amyloid (75). Interestingly, biochemical tests on the brains of AD flies demonstrates the presence of SDS-resistant oligomers of Abeta, mirroring similar findings from examination of the human cerebral cortex and CSF (77, 78) and also from studies of the conditioned medium of Chinese hamster ovary cells that have been genetically modified to produce human APP variants (79, 80).
The powerful genetic tools that are available for the fly also allow us to dissect the biological pathways that mediate the toxic response of neurons to Abeta aggregates. By randomly manipulating the expression level of genes in the fly genome we can assess the effects of these genes on the sensitivity of the fly to Abeta toxicity. Cao and colleagues have reported a genetic screen in which a wide range of genes are implicated in the pathogenesis of the AD-related phenotypes, including those associated with secretion, cholesterol homeostasis, and regulation of chromatin structure and function (81). We have also performed genetic screens that have revealed novel modifiers of Abeta toxicity, allowing us to dissect pathways of toxicity in molecular detail.
We can also combine biophysical studies with Drosophila modeling to assess the relative pathogenic contributions of the intrinsic aggregation properties of the Abeta peptide and the cellular factors present in the in vivo environment (82). Specifically, by performing mutagenic analysis of Abeta behavior in vitro and in vivo, guided by in silico predictions of Ab aggregation propensity, it is possible to link the generation of particular aggregates with in vivo toxicity. Indeed we have found a strong correlation between the propensity of the Abeta42 peptide to form protofibrils and neuronal dysfunction in a Drosophila model of AD (28). This observation supports the notion that it is the early-formed Abeta aggregates that have potent neurotoxic activity and that these species may be the key effectors of neurotoxicity in AD (83). In fact, there is evidence that the cognitive features of early AD, as well as synaptic loss, correlate better with the amount of soluble Abeta aggregates than with the appearance of insoluble plaques in the brains of AD patients and in mouse models (84, 85).
4. LYSOZYME AND SYSTEMIC AMYLOIDOSIS
In the 1960's, lysozyme was the first enzyme to have its X-ray crystallographic structure determined (86). Since then lysozyme has been extensively studied, using a wide variety of biophysical techniques, with the aim of understanding many features of its behavior including the principles that govern both normal protein folding and pathological misfolding and aggregation (for a more detailed review see (87) and references therein). In the early 1990s, it was found that mutations in the human lysozyme gene cause hereditary non-neuropathic systemic amyloidosis (88). Since the first reported case, four single amino acid mutations (I56T, F57I, W64R, D67H) and two double mutations (F57I/T70N, T70N/W112R) have been linked with this rare autosomal-dominant disease (Figure 6) (89-91). In common with Alzheimer's disease, all these cases of familial disease appear to be characterized by amyloid deposits but, in contrast to AD, the pathogenicity of systemic disease appears to be primarily mediated by the disruption of organ function caused by the bulk accumulation of amyloid fibrils (7, 92). Detailed investigation of patients carrying the D67H lysozyme variant has demonstrated that the deposits are composed solely of the full-length, variant protein (92). Circulating levels of lysozyme have been measured in the plasma and urine of a small number of patients and found to be within the normal range but, interestingly, patients possessing the W64R mutation have been reported to have only wild-type (WT) lysozyme in their plasma and urine and no detectable levels of the W64R protein (based on analysis by mass spectrometry) (89).
4.1. In vitro characterization of lysozyme fibril formation
Variants of human lysozyme (I56T and D67H) have been shown to convert in vitro from normally soluble and functional proteins into intractable fibrillar aggregates (92, 93). These fibrillar aggregates resemble those found in vivo and have been characterized by a wide variety of techniques including light-scattering, thioflavin T fluorescence spectroscopy, transmission electron microscopy (TEM), X-ray and electron fibre diffraction and Congo-Red staining (94, 95). The kinetics of in vitro aggregation show a clear lag phase, followed by a rapid exponential growth phase. It has been shown that the lag phase is however abolished, and the overall rates of fibril growth for I56T, D67H and WT lysozyme are greatly accelerated, by seeding with preformed fibrils (93, 96). These results are consistent with a general nucleation-dependent growth process typical of amyloid formation observed in other systems (37).
From detailed structural studies of the I56T and D67H variants, and to a lesser extent the F57I and W64R variants, it is believed that a decrease in native stability and global structural co-operativity (i.e. with respect to the co-operative two-state transition between the folded and unfolded states of WT lysozyme), leads to the population of a transient, partially unfolded species, and eventually to the formation of fibrils (97-99). This transient intermediate can be monitored by measuring the rates of solvent exchange of labile hydrogens in the protein by mass spectrometry under conditions where the exchange rates are fast compared to the local co-operative unfolding/folding process that generates the aggregation prone transient intermediate (i.e. EX1 conditions (100)). Under these conditions all labile hydrogens that are exposed will exchange each time a fluctuation occurs. Such behavior is usually indicative of complete global unfolding of the protein structure from the native state; however, a fluctuation resulting in a co-operative unfolding of a region of the protein molecule would result in a well-resolved multimodal distribution of masses (97). Under physiologically relevant conditions of pH and temperature, a bimodal distribution is observed for the I56T and D67H variants; in contrast, WT lysozyme and the naturally occurring, non-amyloidogenic variant T70N do not display such behavior that is indicative of a well-defined transient intermediate (97, 98, 101). This intermediate is remarkably similar for both the I56T and D67H variants and consists of an unfolded beta-domain and C-helix while the rest of the alpha-domain remains folded. Interestingly, it bears a close resemblance to the dominant intermediate detected during non-oxidative folding (97, 102); moreover, this intermediate species is not unique to amyloidogenic variants, and in fact can be populated under non-physiologically relevant conditions (i.e. at higher temperature) in WT lysozyme and the T70N variant (101). Limited proteolytic digestion of fibrils formed at low pH with the homologous hen egg white lysozyme (HEWL) has revealed that regions corresponding to the beta-domain and the C-helix are resistant to enzymatic digestion (103). These fragments rapidly formed amyloid fibrils after purification, suggesting that the region corresponds to a high intrinsic propensity to aggregate and are located in the fibrillar core. These characteristics strongly suggest that in vivo aggregation is closely coupled to the ability to readily form the intermediate species under physiological condition.
Based on the structural data, we can rationalize that, for the amyloidogenic variants, the mutations result in the destabilization of the interface between the alpha- and beta-domains, allowing a partial unfolding of the beta-domain and the C-helix and thereby exposing a substantial region of the protein that can, in some conditions, result in intermolecular interactions being favored over the intramolecular interactions characteristic of the native structure (87). In such circumstances, the process of aggregation can be initiated which ultimately leads to the formation of amyloid fibrils.
The ability to follow lysozyme fibril formation in vitro provides an excellent opportunity to investigate potential strategies to inhibit aggregation. Our first approach in this direction builds on our understanding that decreased native stability, along with lower co-operativity, results in enhanced amyloidogenicity and has involved the use of a camelid antibody fragment (cAb-HuL-6, raised against WT human lysozyme) that binds to and stabilizes the native protein (96, 98). The binding of cAb-HuL-6 to the native state of D67H or I56T variant lysozyme abolished the formation of the transient intermediate and resulted in the suppression of fibril formation. Structural studies of this complex revealed that the epitope on the lysozyme molecule does not include the sites of mutations or even the region prone to local unfolding (beta-domain). The antibody therefore appears to act by restoring global co-operativity to the native structure and thereby preventing destabilization of the interfacial region through long-range conformational effects. This study demonstrates the crucial nature of maintaining the global co-operative nature within globular proteins (e.g. by preventing the formation of unfolding intermediates) in order to resist aggregation; in this case of lysozyme this study therefore pinpoints the transient intermediate as a key species along the fibril forming pathway (Figure 7) (87).
4.2. Understanding lysozyme aggregation in vivo
In biological systems, the evolutionary design of the amino acid sequence of proteins along with other protective mechanisms such as quality control and degradation processes and the presence of molecular chaperones, have evolved to avoid aggregation under normal circumstances (104). The large majority of molecular chaperones are located in the intracellular environment; however, recently three extracellular chaperones, clusterin, haptoglobin (Hp) and alpha2-macroglobulin (alpha2M) have been identified and found to inhibit protein aggregation in vitro (105-107). Due to the extracellular nature of the lysozyme fibrils found in vivo, lysozyme represented a relevant target for investigating whether the extracellular chaperones could affect fibril formation.
All three proteins are secreted glycoproteins found circulating in blood and other bodily fluids of humans and other mammals. Clusterin, the most well characterized of the three chaperones, is translated as a 449 amino acid residue single polypeptide chain, which has a 22 residue secretory signal that is proteolytically cleaved after biosynthesis (108, 109). Post-translational modifications include N-linked glycosylation and proteolytic cleavage into an alpha- and beta-peptide that are concomitantly linked by five disulfide bonds to form a heterodimer. The precise physiological role of clusterin is unknown, although a diversity of functions has been suggested including its ability to act in a fashion similar to small heat-shock proteins in vitro (110, 111). Reports of in vitro inhibition of fibril formation by clusterin has been published for a number of peptides and proteins linked with aggregation diseases including the Abeta peptide, apo C-II, and the prion peptide 106-126 (105, 112, 113).
