Bio-nanoimaging: Protein Misfolding and Aggregation

China

Chapter 1

Molecular Mechanisms of Protein Misfolding

Leonid Breydo,    Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA

Vladimir N. Uversky,    University of South Florida, College of Medicine, Department of Molecular Medicine, Miami, FL, USA; Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

Abstract

Unlike folded proteins that possess a single native fold that is determined only by their amino acid sequence, protein aggregates (fibrils and oligomers) are structurally highly diverse. The core of amyloid fibrils is composed of stacks of β-sheets, providing their structure with a high level of stability. High variability of packing arrangements within the fibril allows the same polypeptide chain to form amyloid fibrils with different tertiary structures. Fibrils propagate their tertiary structure by seeding; however, this process is imperfect and it results in structural alterations. Amyloid oligomers are the smaller aggregates; they have even higher conformational flexibility. Oligomers usually also rely on β-sheet stacking for intermolecular interactions, although there is a much higher degree of disorder in their structure compared to fibrils. Many oligomers are also capable of self-propagation by seeding. In this chapter we describe the mechanism of formation and the structural features of protein aggregates.

Keywords

protein misfolding; protein aggregation; protein fibrillation; conformational diseases; amyloid fibril; oligomer; amorphous aggregate

Chapter Outline

Introduction  1

The Mechanism of Aggregation  1

Structures of Protein Aggregates  3

Protein Aggregation Pathways  5

Amyloid Beta, Aβ  5

α-Synuclein  6

Yeast and Fungal Prions  7

Mammalian Prions  7

Insulin  8

Functional Amyloids  9

Extracellular Bacterial and Fungal Amyloids  9

Mammalian Functional Amyloids  9

β-2 Microglobulin  10

Other Amyloidogenic Proteins  10

Conclusions  10

Introduction

For decades, our understanding of protein structure has been driven by Anfinsen’s dogma: a protein possesses a single native structure that is determined only by its amino acid sequence [1]. It is now clear that this assumption does not fully describe the variety of structures found in proteins. Many proteins lack a stable tertiary and/or secondary structure in solution, existing instead as dynamic ensembles of interconverting structures. These proteins are generally known as intrinsically disordered proteins (IDPs). In addition, both natively folded proteins and IDPs are capable of aggregation (Fig. 1.1). Due to intermolecular interactions in protein aggregates, the same protein can form aggregates with a variety of different structures and morphologies. In this chapter we examine the process of aggregation and the structural features of protein aggregates.

FIGURE 1.1 Fate of a newly synthesized polypeptide chain in a cell.

The Mechanism of Aggregation

Amyloid fibril formation starts with the protein in a partially unfolded conformation similar to that in the pre-molten globule [2]. In the case of large, multi-domain proteins only the domain directly involved in aggregation needs to be partially unfolded. In order to adopt this conformation, a folded protein needs to partially unfold and an extended IDP needs to partially fold. Thus, partially denaturing conditions (low pH, high temperature, low concentrations of denaturants) promote aggregation of folded proteins.

These partially unfolded monomers usually assemble into oligomers that initially remain unfolded [3,4]. Within these oligomers, the protein molecules initially form metastable β-sheet conformations and, over time, they undergo gradual conformational conversion to β-sheet-rich structures [3–6]. These oligomers can then serve as nucleation seeds for other oligomers or fibrils (Fig. 1.2). The size of the ‘critical nucleus’ (i.e. the smallest oligomer capable of acting as a nucleation seed) can be estimated on the basis of aggregation kinetics; it depends on the protein sequence and conditions. It can range from a monomer for polyglutamine [7] to a hexamer for β-2 microglobulin [8]. However, even for polyglutamine, it appears likely that conversion of the peptide into a β-sheet-rich conformation actually occurs within the confines of a disordered oligomer [4,6].

FIGURE 1.2 Protein aggregation cascade. Adapted from reference 157.

After the stable oligomeric structures have been formed, they rapidly seed conversion of protein monomers to fibrils or other oligomers. Fragmentation of newly formed fibrils is an extremely important process for their propagation as it creates fibril edges that serve as seeds. Because more thermodynamically stable fibrils fragment less efficiently, the efficiency of fibril propagation has been found to be inversely proportional to their thermodynamic stability [5,9,10].

