CHEMICAL BIOLOGY

Protein Misfolding, Amyloid Formation

Andisheh Abedini, Josiin Diabetes Center, Harvard Medical School, Boston, Massachusetts

Daniel P Raleigh, Department of Chemistry and Institute for Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook, New York

doi: 10.1002/9780470048672.wecb484

Amyloids are β-sheet rich fibrillar protein structures that result from the self-assembly of polypeptides and proteins. A wide range of proteins are known to form amyloid fibrils in vivo and an even larger number do so in vitro. More than 20 different human diseases involve amyloid formation, and the amyloid fibril or intermediates populated during its assembly are cytotoxic. All amyloid fibrils share common structural features despite exhibiting considerable variation in primary sequence. These features include a crossed β-sheet organization in which the individual β-strands are arranged perpendicular to the fiber axis such that the hydrogen bonds are oriented parallel to the fibril axis, resistance to proteolysis, and the ability to bind to certain dyes. Normally, the aggregation of proteins into amyloid is a pathological event; however, evidence indicates that naturally occurring amyloids may also play a beneficial biological role in vivo.

A wide range of human diseases result from the inability of specific proteins to fold into their correct biologically active three-dimensional structures or result from the failure of proteins to remain in their properly folded states. These conditions are referred to broadly as protein misfolding diseases, and they result from a variety of causes. In some cases, the efficiency of folding may be compromised by a range of posttranslational events that lead to insufficient production of active proteins; however, many protein misfolding diseases are caused by the transformation of normally soluble proteins or polypeptides into ordered aggregates. The latter diseases are referred to commonly as amyloidoses. They represent a large group of diseases characterized by the deposition of insoluble ordered protein deposits that are known as amyloid fibrils or amyloid plaques (Table 1). The term “amyloid” was used first by Rudolph Virchow in 1854 when he used the word to describe a macroscopic tissue abnormality that exhibited a positive iodine staining reaction. For reviews of the history of amyloid, see References 1 and 2. Today the term amyloid refers to a specific type of protein quaternary cross-β structure that results from the self-assembly of peptides, polypeptides, and proteins into ordered aggregates. The first recognition of amyloid as something more than just an amorphous deposit was made in 1927, when polarized light microscopy was used to show that Congo Red-stained amyloid from a variety of tissues exhibited positive birefringence (3). These early observations of Congo Red birefringence promoted electron microscopic (EM) investigations of human amyloid. The first EM studies revealed a common fibrillar ultra structure among the various amyloid deposits investigated (4). Since then, our understanding of the general architecture of amyloid fibrils has increased dramatically with the advancement and refinement of experimental techniques (5-12). Research into amyloid has exploded in recent years into a large and dynamic field that continues to grow and to develop. Because space limitations prevent a comprehensive discussion of this fascinating area of chemical biology, we will avoid in-depth discussions of specific amyloid diseases, or of the computational and theoretical studies that are emerging. The development of inhibitors of amyloid is an active area of research, but space limitations prevent a detailed discussion. We do note, however, that several amyloid inhibitors are in various stages of clinic trials, and their development bears watching.

In this article, we provide a brief overview of the fundamental aspects of amyloids by focusing on the common features of amyloid fibrils, which include their mechanisms of formation and their cytoxicity. From the vast and ever-growing amyloid literature, we cite several key papers and some interesting historical references, and we provide citations to several recent review articles.

Table 1. Prevalent pathological and functional amyloid and amyloid-like structures, and their major protein components

A Diverse Range of Proteins Form Amyloid

Amyloids are formed when normally soluble proteins or polypeptides aggregate and deposit as amyloid plaques in the tissues of affected individuals. In vivo, amyloid deposits consist of one major protein and, usually, a set of common minor components that are derived largely from building blocks of the basement membrane, which includes proteoglycans (10, 13, 14). Minor protein components of in vivo amyloid deposits include serum amyloid β component, collagen, and apolipoprotein E. In vitro, highly purified proteins can self-assemble into amyloid fibrils in the absence of these other components. A wide range of proteins are known to form amyloid in vivo, and even more proteins can form amyloid in vitro (Table 1). Indeed, it has been proposed that nearly all proteins can form amyloid under appropriate conditions, and the cross-β structure has been hypothesized to represent a default free energy minimum for the conformation of a polypeptide chain (15). Thus, it has been suggested that amyloid was an important early fold in pre-biotic evolution and perhaps has existed for as long as proteins (16).