More recently, the chaperone-like ability of Hp and alpha2M has been reported (106, 107). Alpha2M is an abundant human blood glycoprotein, comprised of ~10% carbohydrate by mass. It is best known for its ability to inhibit a broad range of proteases, but has also been found to interact with a number of proteins and peptides involved with deposition diseases (114); indeed it has recently been shown that alpha2M can inhibit fibril formation of Abeta peptide as well as aggregation of a variety of other proteins (107, 115). Hp is produced mainly in the liver and is found in most bodily fluids of humans and other mammals. Like clusterin, levels of Hp in human plasma are increased during various physiological stresses (e.g. inflammation) (108, 116). Hp has been found to protect many different proteins from stress-induced amorphous precipitation and has been found to be associated with Abeta deposits in vivo (106, 117).
Our initial studies looked at the influence of substoichiometric amounts of clusterin on in vitro fibril formation by the amyloidogenic variant, I56T, as well as a non-amyloidogenic variant, I59T, which possesses many characteristics of the amyloidogenic variants. It was found that clusterin could potently inhibit lysozyme fibril formation even at molar ratios of clusterin-to-lysozyme of 1:80 (Figure 8) (99). It was further observed that clusterin did not detectably interact with the native or fibrillar states of lysozyme, nor did it influence the formation of the monomeric transient intermediate as observed by mass spectrometry. Moreover, addition of substoichiometric amounts of clusterin at the beginning of the aggregation reaction resulted in complete inhibition whereas addition at a later stage (mid-elongation) resulted in a much less effective inhibition. From this study, it appears that the clusterin interacts with oligomeric species that are present at low concentrations during the lag phase of the aggregation reaction and in this way it inhibits fibril formation by lysozyme in vitro, a result that strongly support the role of the transiently populated aggregation intermediates discussed above. These findings have been supported by investigations with other fibril forming proteins (113).
Recently we have also examined the effects of Hp and alpha2M on lysozyme fibril formation in vitro and have found that they too can inhibit the aggregation process (118). The ability of these extracellular chaperones to potently inhibit in vitro fibril formation by human lysozyme variants, as well as other disease related peptides and proteins, is of great significance as this suggests that in a biological system they could play a key role in restricting the potentially pathogenic effects of the aggregation of proteins that are secreted into the extracellular environment.
Until recently, attention has been focused on understanding the mechanism of in vitro fibril formation by the amyloidogenic variants of human lysozyme at a molecular level. However, a full appreciation of the origins of pathogenesis requires more complex model systems and to achieve this goal we have examined the behavior of amyloidogenic lysozyme variants in yeast (P. pastoris) and Drosophila ((119), manuscript in preparation). Our initial experiments have examined the levels of secretion of WT and disease-associated variants of lysozyme. Because the toxicity of lysozyme amyloidosis results from the effect of large, extracellular deposits containing full-length, protein, one might assume that the variant protein is capable of bypassing the normal quality control mechanisms, resulting in high level secretion of these proteins that subsequently deposits systemically. We have found, however, when expressing lysozyme in Pichia pastoris, the level of secreted protein is lower for destabilized variants of lysozyme (Figure 9) (119). In line with these data we have also found, by using Drosophila as a model organism of lysozyme amyloidosis, that the level of soluble lysozyme is much lower for the disease associated variants than for the WT protein (unpublished data). By using a Drosophila model it is possible to undertake tissue selective studies in an age dependent manner to examine how the onset and severity of the disease is affected by the complex interplay of various factors including reduced stability and increased aggregation propensity of the protein and the effectiveness of the quality control system in the cell.
The relationship between stability and secretion level observed in these in vivo models suggests that, in a normally functioning organism, a delicate balance exists between the efficiency of the secretory processes and the nature of the destabilization of the protein, caused by a mutation, that is crucial in determining whether or not specific variants are able to accumulate in the tissue. This suggests that other biological processes may contribute to the development and severity of this disease.
5. SERPINS AND FAMILIAL ENCEPHALOPATHY WITH NEUROSERPIN INCLUSION BODIES
The autosomal dominant dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), is caused by mutations in neuroserpin, a neuronal serine protease inhibitor (serpin) (120). FENIB results from the accumulation of neuroserpin polymers and is remarkable because of the strong genotype-phenotype correlation that is observed in various kindreds, whereby the mutations that most strongly promote neuroserpin aggregation have the most profound clinical sequelae (121). The handling and toxicity of neuroserpin polymers has been investigated using a range of methods both in vitro and in vivo using both cell and animal models of FENIB.
5.1. Serpins and the serpinopathies
The serpins are an abundant and widespread group of proteins characterized by greater than 30% amino acid sequence homology with the archetype alpha1-antitrypsin. Found in all major forms of life from viruses to humans, they fulfill a large number of diverse roles by utilizing a unique mechanism of protease inhibitory action (122, 123). The structure of the serpins is centered on three beta-sheets (A-C) and a flexible reactive centre loop (RCL) which is exposed at the top of the molecule (Figure 10A) (124). This reactive centre loop and the largest beta sheet, beta-sheet A, interact to snare and inactivate the target protease in a unique and dramatic mousetrap action (125). The reactive loop acts as a pseudo-substrate to which the target protease docks. The protease then cleaves the reactive centre loop which triggers a large molecular transformation in the serpin. The reactive centre loop is incorporated as an extra strand in beta-sheet A and swings the protease from one pole of the serpin to the other, irreversibly inactivating it and targeting it for degradation.
The unique mechanism of inhibition exhibited by the serpins is highly effective but the metastability of their native structures and the large molecular transformations that occur during protease inhibition leaves them vulnerable to mutations that destabilize the molecule and result in the formation of polymers. The accumulation of variant serpin polymers is associated with a group of diseases termed the serpinopathies (126). The serpinopathies exhibit two pathological mechanisms, firstly, the accumulation of serpin polymers within the organ of synthesis results in local damage and, secondly, there may be lack of circulating serpin (126). The clinical features of different serpinopathies depend on both the organ of serpin synthesis and its target tissue. For example, alpha1-antitrypsin is synthesized in the liver and inhibits neutrophil elastase in the lung (127). Mutations that cause alpha1-antitrypsin to polymerize result in the retention of the protein within the liver and hence, cirrhosis and emphysema of the lungs develop due to a lack of circulating alpha1-antitrypsin and unchecked protease activity (128).
Two mechanisms for the formation of serpin polymers have been proposed, and both models involve an expansion of beta-sheet A and a domain swap between serpin molecules into beta-sheet A. The first model for the formation of serpin polymers was proposed by Lomas and colleagues in 1992, and recently Huntington and co-workers have solved a new crystal structure suggesting that two beta-strands are involved in the domain swap (129-130). Vitally, both models describe polymers which are composed of correctly folded monomers in contrast to the protein aggregates of other conformational diseases in which the native structures of the individual monomers are partially or completely lost when they are assembled into amyloid fibrils (2).
Under certain circumstances, the RCL of a serpin molecule is incorporated into the centre of its main beta-sheet A, without cleavage of the loop. This results in a "latent" conformation of the protein which is more stable than the active conformation in which the RCL is solvent exposed at the top of the molecule (131-134). Mutations that destabilize this serpin architecture make the protein highly susceptible to intermolecular interactions, in which the RCL of one serpin monomer is incorporated into beta-sheet A of another serpin monomer resulting in chains of loop-beta-sheet polymers.
The molecular basis of the polymerization for the Z variant of alpha1-antitrypsin (E342K) has been studied in detail (128, 129, 135-138). The E342K mutation is located at the C-terminal end of beta-strand 5 of beta-sheet A and at the base of the RCL (Figure 10A). This mutation causes a conformational transition resulting in the formation of an unstable intermediate (denoted M*) where the RCL is only partially inserted into beta-sheet A. This event causes an expansion of beta-sheet A, thereby favoring the incorporation of the RCL of another molecule resulting in the formation of a loop-sheet dimer which extends to form longer chains of loop-sheet polymers (129, 136, 137, 139-142).
5.2. Neuroserpin and FENIB
Neuroserpin is expressed in both developing and adult brains in areas of synaptic plasticity and migrating neurons where it inhibits its target protease tissue-type plasminogen activator (tPA) (143-147). Through its interaction with tPA, and potentially through roles independent of tPA, neuroserpin is involved in modulating synaptogenesis and syntaptic plasticity, in memory, emotion, behavior and in protection from ischemic injury (148-152). However, in FENIB, mutations in neuroserpin (Figure 10B) cause it to gain a toxic function, the propensity to form polymers.