Propagation of fibrils by seeding usually results in the formation of ‘daughter’ fibrils with the same tertiary structure as the ‘mother’ fibrils. However, conformational changes in the fibrils sometimes occur during propagation. This phenomenon has been extensively investigated for yeast and mammalian prions [11–14]. This conformational change may occur either by a rare spontaneous structural change in the fibril or through lack of fidelity during the replication process [11,15]. This process has been called ‘deformed templating’ [15,16]. Fibrils with altered conformations that make them more proficient in replication are then selectively amplified during the seeding process. Eventually, faster replicating fibril conformations will dominate by successfully competing with slower replicating ones for the monomers. Chaperones, such as Hsp104, can contribute to this process by more efficiently fragmenting faster propagating fibrils [17,18]. This fragmentation creates more fibril seeds and further accelerates their propagation.

Interestingly, oligomers – as well as fibrils – can be propagated by seeding. In fact, oligomers may be able to propagate faster than fibrils due to their lower stability and smaller size. For example, for Aβ peptide it has been shown that oligomers indeed propagate faster than fibrils [19]. Moreover, the addition of oligomer seeds interfered with fibril formation [19], and structurally distinct oligomer populations faithfully propagated their respective conformations – resulting in the appearance of at least three distinct ‘strains’ of oligomer [19,20]. In addition to amyloid β, oligomer seeding has also been shown for tau protein [21,22] and it is likely to be a common phenomenon. Seeding was not completely sequence-specific, as oligomers of Aβ peptide were able to seed oligomer formation from tau protein [22].

Structures of Protein Aggregates

Protein aggregates can be divided into three structural classes: amorphous aggregates, amyloid fibrils, and oligomers. Only two of these, amyloid fibrils and oligomers, are considered below.

Amyloid fibrils are repetitive, β-sheet-rich structures. Their high thermodynamic and kinetic stability comes from association of multiple polypeptide molecules in a cross-β structure. Fibrils can be formed either from natively folded proteins or from intrinsically disordered proteins. These structures are organized on at least three levels: (1) β-strands are stacked into β-sheets, (2) β-sheets interact with each other to form protofilaments, and (3) protofilaments stack on, or twist around, one another to form a fibril [23,24].

The first level of organization is the stacking of individual β-strands, derived from different polypeptide molecules, to form a β-sheet. This interaction is driven primarily by the hydrogen bonds between the backbone amides. β-Sheets can be either parallel or antiparallel, depending on the directions of neighboring β-strands. Parallel β-sheets are more common in fibrils formed by longer polypeptides and proteins, although examples of antiparallel β-sheets are also known [23,25].

The second level of fibril organization involves interaction between two β-sheets. Side chains protruding from the two β-sheets interdigitate to give rise to highly ordered structures known as steric zippers [23]. These steric zippers run up and down the fibril axis and exclude water from the interface between the β-sheets. The formation of this interface is primarily driven by van der Waals interactions and hydrogen bonds between the side chains. Steric zippers are formed by stacked β-strands running perpendicular to the fibril axis in either a parallel or an antiparallel arrangement. In this interface the side chains of one β-strand are located between the side chains of another strand. Detailed investigation of various possible arrangements of β-strands in steric zippers with X-ray crystallography allowed Eisenberg and coworkers [23] to propose eight possible structural classes of steric zipper. The steric zippers in these classes differ in the arrangement of β-strands within each β-sheet (parallel vs antiparallel), utilization of the same or different surfaces for packing β-sheets against each other (‘face-to-face’ vs ‘face-to-back’), and whether β-sheets are parallel (‘up-up’) or antiparallel (‘up-down’) with respect to one another (Fig. 1.3). The same polypeptide may be able to form steric zippers belonging to different structural classes depending on the experimental conditions. Aromatic side chains are important for stabilization of steric zippers [26,27] because their van der Waals interactions are especially strong. A variety of steric zippers that can be formed by the amyloidogenic sequences is the important source of polymorphism of fibrillar structures [28].

FIGURE 1.3 Structural classes of steric zippers. Reproduced with permission from reference 23.

Longer polypeptides may contain multiple amyloidogenic regions connected by loops. These proteins assemble into protofibrils by forming β-helical structures. In this structure a single polypeptide chain forms multiple turns of the β-helix. Several examples of β-helices containing either two or three strands per turn are found in amyloid fibrils. In some cases (e.g. amyloid beta) the same polypeptide can form β-helical structures with either two or three turns per helix depending on the reaction conditions [29].

The third level of fibril organization is assembly of protofilaments into a fibril. The interaction of protofilaments with each other is relatively weak. Depending on the structure of protofilaments, they either stack on top of each other, to form flat ribbons, or twist around each other to form twisted fibrils. This assembly process serves to further shield the hydrophobic regions of the proteins from water and contributes to diversity of aggregate morphology.