Despite the vast differences in the amino acid sequence of amyloid-forming polypeptides, all amyloid fibrils share common structural features and ligand binding properties. These properties include a crossed β-sheet organization in which the individual β-strands are arranged perpendicular to the fiber axis such that the hydrogen bonds are oriented parallel to the fibril axis, resistance to proteolysis, and the ability to bind to the dyes Congo Red and thioflavin-T (5-12, 17, 18). Typically, amyloid fibrils are 5 to 10 nm wide, unbranched, and variable in length. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies have shown that amyloid fibrils are usually made up of protofilaments, each of which are typically 2-5 nm in diameter. Normally, the protofilaments twist together into rope-like structures with 2-6 protofilaments per fibril or self associate in a lateral fashion to generate long ribbons (7, 10, 11). The submicroscopic packing of the protofilaments varies according to differences in the primary amino acid sequence of the polypeptide and the environmental conditions in which amyloid formation is triggered. Because of the noncrystalline and insoluble nature of amyloids, it has been difficult to obtain detailed molecular structures of amyloidogenic proteins in their fibril form using conventional biophysical techniques such as X-ray crystallography and solution-state nuclear magnetic resonance (NMR) spectroscopy. Solid state NMR methods have been applied to the study of amyloid fibrils and have proven to be enormously useful, most notably in studies of the AP peptide (8, 19, 20). Solid-state NMR studies of synthetic amyloid fibrils have demonstrated that fibrils formed from peptides can be composed of parallel or antiparallel β-sheets, depends on the length and amphilicity of the peptide (20). Exciting recent work has led to high-resolution X-ray structures of several 7-mer polypeptides derived from amyloidogenic proteins (9, 12). These new structures reveal an extremely well-packed interface between pairs of β-sheets and the expected cross-β structure. It is not yet clear that these exceptionally well-packed structures will be representative of amyloid fibrils formed by larger polypeptides and proteins. One theory is that large globular proteins, like lysozyme (21), β2 microglobulin (22), and cystatins (23, 24) undergo three-dimensional domain swapping during amyloid fibril formation. Studies of amyloid-like fibrils of ribonuclease A with a Q₁₀ hinge-loop expansion demonstrate domain swapping and functional native-like domains. This study indicates that the native-like conformation of the primary sequence can be maintained in the non-cross-β regions of the protein chain (25). It is, however, not clear whether the subunits of other domain-swapped amyloid-like fibrils might also consist of native-like polypeptide structures. How the non-cross-β regions of the protein chain are accommodated in the amyloid fibril is still unknown.

The highly ordered structures of amyloids allow the specific binding of histological dyes like Congo Red and thioflavin-T (3, 17, 18). Dye-binding studies have played a major role in amyloid research and form the basis of simple convenient assays; however, they are not without their pitfalls. For many years, Congo Red birefringence has been the standard test for the presence of amyloid (3, 17). Recent studies argue that Congo Red also binds to native proteins and lacks secondary structure specificity, which indicates that the dye is not specific for amyloid (26). Congo Red can also affect the rate of amyloid formation, either enhancing or inhibiting fibril formation, which emphasizes even more that it should be used with caution as a diagnostic tool for studying amyloid fibrils in vitro (27). Thioflavin-T is also used extensively for characterizing the presence of amyloid fibrils and their rate of formation (18). This dye, which can be detected by fluorescence, is a better alternative for in vitro fibril detection than Congo Red. Studies by confocal microscopy that use polarized light have indicated that thioflavin-T binds to amyloid with the long axis of the dye parallel to that of the fibril axis (i.e., perpendicular to the direction of the individual strands). The dye likely binds by inserting itself into the grooves formed between sidechains at positions i and I + 2 on the surface of the cross-β structure (28). For a flat β-sheet, approximately four strands are sufficient to generate a groove long enough to accommodate one molecule of thioflavin-T. It is thought that the steric constraints imposed by these surface grooves account for the relative specificity of the dye for the crossed-β structure of amyloid fibrils.