Mutations in neuroserpin cause the retention of neuroserpin polymers in neurons resulting in the autosomal dominant dementia FENIB (120). Immunohistochemical staining of brain sections reveals neuroserpin polymers in PAS-positive inclusions (known as Collins bodies) that accumulate in the deeper layers of the cortex and the substantia nigra (120). By using molecular modeling, the five mutations in neuroserpin, that have been linked with FENIB, are predicted to disrupt the structure of neuroserpin and, to differing extents, favor the opening of beta-sheet A and the consequent formation of loop-sheet polymers, in a similar fashion to the polymerization described for Z alpha1-antitrypsin (153). Strikingly, the predicted molecular instability for each neuroserpin variant correlates well with its in vitro polymerization rate, the number of inclusions formed in the brain, the clinical severity of the disease and a decreasing age of onset (121, 153, 154). Specifically, the S49P Syracuse mutation introduces the cyclic, uncharged residue proline beneath beta-sheet A, whilst the S52R Portland mutation introduces the more extended, charged residue arginine in this same region. Individuals carrying the S49P mutation develop dementia and tremors between the ages of 45 and 60 whilst the S52R mutation causes dementia, myoclonus and status epilepticus with onset during the third and fourth decades of life (120). These mutations affect residues beneath beta-sheet A, but a more severe form of the disease is caused by mutations affecting residues within beta-sheet A itself. His338 of strand 5 forms essential hydrogen bonds that stabilize strands 3 and 5 with beta-sheet A in a closed confirmation. The mutation H338R obliterates these bonds, resulting in an opening of beta-sheet A and a more severe disease phenotype, with progressive myoclonus epilepsy and dysarthia at 15 years of age (121). This region is disrupted to yet a greater extent by mutation of residue 392. Normally, the glycine residue at this position allows compact positioning of the strands of beta-sheet A; however, the introduction of glutamic acid with the mutation G392E greatly favors the opening of beta-sheet A, resulting in progressive myoclonus epilepsy, dysarthia and chorea at 13 years of age (121). Finally, the G392R mutation introduces the larger arginine residue at the same position and causes the most severe form of FENIB with the earliest onset, at 8 years of age (154). It is possible that the epilepsy associated with FENIB is caused by a lack of circulating neuroserpin, although neuroserpin-knock out mice show no seizure phenotype (151, 153); however, dementia and neurodegeneration is a result of the accumulation of unique polymers of correctly folded neuroserpin.
5.3. Characterization of neuroserpin polymerization in vitro and in cell culture
The quantitative measurement of the in vitro aggregation rates of neuroserpin variants has allowed us to clearly link the molecular behavior of this specific protein to the clinical consequences of the mutations. Accordingly, the S49P and S52R neuroserpins form polymers in vitro much more rapidly than does the wild type (WT) protein, with the clinically most severe mutation, S52R, exhibiting the fastest rate of polymerization (153, 155). The genotype-phenotype correlation has been further investigated in cell cultures by expressing neuroserpin variants transiently in COS-7 cells and in stably transfected PC-12 cells. Here we see increasing intracellular retention of neuroserpins as we express increasingly polymerogenic variants (156, 157). Expression in PC-12 cells has shown that variant neuroserpins are retained within the endoplasmic reticulum and that polymers co-localize with the ER marker calreticulin, whereas WT neuroserpin is not retained in the ER but instead co-localizes with GM130, in the Golgi, and then chromogranin A, in the dense-core secretory vesicles, demonstrating successful trafficking through the regulated secretion pathway. This conclusion is confirmed by the observation that G392E neuroserpin from PC-12 cell lysates is sensitive to endoglycosidase H treatment, as revealed by a reduction in its apparent molecular mass after such treatment. In contrast, WT neuroserpin is insensitive to this treatment and the S52R neuroserpin exhibits an intermediate behavior with a fraction of the protein being sensitive to the endoglycosidase treatment. Here we see a subcellular genotype-phenotype correlation, with more severe mutations resulting in earlier retention in the secretory pathway.
5.4. Animal models of FENIB
A number of animal models of FENIB have also provided important insights into the disease. The first, a rat model using over-expression of wild type megsin, a kidney serpin, has demonstrated the existence of intraneuronal inclusions, up-regulation of ER stress proteins, activation of caspases-12 and -3, loss of neurons and a loss of motor coordination (158). Arguably, the expression of human neuroserpin is required for a faithful model of FENIB and consequently mice have been generated that express WT, S49P and S52R neuroserpins under the Thy-1 promoter (159). These mice accumulate neuroserpins within intraneuronal inclusions and develop a range of neurological symptoms including ataxia and coordination deficits. Mice expressing variant neuroserpins lose their ability to grasp a horizontal wire and to climb a grid of negative geotaxis, and are found to be more passive during handling. The observed accumulation and clinical symptoms correlate with the genotypes, whereby mice expressing S52R generally accumulate more neuroserpin, and exhibit more severe symptoms, including tremor and seizure-like episodes, earlier in their lives than the mice expressing S49P. Interestingly, it has been found that inclusions are present in the brain prior to clinical symptoms.
We have developed a Drosophila model of FENIB by expressing WT and variant neuroserpins in the central nervous system of the flies using the pan-neuronal driver elav-Gal4 (157). Using a sandwich ELISA assay the accumulation of neuroserpins is seen to recapitulate the genotype-to-phenotype correlation observed in human disease. Expression of increasingly polymerogenic variants of neuroserpin results in an increasing accumulation of neuroserpins in the brains of the flies. Immunohistochemical analysis of neuroserpin accumulation in the flies' brains reveals strong staining within cortical neuronal cell bodies, adjacent to the mushroom bodies and lobula, again with the most severe variants showing the highest level of accumulation (Figure 11). In addition, when a monoclonal antibody specific for polymers of neuroserpin is used in the ELISA assay, it can be seen that polymers are generated in flies expressing variant neuroserpins, but not in flies expressing WT neuroserpin. The accumulation of these polymers is detrimental to the flies and they exhibit an age-dependent loss of locomotor ability that correlates with the accumulation of neuroserpin as measured by ELISA (Figure 12). This model provides an opportunity to perform a genetic screen for modifiers of the accumulation of variant neuroserpins and may help us to understand the pathways and processes that handle these unique protein polymers.
6. PERSPECTIVE
In the crowded macromolecular environment of the cytoplasm, the ability of proteins to fold and function without forming aberrant intra- or intermolecular interactions is of crucial importance. There is increasing evidence that the need to maintain protein solubility provides a major evolutionary pressure on protein-coding DNA (160, 161). One common consequence of protein aggregation is the formation of amyloid deposits and these can cause disease either by the cytotoxic action of soluble aggregates, as in AD, or by the effect of the deposition in vital organs of large quantities of protein, as in the systemic amyloidoses. The serpinopathies represent an alternative pathogenic mechanism, in which the intracellular accumulation of near-natively folded proteins can cause disease. The methods and models that we have described for these representative proteins are applicable to a wider range of protein misfolding problems and provide vital opportunities for exploring therapeutic strategies, in addition to enhancing our knowledge of the delicate balance between the normal and pathological interactions behind protein misfolding.
7. ACKNOWLEDGMENTS
This work was supported by the Swedish Research Council (ACB), Alzheimer's Research Trust (BB), Biotechnology and Biological Sciences Research Council, UK (CMD and JRK), Medical Research Council, UK (DCC, IM, LML, DAL and CMD), Interdisciplinary Research Collaboration in Nanotechnology (TPJK) and Engineering and Physical Sciences Research Council, UK (DCC, IM, TPJK, DAL and CMD).