Intrinsically disordered proteins (IDPs) form fibrils by converting either all or part of the previously unstructured polypeptide into β-sheet-rich secondary structures. Natively folded proteins form amyloid fibrils either by complete refolding or by a limited conformational change resulting in the formation of a fibrillar structure [30]. In the refolding model, proteins such as insulin convert from native structures to fibrils by initially unfolding, and then refolding into a secondary structure that is rich in β-sheets. Fibrils formed in this way are primarily stabilized by backbone hydrogen bonding rather than by side-chain–side-chain interactions. Alternatively, β-sheet-rich natively folded proteins can undergo a limited conformational change to expose a short segment of a previously inaccessible region. This region can then interact with the surfaces of other molecules to form fibrils without causing a major perturbation in the protein’s native structure. Fibrils are usually formed through (1) direct stacking of exposed regions (e.g. TTR and superoxide dismutase) [31,32], (2) the swapping of two β-sheet-containing domains from two monomers to form a cross-β spine (e.g. Sup35 and β2-microglobulin) [24,33], or (3) strand-swapping between two adjacent monomers (e.g. serpins) [34]. Fibrils formed by this mechanism mostly preserve the native structures of the proteins with minimal alterations.

In addition to fibrils, oligomeric amyloid structures are often observed. In some cases, they are essentially the short fragments of fibrils that appear as intermediates during fibril formation. Some oligomers are highly unstable and easily convert to fibrils or larger oligomers. Most oligomers that can be observed or isolated differ in structure from fibrils and have at least some degree of kinetic stability. Structural information about these oligomers is quite limited. In many cases only the secondary structure of the oligomers is known, although there are a few examples of amyloid oligomers with fully characterized 3D structures [35–38]. Amyloid oligomers are generally β-sheet-rich, although they vary from primarily disordered [39] to structures similar to those of fibrils [19,40].

Stable amyloid oligomers have been shown to have at least some cross-β-sheet structure. For example, the structure of Aβ oligomers with a high β-sheet content was analyzed by a combination of experimental and computational methods; the structure was predicted to be similar to that of fibrils with some disruptions in the β-sheet stacking that prevent elongation of oligomers into fibrils [19,40]. In another preparation of Aβ oligomers that appeared disordered upon initial examination [41], a detailed NMR analysis found that one of the two β-sheets present in the fibrils was intact while another was disordered [38]. The crystal structure of an oligomer belonging to another structural class has been determined; it was found to form a β-barrel-like structure [36]. Structurally similar oligomers are produced by many amyloidogenic peptides and proteins [36,42]. β-2 microglobulin oligomers form a cyclic hexamer with β-strands from the neighboring protein molecules forming the intermolecular interface [37]. Significant amounts of β-structure have been detected in oligomers of prion protein [43], β-2 microglobulin [37] and other proteins. We can conclude that β-sheets play an important role in amyloid oligomers, facilitating intermolecular interactions. The β-sheet-rich core in oligomers may be shorter than it is in fibrils and/or may contain packing defects preventing their growth into fibrils. In some cases [44,45], oligomers may form stable β-sheet-rich donut-like structures that do not grow or propagate further.

The kinetic relationship between oligomers and fibrils can be quite complex. Despite containing a β-sheet-rich structure, most stable oligomers are not directly on the pathway to fibrils. In fact, the formation of oligomers often slows down fibril formation [19,43]. In addition, both oligomer-specific and fibril-specific small-molecule inhibitors have been identified [46,47]. Inhibition of fibril formation either had no effect on oligomer formation or lead to its acceleration [39,46]. Oligomers are highly dynamic, and oligomers of different sizes are often in rapid equilibrium. In some cases oligomers are in equilibrium with monomers as well, while in others significant kinetic barriers prevent their conversion to monomers. Structural heterogeneity of amyloid oligomers is highlighted by conformation-specific antibodies that often recognize only small subpopulations of oligomers [42,48–50].

The complexity of protein aggregation allows proteins to give rise to a great variety of conformations in the aggregated state. Here we discuss the mechanisms of aggregation of several proteins for which this process has biologic importance and where information about the structure of the aggregates is available.

Protein Aggregation Pathways

Amyloid Beta, Aβ

Aggregation of the amyloid beta peptide is probably the most extensively studied aggregation process due to an important role played by this peptide in Alzheimer’s disease. Amyloid β peptide is created by proteolytic cleavage of the amyloid precursor protein. Depending on the cleavage site, multiple Aβ variants are present in vivo, with Aβ40 (40 amino acids long) and Aβ42 (42 amino acids long) by far the most common. Aβ40 is the most prevalent variant in vivo, comprising about 90% of the overall Aβ population. Aβ42 is significantly more amyloidogenic than Aβ40 due to the presence of two additional hydrophobic amino acids at the C-terminus. These peptides aggregate readily and form a variety of fibrillar and oligomeric aggregates. The amyloidogenic core of Aβ is formed by residues 9–40 (or 9–42 in the case of Aβ42). These residues usually form two β-strands separated by a loop. The exact location and length of the loop varies, but it is usually located between residues 23 and 30.

