**3. Fibril structure**

All amyloid fibrils share a basic unit comprising a relatively short and (usually) contiguous stretch of amino acids whose three-dimensional contours can be accommodated in a β-pleated sheet conformation (both parallel and antiparallel assemblies are recognized in fibrils). Hydrogen bonds between the amide groups polarize each other. Van der Waals forces also develop between the β-sheets. Water molecules are displaced from between the faces of the sheets. These important features emphasize that protein precursors of amyloid fibrils are not in their native state—the constituent monomer is in an altered conformation, and this state may only be transient prior to nucleation. Thus, there is an array of polypeptide species that may underlie fibril formation; the most (transiently) stable and/or abundant species is most likely to be captured in a stable β-sheet.

As shown by Riek and Eisenberg [12], amyloids derived from different protein constituents may have very different β-sheet domains. In addition, these regions may be rather small in comparison with the length of the parent protein(s). This implies that only a fragment of the parent (i.e., longer) protein may be found in the ultimate amyloid fibril, and this, in turn, implies that cleavage of the parent protein is often part of the process of fibril formation. **Figure 2** presents example details of the particularly well-studied Alzheimer Aβ(1-42) fibril.

The β-pleated sheet topology of the basic interacting region permits individual regions to stack upon one another in a highly ordered manner—thus extending into the long axis of the fibril (see **Figure 2**). The result is stabilization not only between β-sheet domains but also between stacked subunits. Fibrils often show a twist along their long axis.

Once the requisite region for forming β-sheets becomes available, fibril formation begins as a nucleation event. Usually small numbers of monomers interact, but once the primordial oligomeric fibrillary unit is assembled, further extension can occur by aligning new monomers with the growing fibril surface. The kinetics of this process are consistent with this scheme, there generally being a lag in assembling the oligomer followed by more rapid extension as subunits are added.

Most amyloid fibrils share relatively common dimensions although their lengths often vary. Because the fibrils are so tightly associated within their common nuclei of β-sheet domains, they are themselves rather resistant to dissociation (substantial free energy of formation) as well as to attack by proteases (regions susceptible to

#### **Figure 2.**

*Details of Alzheimer Aβ(1-42) fibril emphasizing amino acids 17-42. (A) Ribbon diagram of the core. (B) Salt bridges are indicated by the rectangles. (C) View of the "odd" end of the growing fibril showing the van der Waals surface as well as the amino acid side chains. The fibril axis is shown, corresponding to that in A and B. (D) Simulation of a fibril with four filaments (the adjacent figure shows the filament ends and the dimensions). (E) Cryoelectron micrographs of single fibrils [3]. Copyright 2005, National Academy of science, used by permission.*

proteolysis may not be accessible). These molecular features help explain both the chronicity and the progressive nature of amyloid disorders.

Amyloid fibrils characteristically bind planar dye molecules. Upon binding to fibrils, these dyes can show birefringence when viewed using polarized light microscopy, providing a widely used method for identifying amyloid deposits in biologic tissues. The dyes Congo Red and Thioflavin T are used most frequently.

Another feature of amyloid fibrils is that they are usually found associated with other molecules. These include glycosaminoglycans (which may be responsible for calcium binding) as well as the pentraxin protein—"serum amyloid P" (SAP) [13]. These common features permit some imaging techniques to identify amyloid fibrils radiographically. For example, it is often possible to scan for SAP in order to localize amyloid deposits of various types [14].

Reference to **Table 1** emphasizes the variety of proteins that can form the β-pleated sheet structural units of amyloid fibrils. The proteins generally do not share common sequences or large-scale features because the region(s) essential for nucleating fibrils is(are) small (recall **Figure 2**). The protein constituent(s) of fibrils can frequently be determined using specific antibodies. The choice of antibodies to use is generally aided by the location of the deposit(s) and the clinical and/or pathologic context.