8. REFERENCES
1. M. Knapp and M. Prince, Dementia UK. London: Alzheimer's Society (2007)
2. F. Chiti and C. M. Dobson: Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333-366 (2006)
doi:10.1146/annurev.biochem.75.101304.123901
3. M. Sunde, L. C. Serpell, M. Bartlam, P. E. Fraser, M. B. Pepys and C. C. Blake: Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 273, 729-739 (1997)
doi:10.1006/jmbi.1997.1348
4. L. C. Serpell, M. Sunde, M. D. Benson, G. A. Tennent, M. B. Pepys and P. E. Fraser: The protofilament substructure of amyloid fibrils. J. Mol. Biol. 300, 1033-1039 (2000)
doi:10.1006/jmbi.2000.3908
5. O. S. Makin, E. Atkins, P. Sikorski, J. Johansson and L. C. Serpell: Molecular basis for amyloid fibril formation and stability. Proc. Natl. Acad. Sci. USA 102, 315-320 (2005)
doi:10.1073/pnas.0406847102
6. M. R. Sawaya, S. Sambashivan, R. Nelson, M. I. Ivanova, S. A. Sievers, M. I. Apostol, M. J. Thompson, M. Balbirnie, J. J. W. Wiltzius, H. T. McFarlane, A. O. Madsen, C. Riekel and D. Eisenberg: Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447, 453-457 (2007)
doi:10.1038/nature05695
7. M. B. Pepys: Amyloidosis. Annu. Rev. Med. 57, 223-241 (2006)
doi:10.1146/annurev.med.57.121304.131243
8. C. L. Masters, G. Simms, N. A. Weinman, G. Multhaup, B. L. McDonald and K. Beyreuther: Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. USA 82, 4245-4249 (1985)
doi:10.1073/pnas.82.12.4245
9. S. A. Gravina, L. Ho, C. B. Eckman, K. E. Long, L. Otvos, L. H. Younkin, N. Suzuki and S. G. Younkin: Amyloid beta protein (Abeta) in Alzheimer's disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at Abeta40 or Abeta42 (43). J. Biol. Chem. 270, 7013-7016 (1995)
10. T. Iwatsubo, D. M. Mann, A. Odaka, N. Suzuki and Y. Ihara: Amyloid beta protein (Abeta) deposition: Abeta42 (43) precedes Abeta40 in Down syndrome. Ann. Neurol. 37, 294-299 (1995)
doi:10.1002/ana.410370305
11. M. P. Lambert, A. K. Barlow, B. A. Chromy, C. Edwards, R. Freed, M. Liosatos, T. E. Morgan, I. Rozovsky, B. Trommer, K. L. Viola, P. Wals, C. Zhang, C. E. Finch, G. A. Krafft and W. L. Klein: Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. USA 95, 6448-6453 (1998)
doi:10.1073/pnas.95.11.6448
12. P. N. Lacor, M. C. Buniel, L. Chang, S. J. Fernandez, Y. Gong, K. L. Viola, M. P. Lambert, P. T. Velasco, E. H. Bigio, C. E. Finch, G. A. Krafft and W. L. Klein: Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J. Neurosci. 24, 10191-10200 (2004)
doi:10.1523/JNEUROSCI.3432-04.2004
13. A. T. Petkova, Y. Ishii, J. J. Balbach, O. N. Antzutkin, R. D. Leapman, F. Delaglio and R. Tycko: A structural model for Alzheimer's beta-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99, 16742-16747 (2002)
doi:10.1073/pnas.262663499
14. T. Luhrs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Dobeli, D. Schubert and R. Riek: 3D structure of Alzheimer's amyloid-beta (1-42) fibrils. Proc. Natl. Acad. Sci. USA 102, 17342-17347 (2005)
doi:10.1073/pnas.0506723102
15. C. J. Pike, D. Burdick, A. J. Walencewicz, C. G. Glabe and C. W. Cotman: Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J. Neurosci. 13, 1676-1687 (1993)
16. C. J. Pike, A. J. Walencewicz, C. G. Glabe and C. W. Cotman: In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res. 563, 311-314 (1991)
doi:10.1016/0006-8993(91)91553-D
17. A. Lomakin, D. S. Chung, G. B. Benedek, D. A. Kirschner and D. B. Teplow: On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants. Proc. Natl. Acad. Sci. USA 93, 1125-1129 (1996)
doi:10.1073/pnas.93.3.1125
18. R.B. Merrifield: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149-2154 (1963)
doi:10.1021/ja00897a025
19. A. K. Tickler, C. J. Barrow and J. D. Wade: Improved preparation of amyloid-beta peptides using DBU as Nalpha-Fmoc deprotection reagent. J. Pept. Sci. 7, 488-494 (2001)
doi:10.1002/psc.342
20. E. K. Lee, J. H. Hwang, D. Y. Shin, D. I. Kim and Y. J. Yoo: Production of recombinant amyloid-beta peptide 42 as an ubiquitin extension. Protein Expr. Purif. 40, 183-189 (2005)
doi:10.1016/j.pep.2004.12.014
21. D. B. Teplow: Preparation of amyloid beta-protein for structural and functional studies. Methods Enzymol. 413, 20-33 (2006)
doi:10.1016/S0076-6879(06)13002-5
22. D. M. Walsh, D. M. Hartley, Y. Kusumoto, Y. Fezoui, M. M. Condron, A. Lomakin, G. B. Benedek, D. J. Selkoe and D. B. Teplow: Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. J. Biol. Chem. 274, 25945-25952 (1999)
doi:10.1074/jbc.274.36.25945
23. Y. Fezoui, D. M. Hartley, J. D. Harper, R. Khurana, D. M. Walsh, M. M. Condron, D. J. Selkoe, P. T. Lansbury, A. L. Fink and D. B. Teplow: An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments. Amyloid 7, 166-178 (2000)
doi:10.3109/13506120009146831
24. S. J. Wood, B. Maleeff, T. Hart, and R. Wetzel: Physical, morphological and functional differences between pH 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide Abeta. J. Mol. Biol. 256, 870-877 (1996)
doi:10.1006/jmbi.1996.0133
25. E. Terzi, G. Holzemann and J. Seelig: Self-association of beta-amyloid peptide (1-40) in solution and binding to lipid membranes. J. Mol. Biol. 252, 633-642 (1995)
doi:10.1006/jmbi.1995.0525
26. R. Riek, P. Guntert, H. Dobeli, B. Wipf, and K. Wuthrich: NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, Abeta (1-40) (ox) and Abeta (1-42) (ox). Eur. J. Biochem. 268, 5930-5936 (2001)
doi:10.1046/j.0014-2956.2001.02537.x
27. W. Garzon-Rodriguez, M. Sepulveda-Becerra, S. Milton, and C. G. Glabe: Soluble amyloid Abeta- (1-40) exists as a stable dimer at low concentrations: J. Biol. Chem. 272, 21037-21044 (1997)
doi:10.1074/jbc.272.34.21037
28. L. M. Luheshi, G. G. Tartaglia, A-C. Brorsson, A. P. Pawar, I. E. Watson, F. Chiti, M. Vendruscolo, D. A. Lomas, C. M. Dobson and D. C. Crowther: Systematic in vivo analysis of the intrinsic determinants of amyloid beta pathogenicity. PLoS Biol. 5, 2493-2500 (2007)
doi:10.1371/journal.pbio.0050290
29. H. LeVine: Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404-410 (1993)
30. H. LeVine: Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 309, 274-284 (1999)
doi:10.1016/S0076-6879(99)09020-5
31. H. Naiki, K. Higuchi, M. Hosokawa and T. Takeda: Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal. Biochem. 177, 244-249 (1989)
doi:10.1016/0003-2697(89)90046-8
32. G. Bitan, M. D. Kirkitadze, A. Lomakin, S. S. Vollers, G. B. Benedek and D. B. Teplow: Amyloid beta-protein (Abeta) assembly: Abeta40 and Abeta42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. USA 100, 330-335 (2003)
doi:10.1073/pnas.222681699
33. C. J. Barrow and M. G. Zagorski: Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition. Science 253, 179-182 (1991)
doi:10.1126/science.1853202
34. D. M. Walsh, A. Lomakin, G. B. Benedek, M. M. Condron and D. B. Teplow: Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J. Biol. Chem. 272, 22364-22372 (1997)
doi:10.1074/jbc.272.35.22364
35. M. R. H. Krebs, E. H. C. Bromley and A. M. Donald: The binding of thioflavin-T to amyloid fibrils: localisation and implications. J. Struct. Biol. 149, 30-37 (2005)
doi:10.1016/j.jsb.2004.08.002
36. R. Khurana, C. Coleman, C. Ionescu-Zanetti, S. A. Carter, V. Krishna, R. K. Grover, R. Roy and S. Singh: Mechanism of thioflavin T binding to amyloid fibrils. J. Struct. Biol. 151, 229-238 (2005)