Aβ peptides form several structurally distinct types of amyloid fibril [51–53]. In fact, one study identified at least five different types of Aβ40 fibril [52]. Incubation of the Aβ40 peptide in buffer, with shaking, results in the formation of fibrils consisting of striated ribbons. Each filament of this fibril contains a β-helix formed by two β-strands, as determined by solid-state NMR [54]. Fibrils formed by the same peptide in quiescent conditions have a twisted morphology and consist of three cross-β strands [29]. Both of these types of fibril are formed from parallel, in-register β-sheets (Fig. 1.4). However, it was determined by solid-state NMR that the D23N mutant of the Aβ40 peptide, associated with early-onset Alzheimer’s disease, is capable of forming antiparallel fibrils in addition to parallel ones [25]. Fibrils with antiparallel β-sheets were shorter and more curved compared to the ones with parallel β-sheets, a possible indication of their lower stability. The structure of Aβ42 fibrils was also determined by NMR [55] and was found to be quite similar to the structure of Aβ40 fibrils with two β-strands.

FIGURE 1.4 Amyloid fibrils formed by amyloid beta peptide. (A) Ribbon representation of fibrils with twisted morphology. (B) Atomic representation of fibrils with twisted morphology viewed down the fibril axis. Hydrophobic, polar negatively charged and positively charged amino acids are green, magenta, red and blue, respectively. Unstructured residues 1–8 are omitted. (C) Comparison of twisted (upper) and striated ribbon (lower) fibril morphologies by TEM. (D) Atomic representation of fibrils with striated ribbon morphology viewed down the fibril axis. Reproduced with permission from reference 29.

In addition to fibrils, Aβ peptides aggregate into a variety of oligomeric and protofibrillar structures. Stable Aβ oligomers range in size from dimers [56] to very large aggregates [50,57,58]. Many of these oligomers are cytotoxic and their increased levels correlate with the severity of Alzheimer’s disease. Thus, they are believed to play an important role in the pathogenesis of the disease. Aβ oligomers have been identified by their size, by the method of preparation, and by their structure. Reliance on the oligomer’s size works well for small oligomers (dimers to tetramers) but not for larger ones as their size is highly variable. Preparation of the oligomers using a specific protocol in vitro can provide reproducible oligomer populations but they are often highly heterogeneous [59,60]. Conformational antibodies allowed the researchers to distinguish different structural classes of oligomer and to improve preparation methods to provide relatively pure preparations of structurally similar oligomers [50,61]. None of the stable Aβ oligomers were found to be directly on the pathway to fibrils, and in many cases it was possible to specifically inhibit the formation of either oligomers or fibrils [19,39,46,62].

Aβ dimers isolated from Alzheimer’s disease (AD) brains were shown to cause neurodegeneration [56]. Dimers and slightly larger oligomers (up to tetramers) were trapped by crosslinking [63–65]. All of them were cytotoxic, with cytotoxicity increasing with increased oligomer size from dimers to tetramers [65]. While the structure of these oligomers is unknown, molecular modeling has shown that they are primarily disordered with some elements of β-structure [66,67]. Ion mobility mass spectroscopy indicated that Aβ40 and Aβ42 form different oligomeric intermediates [68]. In addition, Aβ oligomers with the same molecular weight, yet with a different degree of compactness, were detected, an indication of multiple competing aggregation pathways [69,70].

Higher-molecular-weight Aβ oligomers can be separated based on their method of preparation or reactivity with conformation-specific antibodies. Reactivity with conformation-specific antibodies allowed the researchers to separate Aβ oligomers into three broad classes: fibrillar, prefibrillar and annular. Fibrillar Aβ oligomers reacted with fibril-specific conformational antibodies and ranged in size from trimers to high-MW oligomers and had a high β-sheet content; their structure was predicted to be similar to that of fibrils with some disruptions in the β-sheet stacking that prevent elongation of oligomers into fibrils [19,40,71]. Prefibrillar Aβ oligomers were highly cytotoxic and were shown to form a β-barrel-like structure by X-ray crystallography [36]. Cytotoxicity of these oligomers was attributed to out-of-register β-sheets present in their structure [35].

Primarily disordered Aβ oligomers were formed when the peptide was aggregated in the presence of aromatic compounds such as resveratrol or EGCG [39,41,72]. These oligomers were essentially nontoxic. Detailed NMR analysis of oligomers formed in the presence of EGCG found that they were only partially disordered, preserving the β-sheet structure for residues 22–39 [38]. Many other oligomeric preparations of Aβ (ADDLs, globulomers, annular oligomers, etc.) have been identified but very little structural information about them is available [45,61,73].