### **4. Fibril formation**

As noted above, the fundamental event in amyloid formation is highly specific interaction between adjacent (usually) small protein units to form antiparallel (and, in some cases, parallel) β-sheets. The native state of the parent protein is generally thermodynamically more stable than the amyloid state. However, it is important to note that the stability of the amyloid state depends on protein concentration while that of the native precursor is largely independent of protein concentration. There thus arises a concentration above which the stability of the amyloid state can

**181**

**Figure 3.**

*Pathophysiology of Amyloid Fibril Formation DOI: http://dx.doi.org/10.5772/intechopen.81965*

responsible proteases) have been determined.

and at least some oligomer formation (steps C, D).

proteins <150 aa [5].

become the same as or even transiently greater than that of the native state. Not surprisingly, the complex topology of long proteins makes them unlikely to nucleate and extend into stable fibrils. This generally limits formation of amyloid fibrils to

Once the requisite region for forming the β-sheet becomes accessible, fibril formation begins as a stochastic nucleation event. Usually small numbers of monomers interact, but once the primordial oligomeric fibrillary unit is assembled, further extension can occur by aligning new monomers with the growing fibril surface. The kinetics of this process are consistent with this scheme, there generally being a lag in assembling the oligomer followed by more rapid extension as subunits are added. Because different types of amyloid fibrils have different protein constituents and may be found in different locations, it is not always possible to identify common features. An important distinction is between fibrils that have soluble, generally bloodbased, precursors and those derived from specific intracellular or organ-limited proteins. Among the former are immunoglobulins (usually κ or λ light chains) and serum amyloid A. The latter include hormone precursors such as procalcitonin, prolactin, as well as amylin and the β-protein precursor. Prions generally begin intracellularly. As noted earlier, the protein precursors of amyloid fibrils are usually complex with multiple domains, and the critical, nucleating region is often small. Thus, many must undergo proteolysis in order to eliminate domains that are topologically incompatible with fibril dimensions and structure. In the best-studied examples, proteolysis occurs within lysosomes or related organelles although not all sites (or

**Figure 3** presents a scheme for AA fibril formation from circulating SAA. SAA is usually found in association with high-density lipoprotein in the blood, and it must dissociate and enter the cell, often using clathrin-mediated endocytosis. Within the cell (in lysosomes or related structures), the acidic pH leads to unfolding of the precursor as reactive groups are titrated (steps A–C). This likely exposes region(s) with increased susceptibility to proteolysis. The considerable free energy of antiparallel (or, for some other fibril precursors, parallel) β-pleated sheet formation and the relatively high local concentrations of appropriate domains leads to nucleation

Intracellular oligomeric fibril nucleation ultimately causes organelle disruption followed by loss of cellular integrity (step E). The possibility of exosome participation at this stage has not been excluded. The result is a local mixture of nascent and growing amyloid fibrils and cell debris. Having oligomeric "seeds" already formed intracellularly, fibril elongation can then be extended. The result becomes a mixed,

*Scheme for multiple steps in AA fibril formation from circulating SAA (from [16] see text for details).*

#### *Pathophysiology of Amyloid Fibril Formation DOI: http://dx.doi.org/10.5772/intechopen.81965*

*Amyloid Diseases*

**Figure 2.**

proteolysis may not be accessible). These molecular features help explain both the

*Details of Alzheimer Aβ(1-42) fibril emphasizing amino acids 17-42. (A) Ribbon diagram of the core. (B) Salt bridges are indicated by the rectangles. (C) View of the "odd" end of the growing fibril showing the van der Waals surface as well as the amino acid side chains. The fibril axis is shown, corresponding to that in A and B. (D) Simulation of a fibril with four filaments (the adjacent figure shows the filament ends and the dimensions). (E) Cryoelectron micrographs of single fibrils [3]. Copyright 2005, National* 

Amyloid fibrils characteristically bind planar dye molecules. Upon binding to fibrils, these dyes can show birefringence when viewed using polarized light microscopy, providing a widely used method for identifying amyloid deposits in biologic tissues. The dyes Congo Red and Thioflavin T are used most frequently. Another feature of amyloid fibrils is that they are usually found associated with other molecules. These include glycosaminoglycans (which may be responsible for calcium binding) as well as the pentraxin protein—"serum amyloid P" (SAP) [13]. These common features permit some imaging techniques to identify amyloid fibrils radiographically. For example, it is often possible to scan for SAP in order to localize

Reference to **Table 1** emphasizes the variety of proteins that can form the β-pleated sheet structural units of amyloid fibrils. The proteins generally do not share common sequences or large-scale features because the region(s) essential for nucleating fibrils is(are) small (recall **Figure 2**). The protein constituent(s) of fibrils can frequently be determined using specific antibodies. The choice of antibodies to use is generally aided by the location of the deposit(s) and the clinical

As noted above, the fundamental event in amyloid formation is highly specific interaction between adjacent (usually) small protein units to form antiparallel (and, in some cases, parallel) β-sheets. The native state of the parent protein is generally thermodynamically more stable than the amyloid state. However, it is important to note that the stability of the amyloid state depends on protein concentration while that of the native precursor is largely independent of protein concentration. There thus arises a concentration above which the stability of the amyloid state can

chronicity and the progressive nature of amyloid disorders.

amyloid deposits of various types [14].