doi:10.1016/j.jsb.2005.06.006
37. J. D. Harper and P. T. Lansbury: 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 (1997)
doi:10.1146/annurev.biochem.66.1.385
38. P. Hortschansky, V. Schroeckh, T. Christopeit, G. Zandomeneghi and M. Fandrich: The aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation. Protein Sci. 14, 1753-1759 (2005)
doi:10.1110/ps.041266605
39. M. Calamai, G. G. Tartaglia, M. Vendruscolo, F. Chiti and C. M. Dobson: Mutational analysis of the aggregation-prone and disaggregation-prone regions of acylphosphatase. J. Mol. Biol. 387, 965-974 (2009)
doi:10.1016/j.jmb.2008.09.003
40. J. S. Pedersen and D. E. Otzen: Amyloid-a state in many guises: survival of the fittest fibril fold. Protein Sci. 17, 2-10 (2008)
doi:10.1110/ps.073127808
41. D. Hall and H. Edskes: Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection. J. Mol. Biol. 336, 775-786 (2004)
doi:10.1016/j.jmb.2003.12.004
42. M. Tanaka, P. Chien, N. Naber, R. Cooke and J. S. Weissman: Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323-328 (2004)
doi:10.1038/nature02392
43. J. F. Smith, T. P. J. Knowles, C. M. Dobson, C. E. Macphee and M. E. Welland: Characterization of the nanoscale properties of individual amyloid fibrils. Proc. Natl. Acad. Sci. USA 103, 15806-15811 (2006)
doi:10.1073/pnas.0604035103
44. M. Tanaka, S. R. Collins, B. H. Toyama and J. S. Weissman: The physical basis of how prion conformations determine strain phenotypes. Nature 442, 585-589 (2006)
doi:10.1038/nature04922
45. J. Falsig, T. P. J. Knowles, K. P. R. Nilsson and A. Aguzzi: Chemical and biophysical insights into the propagation of prion strains. HFSP J. 1, 70 (2008)
46. A. T. Petkova, R. D. Leapman, Z. Guo, W-M. Yau, M. P. Mattson and R. Tycko: Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils. Science 307, 262-265 (2005)
doi:10.1126/science.1105850
47. M. Meyer-Luehmann, J. Coomaraswamy, T. Bolmont, S. Kaeser, C. Schaefer, E. Kilger, A. Neuenschwander, D. Abramowski, P. Frey, A. L. Jaton, J-M. Vigouret, P. Paganetti, D. M. Walsh, P. M. Mathews, J. Ghiso, M. Staufenbiel, L. C. Walker and M. Jucker: Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313, 1781-1784 (2006)
doi:10.1126/science.1131864
48. T. P. Knowles, A. W. Fitzpatrick, S. Meehan, H. R. Mott, M. Vendruscolo, C. M. Dobson and M. E. Welland: Role of intermolecular forces in defining material properties of protein nanofibrils. Science 318, 1900-1903 (2007)
doi:10.1126/science.1150057
49. C. J. Barrow, A. Yasuda, P. T. Kenny and M. G. Zagorski: Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra. J. Mol. Biol. 225, 1075-1093 (1992)
doi:10.1016/0022-2836(92)90106-T
50. L. Otvos, G. I. Szendrei, V. M. Lee and H. H. Mantsch: Human and rodent Alzheimer beta-amyloid peptides acquire distinct conformations in membrane-mimicking solvents. Eur. J. Biochem. 211, 249-257 (1993)
doi:10.1111/j.1432-1033.1993.tb19893.x
51. S. Tomaselli, V. Esposito, P. Vangone, N. A. J. van Nuland, A. M. J. J. Bonvin, R. Guerrini, T. Tancredi, P. A. Temussi and D. Picone: The alpha-to-beta conformational transition of Alzheimer's Abeta- (1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding. ChemBioChem 7, 257-267 (2006)
doi:10.1002/cbic.200500223
52. M. Bartolini, C. Bertucci, M. L. Bolognesi, A. Cavalli, C. Melchiorre and V. Andrisano: Insight into the kinetic of amyloid beta (1-42) peptide self-aggregation: elucidation of inhibitors' mechanism of action. ChemBioChem 8, 2152-2161 (2007)
doi:10.1002/cbic.200700427
53. Y. Fezoui and D. B. Teplow: Kinetic studies of amyloid beta-protein fibril assembly. Differential effects of alpha-helix stabilization. J. Biol. Chem. 277, 36948-36954 (2002)
doi:10.1074/jbc.M204168200
54. W. P. Esler, E. R. Stimson, J. B. Fishman, J. R. Ghilardi, H. V. Vinters, P. W. Mantyh and J. E. Maggio: Stereochemical specificity of Alzheimer's disease beta-peptide assembly. Biopolymers 49, 505-514 (1999)
doi:10.1002/(SICI)1097-0282(199905)49:6<505::AID-BIP8>3.0.CO;2-I
55. C. Morgan, M. Colombres, M. T. Nunez and N. C. Inestrosa: Structure and function of amyloid in Alzheimer's disease. Prog. Neurobiol. 74, 323-349 (2004)
doi:10.1016/j.pneurobio.2004.10.004
56. R. W. Hepler, K. M. Grimm, D. D. Nahas, R. Breese, E. C. Dodson, P. Acton, P. M. Keller, M. Yeager, H. Wang, P. Shughrue, G. Kinney and J. G. Joyce: Solution state characterization of amyloid beta-derived diffusible ligands. Biochemistry 45, 15157-15167 (2006)
doi:10.1021/bi061850f
57. B. A. Chromy, R. J. Nowak, M. P. Lambert, K. L. Viola, L. Chang, P. T. Velasco, B. W. Jones, S. J. Fernandez, P. N. Lacor, P. Horowitz, C. E. Finch, G. A. Krafft and W. L. Klein: Self-assembly of Abeta (1-42) into globular neurotoxins. Biochemistry 42, 12749-12760 (2003)
doi:10.1021/bi030029q
58. A-S. Johansson, F. Berglind-Dehlin, G. Karlsson, K. Edwards, P. Gellerfors and L. Lannfelt: Physiochemical characterization of the Alzheimer's disease-related peptides Abeta1-42arctic and Abeta1-42wt. FEBS J. 273, 2618-2630 (2006)
doi:10.1111/j.1742-4658.2006.05263.x
59. C. Nilsberth, A. Westlind-Danielsson, C. B. Eckman, M. M. Condron, K. Axelman, C. Forsell, C. Stenh, J. Luthman, D. B. Teplow, S. G. Younkin, J. Naslund and L. Lannfelt: The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation. Nat. Neurosci. 4, 887-893 (2001)
doi:10.1038/nn0901-887
60. V. H. Finder and R. Glockshuber: Amyloid-beta aggregation. Neurodegener. Dis. 4, 13-27 (2007)
doi:10.1159/000100355
61. A. K. Paravastu, R. D. Leapman, W-M. Yau and R. Tycko: Molecular structural basis for polymorphism in Alzheimer's beta-amyloid fibrils. Proc. Natl. Acad. Sci. USA 105, 18349-18354 (2008)
doi:10.1073/pnas.0806270105
62. J. Kim, B. Kleizen, R. Choy, G. Thinakaran, S. S. Sisodia and R. W. Schekman: Biogenesis of gamma-secretase early in the secretory pathway. J. Cell. Biol. 179, 951-963 (2007)
doi:10.1083/jcb.200709012
63. S. S. Sisodia and P. H. St. George-Hyslop: gamma-Secretase, Notch, Abeta and Alzheimer's disease: where do the presenilins fit in? Nat. Rev. Neurosci. 3, 281-290 (2002)
doi:10.1038/nrn785
64. S. S. Sisodia, W. Annaert, S. H. Kim and B. De Strooper: gamma-Secretase: never more enigmatic. Trends Neurosci. 24, S2-S6 (2001)
doi:10.1016/S0166-2236(00)01987-1
65. O. M. Andersen, J. Reiche, V. Schmidt, M. Gotthardt, R. Spoelgen, J. Behlke, C. A. F. von Arnim, T. Breiderhoff, P. Jansen, X. Wu, K. R. Bales, R. Cappai, C. L. Masters, J. Gliemann, E. J. Mufson, B. T. Hyman, S. M. Paul, A. Nykjaer and T. E. Willnow: Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein. Proc. Natl. Acad. Sci. USA 102, 13461-13466 (2005)
doi:10.1073/pnas.0503689102
66. K. Offe, S. E. Dodson, J. T. Shoemaker, J. J. Fritz, M. Gearing, A. I. Levey and J. J. Lah: The lipoprotein receptor LR11 regulates amyloid beta production and amyloid precursor protein traffic in endosomal compartments. J. Neurosci. 26, 1596-1603 (2006)
doi:10.1523/JNEUROSCI.4946-05.2006
67. E. Rogaeva, Y. Meng, J. H. Lee, Y. Gu, T. Kawarai, F. Zou, T. Katayama, C. T. Baldwin, R. Cheng, H. Hasegawa, F. Chen, N. Shibata, K. L. Lunetta, R. Pardossi-Piquard, C. Bohm, Y. Wakutani, L. A. Cupples, K. T. Cuenco, R. C. Green, L. Pinessi, I. Rainero, S. Sorbi, A. Bruni, R. Duara, R. P. Friedland, R. Inzelberg, W. Hampe, H. Bujo, Y-Q. Song, O. M. Andersen, T. E. Willnow, N. Graff-Radford, R. C. Petersen, D. Dickson, S. D. Der, P. E. Fraser, G. Schmitt-Ulms, S. Younkin, R. Mayeux, L. A. Farrer and P. St. George-Hyslop: The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease. Nat. Genet. 39, 168-177 (2007)