α-Synuclein

The structure of α-synuclein fibrils was first determined by X-ray diffraction of synthetic human synuclein filaments and filaments extracted from DLB and MSA brains [74]. More detailed structural information was obtained by X-ray crystallography of microcrystals of fibrils formed by fragments of α-synuclein [23,24]. Together, these studies revealed that α-synuclein fibrils, similar to most fibrils formed by IDPs, are composed of several protofilaments containing a cross-β structure in which β-strands are arranged in parallel and the β-sheets are in-register with highly ordered amino acid side-chain patterns exposed on the surface of the β-sheets.

Subtle changes in buffer conditions such as the pH, temperature, ion concentration – and external variables such as agitation or toxins – can drastically influence the folding and aggregation processes of α-synuclein. Cryo-electron microscopy and solid-state NMR revealed that the morphology of both recombinant α-synuclein (30–110 fragment) fibrils and filaments extracted from PD patient brains can be classified as either (1) straight or (2) twisted ribbons [75,76]. At the molecular level, both types of fibril share a common core of five-layered, parallel, in-register β-sheets that consists of a five-layered β-sandwich [75,77]. However, these two types of fibril differ significantly in the arrangements of protofilaments. Solid-state and quenched H/D exchange NMR further proposes that the straight fibrils have protofilaments (β4 and β5) aligned unidirectionally with each other to form a fibril, whereas in the twisted fibril type, two protofilaments (residues 20–30) twist around each other, giving rise to a sub-protofilament that can twist again with another sub-protofilament to form a fibril. These results show how the differences in the fibril architecture at the molecular level are translated into differences in their morphology. Propagation and spreading of misfolded synuclein was reported in cases where human PD patients had received embryonic nigral transplants. In these cases, the embryonic dopaminergic neurons grafted into PD patients developed α-synuclein inclusions and had reduced dopamine transporter activity over a period of 14 years [78]. The addition of amyloid fibrils prepared from recombinant α-synuclein induced the PD-like pathology in wild-type mice, indicating the ability of α-synuclein fibrils to propagate in vivo [79].

Oligomers of α-synuclein, similar to those of other amyloidogenic proteins, are structurally highly diverse. Recent studies examined several preparations of α-synuclein oligomers and obtained some structural information for them. For example, Giehm and coworkers identified wreath-like oligomers with a diameter of approximately 18 nm [80]. These oligomers were able to disrupt the cell membranes and they assembled easily into fibrils. Hong et al [81] identified the oligomers that formed in parallel with fibril formation. Their morphology depended on the salt concentration in solution. These oligomers did not incorporate into fibrils but disrupted the lipid membranes. Apetri et al [82] found that oligomers formed at the early stages of α-synuclein aggregation are primarily α-helical. Seeding of endogenous α-synuclein is not specific to the fibrils, but was also previously described for α-synuclein oligomers, in which three distinct types of oligomer promoted intracellular oligomer seeding of endogenous α-synuclein in cultured human neuroblastoma (SH-SY5Y) cells [83].

Yeast and Fungal Prions

Several yeast and fungal proteins (e.g. Sup35, Ure2) have been found to exhibit prion-like behavior where the changes in phenotype were transmitted from mother to daughter cells in non-Mendelian fashion. These proteins have a variety of functions, but many of them are involved in transcription. For example, Sup35p is part of a transcription termination complex, and Ure2p is a transcription repressor [84]. Non-Mendelian inheritance is driven by conversion of these proteins into self-propagating amyloid fibrils. Aggregation of these proteins into the fibrillar form leads to loss of their biologic activity, resulting in the observable phenotype. Fungal prion proteins contain prion domains that participate in the formation of the fibril core. These domains are usually large (60–100 residues), intrinsically disordered, and rich in N and Q residues. However, one of the prions (Mod5) has a prion domain of only 24 amino acids in length and it is not enriched in N or Q amino acids [85]. Some fungal prions (e.g. HetS) are beneficial for the organism’s survival [86] while others (e.g. Sup35) do not appear to have an effect [84].