*Academy of science, used by permission.*

and/or pathologic context.

**4. Fibril formation**

**180**

become the same as or even transiently greater than that of the native state. Not surprisingly, the complex topology of long proteins makes them unlikely to nucleate and extend into stable fibrils. This generally limits formation of amyloid fibrils to proteins <150 aa [5].

Once the requisite region for forming the β-sheet becomes accessible, fibril formation begins as a stochastic nucleation event. Usually small numbers of monomers interact, but once the primordial oligomeric fibrillary unit is assembled, further extension can occur by aligning new monomers with the growing fibril surface. The kinetics of this process are consistent with this scheme, there generally being a lag in assembling the oligomer followed by more rapid extension as subunits are added.

Because different types of amyloid fibrils have different protein constituents and may be found in different locations, it is not always possible to identify common features. An important distinction is between fibrils that have soluble, generally bloodbased, precursors and those derived from specific intracellular or organ-limited proteins. Among the former are immunoglobulins (usually κ or λ light chains) and serum amyloid A. The latter include hormone precursors such as procalcitonin, prolactin, as well as amylin and the β-protein precursor. Prions generally begin intracellularly.

As noted earlier, the protein precursors of amyloid fibrils are usually complex with multiple domains, and the critical, nucleating region is often small. Thus, many must undergo proteolysis in order to eliminate domains that are topologically incompatible with fibril dimensions and structure. In the best-studied examples, proteolysis occurs within lysosomes or related organelles although not all sites (or responsible proteases) have been determined.

**Figure 3** presents a scheme for AA fibril formation from circulating SAA. SAA is usually found in association with high-density lipoprotein in the blood, and it must dissociate and enter the cell, often using clathrin-mediated endocytosis. Within the cell (in lysosomes or related structures), the acidic pH leads to unfolding of the precursor as reactive groups are titrated (steps A–C). This likely exposes region(s) with increased susceptibility to proteolysis. The considerable free energy of antiparallel (or, for some other fibril precursors, parallel) β-pleated sheet formation and the relatively high local concentrations of appropriate domains leads to nucleation and at least some oligomer formation (steps C, D).

Intracellular oligomeric fibril nucleation ultimately causes organelle disruption followed by loss of cellular integrity (step E). The possibility of exosome participation at this stage has not been excluded. The result is a local mixture of nascent and growing amyloid fibrils and cell debris. Having oligomeric "seeds" already formed intracellularly, fibril elongation can then be extended. The result becomes a mixed,

**Figure 3.** *Scheme for multiple steps in AA fibril formation from circulating SAA (from [16] see text for details).*

**Figure 4.** *Scheme for nucleation of TTR amyloid oligomers.*

largely acellular, region where amyloid fibrils become the predominant structured species (step F) [15, 16].

A contrasting situation can occur in situations where the fibril precursor is not only soluble but also intrinsically capable of β structure formation without cleavage. **Figure 4** shows events for transthyretin (TTR). TTR is a 127 amino acid protein circulating in the blood as a stable tetramer that binds thyroid hormone and retin A (hence, its name). The TTR monomer itself contains prominent β-sheet domains. If the tetramer dissociates, the free monomers can misfold into various forms. Among these, some can associate as oligomers which then can be extended into fibrils. TTR amyloid fibrils are particularly prone to cause dysfunction in nerves and the heart. Interestingly, over 100 amino acid substitutions (i*.*e., mutations) have been identified in TTR [17]. Mutations differentially affect tetramer stability—some increase it, while others reduce it. Among the latter are several that are associated with inherited amyloid diseases, and the Val30Met and Val122Ile mutations have been particularly well-studied (affecting nerves and/or the heart). Kinetic and other evidence implicates the oligomer form(s) as directly involved in organ dysfunction. Amyloid fibrils become detectable by microscopy as the disease progresses. One proposed therapeutic strategy involves developing small molecules that stabilize the circulating tetramer, hence reducing (or eliminating) dissociation, oligomer formation, and tissue toxicity [18].