doi:10.1038/ng1943
68. S. Oddo, A. Caccamo, J. D. Shepherd, M. P. Murphy, T. E. Golde, R. Kayed, R. Metherate, M. P. Mattson, Y. Akbari and F. M. LaFerla: Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39, 409-421 (2003)
doi:10.1016/S0896-6273(03)00434-3
69. S. Oddo, A. Caccamo, M. Kitazawa, B. P. Tseng and F. M. LaFerla: Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease. Neurobiol. Aging 24, 1063-1070 (2003)
doi:10.1016/j.neurobiolaging.2003.08.012
70. C. D. Link: Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 92, 9368-9372 (1995)
doi:10.1073/pnas.92.20.9368
71. E. Cohen, J. Bieschke, R. M. Perciavalle, J. W. Kelly and A. Dillin: Opposing activities protect against age-onset proteotoxicity. Science 313, 1604-1610 (2006)
doi:10.1126/science.1124646
72. K. Iijima, H-P. Liu, A-S. Chiang, S. A. Hearn, M. Konsolaki and Y. Zhong: Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer's disease. Proc. Natl. Acad. Sci. USA 101, 6623-6628 (2004)
doi:10.1073/pnas.0400895101
73. M. A. Joiner, Z. Asztalos, C. J. Jones, T. Tully and C-F. Wu: Effects of mutant Drosophila K+ channel subunits on habituation of the olfactory jump response. J. Neurogenet. 21, 45-58 (2007)
doi:10.1080/01677060701247375
74. Z. Asztalos, J. von Wegerer, G. Wustmann, V. Dombrádi, J. Gausz, H. C. Spatz and P. Friedrich: Protein phosphatase 1-deficient mutant Drosophila is affected in habituation and associative learning. J. Neurosci. 13, 924-930 (1993)
75. D. C. Crowther, K. J. Kinghorn, E. Miranda, R. Page, J. A. Curry, F. A. I. Duthie, D. C. Gubb and D. A. Lomas: Intraneuronal Abeta, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer's disease. Neuroscience 132, 123-135 (2005)
doi:10.1016/j.neuroscience.2004.12.025
76. I. Greeve, D. Kretzschmar, J-A. Tschape, A. Beyn, C. Brellinger, M. Schweizer, R. M. Nitsch and R. Reifegerste: Age-dependent neurodegeneration and Alzheimer-amyloid plaque formation in transgenic Drosophila. J. Neurosci. 24, 3899-3906 (2004)
doi:10.1523/JNEUROSCI.0283-04.2004
77. D. M. Walsh, B. P. Tseng, R. E. Rydel, M. B. Podlisny and D. J. Selkoe: The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry 39, 10831-10839 (2000)
doi:10.1021/bi001048s
78. M. Enya, M. Morishima-Kawashima, M. Yoshimura, Y. Shinkai, K. Kusui, K. Khan, D. Games, D. Schenk, S. Sugihara, H. Yamaguchi and Y. Ihara: Appearance of sodium dodecyl sulfate-stable amyloid beta-protein (Abeta) dimer in the cortex during aging. Am. J. Pathol. 154, 271-279 (1999)
79. M. B. Podlisny, B. L. Ostaszewski, S. L. Squazzo, E. H. Koo, R. E. Rydell, D. B. Teplow and D. J. Selkoe: Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture. J. Biol. Chem. 270, 9564-9570 (1995)
doi:10.1074/jbc.270.16.9564
80. D. M. Walsh, I. Klyubin, J. V. Fadeeva, M. J. Rowan and D. J. Selkoe: Amyloid-beta oligomers: their production, toxicity and therapeutic inhibition. Biochem. Soc. Trans. 30, 552-557 (2002)
doi:10.1042/BST0300552
81. W. Cao, H-J. Song, T. Gangi, A. Kelkar, I. Antani, D. Garza and M. Konsolaki: Identification of novel genes that modify phenotypes induced by Alzheimer's beta-amyloid overexpression in Drosophila. Genetics 178, 1457-1471 (2008)
doi:10.1534/genetics.107.078394
82. L. M. Luheshi, D. C. Crowther and C. M. Dobson: Protein misfolding and disease: from the test tube to the organism. Curr. Opin. Chem. Biol. 12, 25-31 (2008)
doi:10.1016/j.cbpa.2008.02.011
83. W. L. Klein, G. A. Krafft and C. E. Finch: Targeting small Abeta oligomers: the solution to an Alzheimer's disease conundrum? Trends Neurosci. 24, 219-224 (2001)
doi:10.1016/S0166-2236(00)01749-5
84. D. J. Selkoe: Alzheimer's disease is a synaptic failure. Science 298, 789-791 (2002)
doi:10.1126/science.1074069
85. L. A. Holcomb, M. N. Gordon, P. Jantzen, K. Hsiao, K. Duff and D. Morgan: Behavioral changes in transgenic mice expressing both amyloid precursor protein and presenilin-1 mutations: lack of association with amyloid deposits. Behav. Genet. 29, 177-185 (1999)
doi:10.1023/A:1021691918517
86. C. C. Blake, D. F. Koenig, G. A. Mair, A. C. North, D. C. Phillips and V. R. Sarma: Structure of hen-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution. Nature 206, 757-761 (1965)
doi:10.1038/206757a0
87. M. Dumoulin, J. R. Kumita and C. M. Dobson: Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants. Acc. Chem. Res. 39, 603-610 (2006)
doi:10.1021/ar050070g
88. M. B. Pepys, P. N. Hawkins, D. R. Booth, D. M. Vigushin, G. A. Tennent, A. K. Soutar, N. Totty, O. Nguyen, C. C. Blake and C. J. Terry: Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 362, 553-557 (1993)
doi:10.1038/362553a0
89. S. Valleix, S. Drunat, J-B. Philit, D. Adoue, J.-C. Piette, D. Droz, B. MacGregor, D. Canet, M. Delpech and G Grateau: Hereditary renal amyloidosis caused by a new variant lysozyme W64R in a French family. Kidney Int. 61, 907-912 (2002)
doi:10.1046/j.1523-1755.2002.00205.x
90. M. Yazaki, S. A. Farrell and M. D. Benson: A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis. Kidney Int. 63, 1652-1657 (2003)
doi:10.1046/j.1523-1755.2003.00904.x
91. C. Rocken, K. Becker, M. Fandrich, V Schroeckh, B. Stix, T. Rath, T. Kahne, J. Dierkes, A. Roessner and F. W. Albert: Alys amyloidosis caused by compound heterozygosity in exon 2 (Thr70Asn) and exon 4 (Trp112Arg) of the lysozyme gene. Hum. Mutat. 27, 119-120 (2006)
doi:10.1002/humu.9393
92. D. R. Booth, M. Sunde, V. Bellotti, C. V. Robinson, W. L. Hutchinson, P. E. Fraser, P. N. Hawkins, C. M. Dobson, S. E. Radford, C. C. Blake and M. B. Pepys: Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385, 787-793 (1997)
doi:10.1038/385787a0
93. L. A. Morozova-Roche, J. Zurdo, A. Spencer, W. Noppe, V. Receveur, D. B. Archer, M. Joniau and C. M. Dobson: Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants. J. Struct. Biol. 130, 339-351 (2000)
doi:10.1006/jsbi.2000.4264
94. M. Dumoulin, V. Bellotti and C. M. Dobson, Amyloid proteins - The beta pleated sheet conformation and disease. Wiley-VCH Verlag GmbH and Co. Kga publisher, Weinheim (2005)
95. M. Dumoulin, R. J. K. Johnson, V. Bellotti and C. M. Dobson, Human lysozyme. Kluwer Academic, Dordrecht, The Netherlands (2005)
96. M. Dumoulin, A. M. Last, A. Desmyter, K. Decanniere, D. Canet, G. Larsson, A. Spencer, D. B. Archer, J. Sasse, S. Muyldermans, L. Wyns, C. Redfield, A. Matagne, C. V. Robinson and C. M. Dobson: A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature 424, 783-788 (2003)
doi:10.1038/nature01870
97. D. Canet, A. M. Last, P. Tito, M. Sunde, A. Spencer, D. B. Archer, C. Redfield, C. V. Robinson and C. M. Dobson: Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme. Nat. Struct. Biol. 9, 308-315 (2002)
doi:10.1038/nsb768
98. M. Dumoulin, D. Canet, A. M. Last, E. Pardon, D. B. Archer, S. Muyldermans, L. Wyns, A. Matagne, C. V. Robinson, C. Redfield and C. M. Dobson: Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations. J. Mol. Biol. 346, 773-788 (2005)
doi:10.1016/j.jmb.2004.11.020