Solid-state NMR and EPR data have shown that fungal prions form parallel, in-register fibrils. The prion domain is converted to the β-structure while the structure of the rest of the protein is preserved [87]. It has been proposed that Sup35 forms β-helical fibrils instead with head-to-head and tail-to-tail linkage of the monomers [5], but more recent data make this model unlikely [53]. Amyloid fibrils formed from these proteins in vitro preserve a significant proportion of infectivity of prions isolated from the yeast cells [88]. Yeast prions form distinct strains that differ in the efficiency of their propagation. ‘Strong’ strains (e.g. those propagating more efficiently) are generated by fibrils with lower stability towards denaturation and a shorter amyloid core [5,89]. Faster propagation of fibrils belonging to strong strains has been attributed primarily to their much higher fragmentation rate compared to more stable fibrils [89]. Chaperones (Hsp104, Hsp40 and Hsp70) also play an important role in the yeast prion propagation by disaggregating fibrils into short fragments required for seeding [17,18].

Oligomers of fungal prions have been proposed to play an important role in prion formation and propagation. It has been proposed that the formation of the initial fibril seeds from Sup35 occurs via a primarily disordered oligomeric intermediate [5,90]. In addition, it has been reported that oligomers, rather than fibrils, serve as seeds for prion propagation in live yeast [91–93]. However, the structure of these oligomers is unknown, and it is possible that they may be either small fibril fragments or β-sheet-rich self-propagating oligomers described for other proteins [19,40].

Mammalian Prions

Prions became famous as the first identified protein-based infectious agents, causative agents of kuru and transmissible spongiform encephalopathy (‘mad cow disease’). Later it was shown that these diseases are transmitted by the aggregates of prion protein (PrP), a ubiquitous membrane-associated protein believed to play a role in copper metabolism [94]. Amyloid fibrils prepared from recombinant PrP have been shown to initiate the infectious disease in animal models, confirming that this disease is spread by PrP aggregation [95–98]. While it was initially believed that brain-derived cofactors are necessary for prion propagation in vivo, it was eventually discovered that recombinant PrP fibrils can become highly infectious either after multiple passages in vivo [96,99] or in the presence of cofactors such as RNA [95] or phosphatidylethanolamine [97].

PrP consists of a disordered N-terminal region of about 100 amino acids in length and a structured C-terminal region 120–160 amino acids in length. During PrP aggregation, primarily the α-helical C-terminal domain of PrP has been shown to unfold completely and to refold into a β-sheet-rich fibrillar structure [100,101]. Protease digestion, EPR and H/D exchange have shown that PrP fibrils have a parallel, in-register β-sheet-rich core extending to residues 160–220 [102–104] (Fig. 1.5). Interestingly, residues 90–120 and 220–240, located just outside the fibril core, still preserve some degree of stable structure and were found to initiate the nucleation process [105]. H/D exchange NMR [106] and other methods [107] have shown structural similarity between the infectious PrP aggregates isolated from the brain (PrPSc) and amyloid fibrils formed by this protein. While the amyloid core in recombinant PrP fibrils covers residues 160–220, in PrPSc the amyloid core extends further into the N-terminal domain [106]. However, PrP fibrils prepared in less denaturing conditions also have an extended amyloid core, indicating a possible similarity of the structures of PrPSc and these fibrils [108]. While PrP fibrils were more stable towards denaturation compared to PrPSc, upon their passage in animals their stability decreased to match that of PrPSc [109].

FIGURE 1.5 Parallel, in-register model of mammalian prion fibrils. Adapted with permission from reference 158.

A variety of PrP oligomers have been identified as well. PrP oligomers formed at low pH were β-sheet rich but had lower stability and a lower extent of β-structure compared to fibrils [43,110,111]. These oligomers were not on the pathway for fibril formation. Thermal unfolding of PrP resulted in the formation of oligomers in which parts of the C-terminal domain (residues 148–175) were unfolded and then refolded into a β-structure [112,113]. PrP oligomers were also detected in vivo but their structure is unclear [114]. Both PrP oligomers and fibrils are cytotoxic, although their toxicity is highly dependent on PrPC expression by the target cells [115]. Oligomers were also shown to activate the complement pathway by interacting with the C1q protein [116].

Insulin

Insulin is resistant to aggregation under physiologic conditions. However, a high concentration of insulin, a low pH and/or a high temperature can trigger its conversion to amyloid fibrils. Insulin fibrils have been found at the insulin injection sites in diabetic patients [117,118]. Aggregation is also a problem for pharmaceutical preparations of insulin. Amyloid fibrils have been prepared from insulin under a variety of conditions. It is important to note that the structure of insulin, prior to aggregation, is strongly condition-dependent. At neutral pH, and in the presence of Zn²+, insulin is primarily hexameric. At low pH it dissociates into dimers and eventually monomers [119,120]. Like many other folded proteins, the insulin molecule completely unfolds before incorporation into a fibril [119]. The resulting fibrils do not preserve any of the structure of the insulin monomer and are parallel, in-register stacks of β-sheets [121]. Residues from both the A and B chains of insulin (A13–A19 and B9–B19) are believed to form the fibrillar core [121,122]. Small differences in insulin conformation in the fibrillar fold have been shown to lead to the formation of insulin fibrils belonging to separate, self-replicating strains [123–125].