Medically, amyloid diseases can be divided into three broad classes: neurodegenerative, systematic, and local amyloidosis (10). In neurodegenerative diseases, amyloids are deposited in the brain. Important examples in this class include the spongiform encephalopathies, Huntington’s, Alzheimer’s, and Parkinson’s disease. In systemic amyloidoses, aggregation occurs in multiple organs and tissues. A subset of systemic amyloidoses includes senile systemic amyloidosis; lysozyme amyloidosis; AL amylodosis, which is caused by the aggregation of immunoglubin light chains or fragments thereof; familial transthyretin-associated amyloidosis, which develops from deposition of wild-type or one of more than 50 mutated forms of transthyretin (TTR); and diseases of chronic inflammation, in which an N-terminal fragment of the acute phase protein serum amyloid A forms amyloid deposits. In the non-neurological, localized amyloidoses, amyloid deposition occurs in one target organ, which is usually proximal to the production site of the amyloidogenic peptide. Common examples of this third class include medullary thyroid carcinoma, which is associated with amyloid deposition of (pro)calcitonin; artial amyloid, which is caused by artial natriuretic factor; amyloid formation by the γ-crystallins associated with cataract; type 2 diabetes, which is characterized by deposition of islet amyloid polypeptide (IAPP also known as amylin); as well as many others (Table 1). Some diseases are sporadic whereas others are hereditary. The association between amyloid fibril formation and disease pathogenesis is common for all amyloidoses, and the cytotoxic properties of amyloidogenic peptides are well documented (29, 30). The exact mechanism of amyloid-induced cell death is, however, still not completely clear.

The proteins and polypeptides that form amyloid can be divided into two broad structural classes: namely those that adopt a well-defined tertiary fold in their normally soluble state and those that are flexible and intrinsically disordered in their unaggregated state. Normally, proteins that adopt a well-defined globular fold in their unaggregated state require a partial unfolding event to become aggregation competent. Particularly well-characterized examples include TTR, β2 microglobulin (responsible for dialysis-related amyloid), and lysozyme (21, 22, 31-35). Molecules that bind and stabilize the native states are potential therapeutic agents for amyloid diseases that develop from folded proteins, because they reduce the tendency to populate the amyloidogenic precursor states (31). In some cases, natively unfolded polypeptides also seem to require a partial fold to form amyloid. Studies on tau (associated with neurofibrillar tangles) and AP (which forms amyloid deposits in Alzheimer’s disease) suggest that amyloid formation in these systems is preceded by formation of a helical intermediate (36, 37).

Common Features of Amyloid Formation

An increasing number of studies on a variety of amyloidogenic peptides and proteins argue that underlying commonalties exist in mechanisms of amyloid formation at least in vitro, which are independent of the details of the polypeptide sequence. This finding is true for both of the structural classes of amyloids defined above. The exact mechanism of amyloid formation has not been determined fully; however, extensive experimental evidence indicates that amyloid formation proceeds by a variation of the so-called nucleation-dependent polymerization pathway (38, 39). The kinetics of amyloid formation are complex and typically exhibit a lengthy lag phase during which little or no amyloid is formed, which is followed by a much more rapid growth phase. Oligomeric nuclei are formed during the slow lag phase. Once a critical assembly of precursors form an active seed, a second, more rapid phase of fibril polymerization occurs, which leads to the classic amyloid morphology. Amyloid formation can be accelerated substantially by the addition of preformed seeds (40). Often, the lag phase can be abolished by seeding a solution of unaggregated peptide with a small amount of preformed fibrils (Fig. 1). Seeding is generally, although not completely, specific. Fibrils often preferentially, but not always, seed reactions that contain the same polypeptides from which they were formed. This finding indicates that primary structure similarity is important for binding interactions between seeds and polypeptide monomers. Although the general ultrastructure of all amyloids seem similar, clear morphological differences exist between amyloid fibrils formed from different proteins. These variations in morphology are caused by differences in the molecular packing and the organization of fibril subunits, as influenced by the polypeptide primary sequence. Other external environmental factors such as solution pH, ionic strength, temperature, and agitation have also been shown to affect fibrillar morphologies of amyloids produced in vitro.