99. J. R. Kumita, S. Poon, G. L. Caddy, C. L. Hagan, M. Dumoulin, J. J. Yerbury, E. M. Stewart, C. V. Robinson, M. R. Wilson and C. M. Dobson: The extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species. J. Mol. Biol. 369, 157-167 (2007)
doi:10.1016/j.jmb.2007.02.095
100. H. Roder, G. Wagner and K. Wuthrich: Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature. Biochemistry 24, 7396-7407 (1985)
doi:10.1021/bi00346a055
101. R. J. K. Johnson, J. Christodoulou, M. Dumoulin, G. L. Caddy, M. J. C. Alcocer, G. J Murtagh, J. R. Kumita, G. Larsson, C. V. Robinson, D. B. Archer, B. Luisi and C. M. Dobson: Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme. J. Mol. Biol. 352, 823-836 (2005)
doi:10.1016/j.jmb.2005.07.040
102. S. D. Hooke, S. E. Radford and C. M. Dobson: The refolding of human lysozyme: a comparison with the structurally homologous hen lysozyme. Biochemistry 33, 5867-5876 (1994)
doi:10.1021/bi00185a026
103. E. Frare, M. F. Mossuto, P. Polverino de Laureto, M. Dumoulin, C. M. Dobson and A. Fontana: Identification of the core structure of lysozyme amyloid fibrils by proteolysis. J. Mol. Biol. 361. 551-561 (2006)
doi:10.1016/j.jmb.2006.06.055
104. P. J. Muchowski and J. L. Wacker: Modulation of neurodegeneration by molecular chaperones. Nat. Rev. Neurosci. 6, 11-22 (2005)
doi:10.1038/nrn1587
105. J. J. Yerbury, E. M. Stewart, A. R. Wyatt and M. R. Wilson: Quality control of protein folding in extracellular space. EMBO Rep. 6, 1131-1136 (2005)
doi:10.1038/sj.embor.7400586
106. J. J. Yerbury, M. S. Rybchyn, S. B. Easterbrook-Smith, C. Henriques and M. R. Wilson: The acute phase protein haptoglobin is a mammalian extracellular chaperone with an action similar to clusterin. Biochemistry 44, 10914-10925 (2005)
doi:10.1021/bi050764x
107. K. French, J. J. Yerbury and M. R. Wilson: Protease activation of alpha2-macroglobulin modulates a chaperone-like action with broad specificity. Biochemistry 47, 1176-1185 (2008)
doi:10.1021/bi701976f
108. S. E. Jones and C. Jomary: Clusterin. Int. J. Biochem. Cell. Biol. 34, 427-431, (2002)
doi:10.1016/S1357-2725(01)00155-8
109. D. E. Jenne, B. Lowin, M. C. Peitsch, A. Bottcher, G. Schmitz and J. Tschopp: Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma. J. Biol. Chem. 266, 11030-11036 (1991)
110. D. T. Humphreys, J. A. Carver, S. B. Easterbrook-Smith and M. R. Wilson: Clusterin has chaperone-like activity similar to that of small heat shock proteins. J. Biol. Chem. 274, 6875-6881 (1999)
doi:10.1074/jbc.274.11.6875
111. J. A. Carver, A. Rekas, D. C. Thorn and M. R. Wilson: Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function? IUBMB Life 55, 661-668 (2003)
doi:10.1080/15216540310001640498
112. D. M. Hatters, M. R. Wilson, S. B. Easterbrook-Smith and G. J. Howlett: Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin. Eur. J. Biochem. 269, 2789-2794 (2002)
doi:10.1046/j.1432-1033.2002.02957.x
113. J. J. Yerbury, S. Poon, S. Meehan, B. Thompson, J. R. Kumita, C. M. Dobson and M. R. Wilson: The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures. FASEB J. 21, 2312-2322 (2007)
doi:10.1096/fj.06-7986com
114. L. Sottrup-Jensen: alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation. J. Biol. Chem. 264, 11539-11542 (1989)
115. S. R. Hughes, O. Khorkova, S. Goyal, J. Knaeblein, J. Heroux, N. G. Riedel and S. Sahasrabudhe. alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation. Proc. Natl. Acad. Sci. USA 95, 3275-3280 (1998)
doi:10.1073/pnas.95.6.3275
116. W. Dobryszycka: Biological functions of haptoglobin-new pieces to an old puzzle. Eur. J. Clin. Chem. Clin. Biochem. 35, 647-654 (1997)
117. J. M. Powers, W. W. Schlaepfer, M. C. Willingham and B. J. Hall: An immunoperoxidase study of senile cerebral amyloidosis with pathogenetic considerations. J. Neuropathol. Exp. Neurol. 40, 592-612 (1981)
doi:10.1097/00005072-198111000-00002
118. J.J. Yerbury, J.R. Kumita, S. Meehan, C. M Dobson and M. R. Wilson: alpha2-macroglobulin and haptoglobin suppress amyloid formation by interacting with prefibrillar protein species. J. Biol. Chem. 284, 4246-4254 (2009)
doi:10.1074/jbc.M807242200
119. J. R. Kumita, R. J. K. Johnson, M. J. C. Alcocer, M. Dumoulin, F. Holmqvist, M. G. McCammon, C. V. Robinson, D. B. Archer and C. M. Dobson: Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris. FEBS J. 273, 711-720 (2006)
doi:10.1111/j.1742-4658.2005.05099.x
120. R. L. Davis, A. E. Shrimpton, P. D. Holohan, C. Bradshaw, D. Feiglin, G. H. Collins, P. Sonderegger, J. Kinter, L. M. Becker, F. Lacbawan, D. Krasnewich, M. Muenke, D. A. Lawrence, M. S. Yerby, C. M. Shaw, B. Gooptu, P. R. Elliott, J. T. Finch, R. W. Carrell and D. A. Lomas: Familial dementia caused by polymerization of mutant neuroserpin. Nature 401, 376-379 (1999)
doi:10.1038/43894
121. R. L. Davis, A. E. Shrimpton, R. W. Carrell, D. A. Lomas, L. Gerhard, B. Baumann, D. A. Lawrence, M. Yepes, T. S. Kim, B. Ghetti, P. Piccardo, M. Takao, F. Lacbawan, M. Muenke, R. N. Sifers, C. B. Bradshaw, P. F. Kent, G. H. Collins, D. Larocca and P. D. Holohan: Association between conformational mutations in neuroserpin and onset and severity of dementia. Lancet 359, 2242-2247 (2002)
doi:10.1016/S0140-6736(02)09293-0
122. J. A. Irving, R. N. Pike, A. M. Lesk and J. C. Whisstock: Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res. 10, 1845-1864 (2000)
doi:10.1101/gr.GR-1478R
123. G. A. Silverman, P. I. Bird, R. W. Carrell, F. C. Church, P. B. Coughlin, P. G. Gettins, J. A. Irving, D. A. Lomas, C. J. Luke, R. W. Moyer, P. A. Pemberton, E. Remold-O'Donnell, G. S. Salvesen, J. Travis and J. C. Whisstock: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276, 33293-33296 (2001)
doi:10.1074/jbc.R100016200
124. P. R. Elliott, D. A. Lomas, R. W. Carrell and J. P. Abrahams: Inhibitory conformation of the reactive loop of alpha1-antitrypsin. Nat. Struct. Biol. 3, 676-681 (1996)
doi:10.1038/nsb0896-676
125. J. A. Huntington, R. J. Read and R. W. Carrell: Structure of a serpin-protease complex shows inhibition by deformation. Nature 407, 923-926 (2000)
doi:10.1038/35038119
126. D. A. Lomas and R. W. Carrell: Serpinopathies and the conformational dementias. Nat. Rev. Genet. 3, 759-768 (2002)
doi:10.1038/nrg907
127. K. Beatty, J. Bieth and J. Travis: Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin. J. Biol. Chem. 255, 3931-3934 (1980)
128. H. L. Sharp, R. A. Bridges, W. Krivit and E. F. Freier: Cirrhosis associated with alpha-1-antitrypsin deficiency: a previously unrecognized inherited disorder. J. Lab. Clin. Med. 73, 934-939 (1969)
129. D. A. Lomas, D. L. Evans, J. T. Finch and R. W. Carrell: The mechanism of Z alpha1-antitrypsin accumulation in the liver. Nature 357, 605-607 (1992)
doi:10.1038/357605a0
130. M. Yamasaki, W. Li, D. J. D. Johnson and J. A. Huntington: Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature 455, 1255-1258 (2008)
doi:10.1038/nature07394
131. J. Mottonen, A. Strand, J. Symersky, R. M. Sweet, D. E. Danley, K. F. Geoghegan, R. D. Gerard and E. J. Goldsmith: Structural basis of latency in plasminogen activator inhibitor-1. Nature 355, 270-273 (1992)
doi:10.1038/355270a0
132. C. M. Hekman and D. J. Loskutoff: Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J. Biol. Chem. 260, 11581-11587 (1985)
133. Z. Wang, J. Mottonen, and E. J. Goldsmith: Kinetically controlled folding of the serpin plasminogen activator inhibitor 1. Biochemistry 35, 16443-16448 (1996)