Insulin oligomers have been detected as intermediates in the fibril formation process [126,127] but they have not been well characterized. Insulin oligomers have been shown to disrupt neurite outgrowth from PC-12 cells. Their secondary structure was reported to consist of approximately equal proportions of β-sheets and random coil [127]. At high pH, insulin fibrils disaggregated into a mixture of native-like monomers and oligomers with a partially disordered structure [128]. These oligomers were capable of seeding insulin fibril formation.

Functional Amyloids

Ease of assembly and high stability of amyloid fibrils make them attractive biomaterials. Recent discoveries have shown that amyloid fibrils are useful in biologic systems for protein sequestration, surface adhesion and other purposes.

Extracellular Bacterial and Fungal Amyloids

Curli are extracellular protein fibers produced by enteric bacteria. They play an important role in surface adhesion and biofilm formation. Curli are amyloid fibrils with a cross-β structure. However, they do not possess a parallel, in-register β-sheet architecture but are instead β-helical [129]. Curli amyloid formation is tightly controlled to prevent accumulation of potentially cytotoxic oligomeric intermediates [130]. Outer-cell-membrane-associated protein CsgB serves as a seed for curli fibrils. Fibrils themselves are composed of the related protein, CsgA. Other proteins (CsgE–G) are required for transport of curli components to the extracellular membrane and their efficient polymerization. The amyloidogenic domain of CsgA contains five N/Q-rich repeats of about 20 amino acids in length with highly similar sequences. Each repeat is predicted to form a β-strand-loop-strand motif. Both N-terminal and C-terminal repeats are required for aggregation in the presence of CsgB, while repeats in the middle of the sequence are optional [131]. Both CsgA and CsgB subunits can seed aggregation of CsgA from other bacteria despite significant sequence differences [132]. This property is likely to be important in biofilm formation because many types of bacteria often form a biofilm together. Other bacterial proteins (FapC, TasA) are involved in bacterial biofilm formation [130]; they appear to be structurally similar to curli proteins.

Other bacterial and fungal amyloids are used by the producing organisms for surface modification. Hydrophobins are the proteins produced by filamentous fungi. They self-assemble at air–water interfaces and form aggregates (rodlets) that are structurally similar to amyloid fibrils [133]. The presence of these aggregates protects fungal spores from wetting and also facilitates their dispersion. As monomers, these proteins form β-barrels stabilized by multiple disulfide bonds [133]. Upon aggregation of a hydrophobin, EAS residues 64–75 convert from the initially disordered conformation to an antiparallel β-sheet core. The rest of the protein largely preserves its original structure [134,135]. Other types of fungi produce amyloidogenic proteins called repellents that perform a function similar to that of hydrophobins despite their lack of sequence similarity [136].

Some functional amyloids utilize the inherent toxicity of the amyloid oligomers. Some of the microcins (anti-microbial peptides produced by bacteria) function by forming oligomeric pores on the bacterial membrane leading to cell death [137]. These toxic oligomers have been proposed to be pentameric [138]. For example, microcin E492 can reversibly convert from amyloid fibrils into soluble, cytotoxic oligomers depending on the environmental conditions such as peptide concentration, pH and temperature [139]. Amyloid fibrils may then serve as storage vehicles for toxic oligomers. Interestingly, mammalian anti-microbial peptides such as LL-37 may act by the same mechanism as they form both toxic oligomers and non-toxic fibrils [140,141]. In addition, a similar role has been suggested for amyloid beta peptides [141].

Mammalian Functional Amyloids

Neuronal cytoplasmic polyadenylation element binding protein (CPEB) plays an important role in memory formation. It has been shown that this protein can form prion-like self-propagating aggregates in vivo. It has been proposed that the aggregated form of CPEB induces mRNA translation, a process which is important for long-term synaptic facilitation [142]. The N-terminal domain of this protein is enriched in glutamines and is highly amyloidogenic [143]. Both full-length CPEB and its amyloidogenic domain have been shown to form multiple distinct strains of amyloid fibril that were capable of self-propagation by seeding in vivo [144]. This process may serve as a basis for long-term memory formation.

Pmel17 is a protein present within melanosomes. It forms amyloid fibrils that are believed to serve as templates for melanin deposition. One of the domains of this protein, containing 10 imperfect repeats of the same sequence, is believed to be involved in amyloid formation [145] although other regions of the protein have been implicated as well [146]. Fibrils formed by this domain are parallel, in-register β-structures. However, they are highly polymorphic, presumably due to the presence of a long amyloidogenic domain that allows the size and location of the amyloid core to vary significantly between the fibril strains [147,148].