Figure 1. Schematic representation of the progress of fibril formation. The solid line represents an unseeded reaction. A distinct lag phase is observed followed by a rapid growth phase. Seeding (dashed line) can by-pass the lag phase.

There is a rich experimental and theoretical literature exists on protein assembly and aggregation, and various kinetic models have been used to rationalize the time course of amyloid formation (38-41). Perhaps the classic example of a protein assembly reaction is that of actin, which is described by a nucleation-dependent mechanism. The nucleation-dependent model comprises three steps: 1) association of monomers into oligomers, 2) conversion of the oligomers to a nucleus, and 3) growth of the final fibril by addition of monomers to the end. The nucleation-dependent polymerization model cannot explain all amyloid formation reactions. The existence of the characteristic lag phase is not well predicted. Double nucleation schemes, which were developed initially by Mozzarelli et al. (42) during their pioneering studies of hemoglobin S polymerization, have been invoked to explain the existence of a lag time. Double nucleation models include primary nucleation steps similar to the nucleation-dependent polymerization model, but they also invoke a second nucleation step that is dependent on the presence of fibrils (i.e., the fibrils can act as secondary nucleation sites). The second step is unimportant before fibrils are formed but becomes increasingly important as the amount of fibrils increases. This mechanism was used early on to model amyloid formation kinetics; however, the dependence of the lag phase on protein concentration in amyloid formation is not always well predicted by this model. In particular, the model predicts a lag time that scales much more strongly with monomer concentration than is sometimes observed. Variants of the original double nucleation scheme have been developed that can account for the weakened concentration dependence. Irrespective of the details, the view that amyloid formation proceeds by a cooperative, nucleation-polymerization mechanism is widely accepted (10, 40).

The structure shared by amyloid fibrils and the common themes in its assembly provide a unifying mechanism of cell toxicity; thus, a better understanding of the amyloid assembly process at the molecular level should give invaluable insight into the identification and the development of effective therapeutic innovations for a wide variety of human amyloid diseases.

Amyloid fibrils or their precursors are toxic and are directly responsible for cell death. The majority view in the field is that partially structured oligomers that appear as intermediates in the aggregation process may be the toxic entities, rather than the mature fibrils (43-49). In this view, amyloid may represent a relatively benign state that sequesters the protein, and if true, may suggest that several compounds that have been developed to inhibit the assembly of amyloid fibrils could actually be harmful if they lead to the build up of toxic prefibril intermediates (50). It is important to realize, however, that good evidence suggests both fibrils and prefibril species can be toxic, at least in some cases. For example, the pathological effects in the systemic amyloidoses are caused by disruption of organ function by the formation of large amyloid deposits. In addition, the results of several recent studies are very difficult to reconcile with the “only oligomers are toxic” model. For example, the Tottori and English Familial Alzheimer disease mutations are associated with aggressive early onset of Alzheimer’s disease, yet their effect is to increase the rate of fibril formation without increasing meta-stable intermediates. Thus, a detailed understanding of the mechanism of amyloid fibril formation and the identification of the toxic species and their mode of action are subjects of major importance and are currently the focus of considerable research.

Until very recently, the aggregation of proteins into ordered amyloid fibrils was thought to be a pathological event that leads to cytoxicity and highly debilitating diseases; however, amyloid need not always be deleterious. Amyloid-like structures may have applications in bio-nanotechnology, which includes roles as templates for the assembly of novel structures, as materials, and as cell-supporting matrices (51, 52). In addition, evidence now suggests that naturally occurring amyloids can play beneficial biological roles in vivo. Several functional amyloid fibrils derived from a diverse range of single and multi-cellular organisms have been identified and described recently (49). A list of functional amyloid and amyloid-like structures is provided in Table 1.