doi:10.1021/bi961214p
134. H. Im, H. Y. Ahn and M. H. Yu: Bypassing the kinetic trap of serpin protein folding by loop extension. Protein Sci. 9, 1497-1502 (2000)
doi:10.1110/ps.9.8.1497
135. S. Janciauskiene, R. Dominaitiene, N. H. Sternby, E. Piitulainen and S. Eriksson: Detection of circulating and endothelial cell polymers of Z and wild type alpha1-antitrypsin by a monoclonal antibody. J. Biol. Chem. 277, 26540-26546 (2002)
doi:10.1074/jbc.M203832200
136. T. R. Dafforn, R. Mahadeva, P. R. Elliott, P. Sivasothy and D. A. Lomas: A kinetic mechanism for the polymerization of alpha1-antitrypsin. J. Biol. Chem. 274, 9548-9555 (1999)
doi:10.1074/jbc.274.14.9548
137. P. Sivasothy, T. R. Dafforn, P. G. Gettins and D. A. Lomas: Pathogenic alpha1-antitrypsin polymers are formed by reactive loop-beta-sheet a linkage. J. Biol. Chem. 275, 33663-33668 (2000)
doi:10.1074/jbc.M004054200
138. D. A. Lomas, D. Belorgey, M. Mallya, E. Miranda, K. J. Kinghorn, L. K. Sharp, R. L. Phillips, R. Page, A. S. Robertson and D. C. Crowther: Molecular mousetraps and the serpinopathies. Biochem. Soc. Trans. 33, 321-330 (2005)
doi:10.1042/BST0330321
139. S. Janciauskiene, S. Eriksson, F. Callea, M. Mallya, A. Zhou, K. Seyama, S. Hata and D. A. Lomas: Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin. Hepatology 40, 1203-1210 (2004)
doi:10.1002/hep.20451
140. E. L. James and S. P. Bottomley: The mechanism of alpha1-antitrypsin polymerization probed by fluorescence spectroscopy. Arch. Biochem. Biophys. 356, 296-300 (1998)
doi:10.1006/abbi.1998.0751
141. P. Purkayastha, J. W. Klemke, S. Lavender, R. Oyola, B. S. Cooperman and F. Gai: alpha1-antitrypsin polymerization: a fluorescence correlation spectroscopic study. Biochemistry 44, 2642-2649 (2005)
doi:10.1021/bi048662e
142. D. Belorgey, P. Hagglof, S. Karlsson-Li and D. A. Lomas: Protein misfolding and the serpinopathies. Prion 1, 15-20 (2007)
143. M. Yepes and D. A. Lawrence: Tissue-type plasminogen activator and neuroserpin: a well-balanced act in the nervous system? Trends Cardiovasc. Med. 14, 173-180 (2004)
doi:10.1016/j.tcm.2004.03.004
144. J. H. Ware, A. J. DiBenedetto and R. N. Pittman: Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration. J. Neurobiol. 28, 9-22 (1995)
doi:10.1002/neu.480280103
145. T. Osterwalder, J. Contartese, E. T. Stoeckli, T. B. Kuhn and P. Sonderegger: Neuroserpin, an axonally secreted serine protease inhibitor. EMBO J. 15, 2944-2953 (1996)
146. G. A. Hastings, T. A. Coleman, C. C. Haudenschild, S. Stefansson, E. P. Smith, R. Barthlow, S. Cherry, M. Sandkvist and D. A. Lawrence: Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival. J. Biol. Chem. 272, 33062-33067 (1997)
doi:10.1074/jbc.272.52.33062
147. S. R. Krueger, G. P. Ghisu, P. Cinelli, T. P. Gschwend, T. Osterwalder, D. P. Wolfer and P. Sonderegger: Expression of neuroserpin, an inhibitor of tissue plasminogen activator, in the developing and adult nervous system of the mouse. J. Neurosci. 17, 8984-8996 (1997)
148. Z. Qian, M. E. Gilbert, M. A. Colicos, E. R. Kandel and D. Kuhl: Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation. Nature 361, 453-457 (1993)
doi:10.1038/361453a0
149. M. Yepes, M. Sandkvist, T. A. Coleman, E. Moore, J-Y. Wu, D. Mitola, T. H. Bugge and D. A. Lawrence: Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent. J. Clin. Invest. 109, 1571-1578 (2002)
doi:10.1172/JCI200214308
150. Z. Zhang, L. Zhang, M. Yepes, Q. Jiang, Q. Li, P. Arniego, T. A. Coleman, D. A. Lawrence and M. Chopp: Adjuvant treatment with neuroserpin increases the therapeutic window for tissue-type plasminogen activator administration in a rat model of embolic stroke. Circulation 106, 740-745 (2002)
151. R. Madani, S. Kozlov, A. Akhmedov, P. Cinelli, J. Kinter, H-P. Lipp, P. Sonderegger and D. P. Wolfer: Impaired explorative behavior and neophobia in genetically modified mice lacking or overexpressing the extracellular serine protease inhibitor neuroserpin. Mol. Cell. Neurosci. 23, 473-494 (2003)
doi:10.1016/S1044-7431(03)00077-0
152. N. W. Seeds, M. E. Basham and J. E. Ferguson: Absence of tissue plasminogen activator gene or activity impairs mouse cerebellar motor learning. J. Neurosci. 23, 7368-7375 (2003)
153. D. Belorgey, L. K. Sharp, D. C. Crowther, M. Onda, J. Johansson and D. A. Lomas: Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers: implications for the epilepsy seen in the dementia FENIB. Eur. J. Biochem. 271, 3360-3367 (2004)
doi:10.1111/j.1432-1033.2004.04270.x
154. M. Coutelier, S. Andries, S. Ghariani, B. Dan, C. Duyckaerts, K. van Rijckevorsel, C. Raftopoulos, N. Deconinck, P. Sonderegger, F. Scaravilli, M. Vikkula and C. Godfraind: Neuroserpin mutation causes electrical status epilepticus of slow-wave sleep. Neurology 71, 64-66 (2008)
doi:10.1212/01.wnl.0000316306.08751.28
155. D. Belorgey, D. C. Crowther, R. Mahadeva and D. A. Lomas: Mutant neuroserpin (S49P) that causes familial encephalopathy with neuroserpin inclusion bodies is a poor proteinase inhibitor and readily forms polymers in vitro. J. Biol. Chem. 277, 17367-17373 (2002)
doi:10.1074/jbc.M200680200
156. E. Miranda, K. Romisch and D. A. Lomas: Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum. J. Biol. Chem. 279, 28283-28291 (2004)
doi:10.1074/jbc.M313166200
157. E. Miranda, I. MacLeod, M. J. Davies, J. Pérez, K. Romisch, D. C. Crowther and D. A. Lomas: The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB. Hum. Mol. Genet. 17, 1527-1539 (2008)
doi:10.1093/hmg/ddn041
158. K. Takano, Y. Kitao, R. Inagi, T. Momoi, T. Matsuyama, T. Miyata, Y. Yoneda, H. Iso, D. M. Stern, O. Hori and S. Ogawa: A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies). Biochem. Biophys. Res. Commun. 346, 1040-1047 (2006)
doi:10.1016/j.bbrc.2006.06.016
159. G. Galliciotti, M. Glatzel, J. Kinter, S. V. Kozlov, P. Cinelli, T. Rulicke and P. Sonderegger: Accumulation of mutant neuroserpin precedes development of clinical symptoms in familial encephalopathy with neuroserpin inclusion bodies. Am. J. Pathol. 170, 1305-1313 (2007)
doi:10.2353/ajpath.2007.060910
160. G. G. Tartaglia, S. Pechmann, C. M. Dobson and M. Vendruscolo: Life on the edge: a link between gene expression levels and aggregation rates of human proteins. Trends Biochem. Sci. 32, 204-206 (2007)
doi:10.1016/j.tibs.2007.03.005
161. D. A. Drummond and C. O. Wilke: Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell 134, 341-352 (2008)
doi:10.1016/j.cell.2008.05.042
162. M. G. Zagorski, J. Yang, H. Shao, K. Ma, H. Zeng and A. Hong: Methodological and chemical factors affecting amyloid beta peptide amyloidogenicity. Methods Enzymol. 309, 189-204 (1999)
doi:10.1016/S0076-6879(99)09015-1
163. S-C. Jao, K. Ma, J. Talafous, R. Orlando and M. G. Zargorski: Trifluoroacetic acid pretreatment reproducibly disaggregate the amyloid-peptide. Amyloid 4, 240-252 (1997)
164. C. L. Shen and R. M. Murphy: Solvent effects on self-assembly of beta-amyloid peptide. Biophys. J. 69, 640-651 (1995)
doi:10.1016/S0006-3495(95)79940-4
Key Words: Protein aggregation, Amyloid beta peptide, Neuroserpin, Lysozyme, Review
Send correspondence to: Damian C. Crowther, Department of Genetics, Downing Site, Cambridge, CB2 3EH, Tel: 01223-760346, Fax: 01223-654400, E-mail:dcc26@cam.ac.uk