Many secretory peptide hormones are stored in secretory granules at high concentration. Maji and coworkers have shown that these peptides (e.g. ACTH, β-endorphin and bombesin) are stored as amyloid fibrils [149]. Hormone aggregation may be either initiated spontaneously, at high peptide concentration, or accelerated by glucosaminoglycans present in these vehicles. Because multiple hormones are often present in the same vehicle, more amyloidogenic peptides may seed aggregation of less amyloidogenic ones. Amyloid formation is fully reversible, and upon release from the granules the peptides dissociate into the active monomers.

β-2 Microglobulin

β-2 microglobulin is a light chain of the major histocompatibility complex; it contains 99 amino acids and has a β-sandwich fold with seven antiparallel β-strands. Aggregation of this protein is often a problem during kidney dialysis. Aggregation of β-2 microglobulin at neutral pH depends on isomerization of the His31–Pro32 bond from cis to trans conformation, leading to partial unfolding of one of the β-strands and exposing a highly aggregation-prone region of the protein [150]. Cu²+ ions are especially effective in catalyzing this rearrangement because they bind to His31 with micromolar affinity and can stabilize partially unfolded conformations [151,152]. The role of Cu²+ ions in β-2 microglobulin aggregation has been confirmed by the structure of a Cu²+-bound β-2 microglobulin hexamer [37]. In these hexamers, the His31–Pro32 bond is in the trans conformation, resulting in a partial rearrangement of the protein structure and formation of the intermolecular interface (Fig. 1.6). Aggregation of β-2 microglobulin at acidic pH involves more extensive unfolding of the protein. Oligomers of up to hexamer size were observed, and in fact a hexamer has been proposed to serve as a critical nucleus for fibril formation in these conditions [8].

FIGURE 1.6 Hexamer of β-2 microglobulin. Reproduced with permission from reference 37.

Oligomers of β-2 microglobulin give rise to fibrils. Fibril morphology is highly variable, depending on the conditions of their formation. At least two fibril morphologies have been identified for β-2 microglobulin: long straight fibrils (formed at pH below 3 or above 7) and worm-like fibrils (formed at pH 3–5). NMR data indicate that both types of fibril contain an extensive parallel in-register β-sheet core with a trans-P32 conformation [153]. Protein conformations were substantially similar in long straight fibrils formed at either pH 7.5 or pH 2.5 [153–155]. However, for worm-like fibrils, protein conformation was somewhat different, with a shorter amyloid core and a more flexible overall molecular structure [156]. EPR data indicated that β-sheets in worm-like fibrils are likely to be antiparallel [154]. NMR and EPR data show some differences between the arrangement of β-sheets in the long straight fibrils and in the monomeric form of β-2 microglobulin. Some structural rearrangement is required to accommodate the transition from antiparallel β-sheets in the monomer to parallel in fibrils. However, the data are inconclusive on the extent of this rearrangement [153–155].

Other Amyloidogenic Proteins

Protein aggregation plays an important role in many diseases and biologic processes. We have not discussed aggregation of many biologically important proteins in this chapter owing to lack of space and limited information available on the structure of aggregates and the mechanism of aggregation for these proteins.

Conclusions

Intermolecular interactions in the aggregated state allow proteins to form stable aggregates with a variety of different structures and morphologies. While fibrils are composed of stacks of β-sheets, even minor variations in sheet packing may result in the formation of different strains of fibril with often significantly different morphology and other properties. Oligomers are even more structurally diverse, although they also usually rely on β-sheets for intermolecular interactions.

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Part I

Nanoimaging and Nanotechnology of Aggregating Proteins: A. In Vitro Approaches

Outline

Chapter 2 Amyloid Fibril Length Quantification by Atomic Force Microscopy

Chapter 3 Imaging Nucleation, Growth and Heterogeneity in Self-Assembled Amyloid Phases

Chapter 4 Molecular-Level Insights into Amyloid Polymorphism from Solid-State Nuclear Magnetic Resonance

Chapter 5 Single-Molecule Imaging of Amyloid-β Protein (Aβ) of Alzheimer’s Disease: From Single-Molecule Structures to Aggregation Mechanisms and Membrane Interactions

Chapter 6 Nanomechanics of Neurotoxic Proteins: Insights at the Start of the Neurodegeneration Cascade

Chapter 7 Reporters of Amyloid Structural Polymorphism

Chapter 8 Conformation-Dependent Antibodies