A particularly well studied example of functional amyloid is provided by Curli assembly (53). Curli amyloids are assembled by bacteria such as Escherichia coli and Salmonella. Once assembled on the extracellular surface, Curli amyloid fibers function as natural cell adhesion molecules that link together bacterial cells into robust cellular networks of biofilms. Other examples of functional amyloids include the silk fibers observed commonly in spider webs; the Chorion proteins of egg shells; Factor XII, which is an activator of the hemostatic system; and other naturally produced adhesives and materials (54).

In humans, the formation of Pmel17 amyloid has been shown to be important for melanin formation (55). Melanin is a natural pigment for skin coloration, UV protection, and chemical detoxification, and it is synthesized in melanosomes. Pmel17 amyloid fibrils have been shown to protect melanosomes against pigment melanin toxicity by decreasing the diffusion of toxic melanin precursor molecules out of the cell. The formation and the degradation of functional amyloids seem to be highly regulated, which is hardly surprising given the toxicity associated with nonfunctional amyloids (54, 55).

Amyloid structures can also act as nonchromosomal genetic elements, which lead to non-Mendelian inheritance. This finding can have drastic and disastrous effects in the prion diseases. However, several amyloid forming prions have been suggested to be potentially functional, which include the yeast proteins Het-s and β-prion. The Het-s prion, which is found in 80% of wild isolates, carries out heterokaryon incompatibility to presumably prevent infection by incapacitating fungal viruses (56, 57). The β-prion, which has been shown to be necessary for meiosis and for cell survival in the stationary phase (58), has also been proposed to be potentially beneficial for fungal cells. Other prion proteins, like Sup35p, URE2p and Rnqlp, which have been demonstrated to form amyloid reversibly in the cytoplasm in a non-Medelian epigenetic fashion, have been argued to provide evolutionary advantages for host cells (59-61). This hypothesis is intriguing and will no doubt be the focus of much attention.

The study of the assembly, degradation, and regulation of functional amyloids may well provide important clues for controlling aberrant amyloid formation, and a greater understanding of the diverse physiological applications of the amyloid fold may open potential new avenues for the treatment of amyloid diseases. The discovery of functional amyloids has interesting and possibly worrying implications for the treatment of amyloidoses. A common strategy for the treatment of amyloid diseases is to develop inhibitors of amyloid formation. Functional amyloids share a common structure with pathological amyloids, thus, inhibitors could have the unintended side effects of disrupting the assembly of functional amyloids. Whether this issue is critical remains to be determined.

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Further Reading

Dobson CM. In the footsteps of alchemists. Science 2004; 304:1259-1262.

Sipe JS, eds. Amyloid Proteins; the Beta Sheet Conformation and Disease, Volume 1. 2005. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany.

Sipe JS, eds. Amyloid Proteins; The Beta Sheet Conformation and Disease, Volume 2. 2005. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany.

Uversky VN, Fink AL. Protein Misfolding, Aggregation and Conformational Diseases; Part A: Protein Aggregation and Conformational Diseases Series: Protein Reviews. Volume 4. 2006. Springer, New York.

Wetzel R, eds. Amyloid assembly and structure. Special Issue, Acc. Chem. Res. 2006; 39:567-679.

Kheterpal I, Wetzel R, eds. Amyloid, prions, and other protein aggregates. Part C Methods in Enzymology 412. 2006. Elsevier Academic Press Inc., San Diego, CA.

Kheterpal I, Wetzel R, eds. Amyloid, Prions, and Other Protein Aggregates. Part C Methods in Enzymology 413. 2006. Elsevier Academic Press Inc., San Diego, CA.

Smith HJ, Simons C, Sewell RDE. Protein Misfolding In Neurodegenerative Diseases: Mechanism and Therapeutic Strategies. 2007. CRC Press, Boca Raton, FL.

See Also

Prion Diseases: Chemical Biology of

Protein Misfolding: Amyloid Formation

Proteins: Computational Analysis of Structure, Function and Stability

Protein Folding: Chemical Biology of Diseases Related to

Chaperones, Molecular