The term amyloid was originally introduced with reference to proteinaceous fibrillar structures that accumulate in the extracellular space in various diverse diseases – prominent examples include type 2 diabetes mellitus (pancreatic amyloid deposits), dialysis-related amyloidosis (amyloid deposits in vasculature), and Alzheimer’s disease (amyloid deposits in brain parenchyma and vessels). There are also examples of amyloid fibrils that accumulate intracellularly in certain neurodegenerative diseases – like Parkinson’s disease, Huntington’s disease and other so-called poly-glutamine extension diseases as well as Alzheimer’s disease and frontotemporal dementias (Ross and Poirier, 2004, Mandelkow et al., 2007). Both plasma-derived and cellular proteins form systemic (in many different organs) or organ-specific amyloid deposits that characterize the different forms of amyloidosis. Amyloid fibrils also afford the molecular mechanism that underlies infectious self-propagating protein polymers called prions in mammals (e.g., leading to Creutzfeldt–Jakob disease in humans and scrapie in sheep) and fungal systems (Baxa et al., 2005, Baxa et al., 2006, Baxa, 2008).
Correlation with particular disease states affords a clinical basis for distinguishing various types of amyloid. Interestingly, the proteins that form the major components of these amyloids are relatively few in number (about 20). More generally, many different polypeptides are capable of forming structurally related amyloid fibrils or amyloid-like structures in vitro (Kodali and Wetzel, 2007, Fändrich, 2007, Baxa, 2008). The hallmark of amyloid fibrils is a cross-β structure in which the β-strands run approximately perpendicular to the fibril axis. Further defining characteristics are that they are unbranched, smooth-surfaced, physically and chemically robust (e.g., protease-resistant) and that they bind the dyes Congo Red and thioflavin T.
Cytotoxicity and organ failure in amyloid-associated diseases is attributed to misfolding of the normally soluble polypeptides into aberrant β-sheet conformations (Cardoso et al., 2002, Ross and Poirier, 2004, Novitskaya et al., 2006, Bitan et al., 2005, Konarkowska et al., 2006, Mocanu et al., 2008). Resultant fibril assemblies, metastable intermediates and off-pathway oligomeric aggregates have all been proposed to be cytotoxic (Wogulis et al., 2005, Lesné et al., 2006, Lambert et al., 2007, Novitskaya et al., 2006, Konarkowska et al., 2006, Lansbury and Lashuel, 2006). This has initiated many investigations into fibrillar and oligomer assembly, which have revealed remarkably complex assembly pathways and polymorphic structures (Kodali and Wetzel, 2007, Fändrich, 2007, Baxa, 2008). Due to the complexity of nomenclature in the literature for various amyloid-related aggregates, a glossary of terminology is provided in Box 1.Glossary of terms
Amyloid: Protein deposits in the form of filamentous aggregates that are implicated in a broad spectrum of diseases. Although the deposits share conserved tertiary cross β-sheet structural motifs, the fibril/filament (terms used interchangeably) forming protein and organ involved is disease specific; for example, deposits of Aβ form amyloid plaques in Alzheimer’s disease brains and deposits of amylin (Islet Amyloid Polypeptide: IAPP) form in the islets of Langerhans in the pancreas in the course of type 2 diabetes mellitus. Fibrils can be straight or twisted and sometimes contain multiple protofilaments that align parallel to the fibril axis or coil around each other. The term “amyloid” has been extended by structural biologists to additionally encompass the fibrillization of synthetic and recombinant polypeptides from various sources that also assemble cross β-sheet structure (Kajava et al., 2009).
Protofibril: Intertwining strands that make up mature amyloid fibrils. This term is used interchangeably with the term “protofilament”. However, in the case of Aβ, “protofibril” has additionally been used to describe metastable fibrils that appear early in solutions of this polypeptide in vitro before mature amyloid fibrils appear (Walsh et al., 1997, Harper et al., 1997). These are short flexible fibrils, rods or globular structures with no clear axial periodicities. Although the term implies it, these transient “protofibrils” are not necessarily on-pathway intermediate structures and may represent semi-stable off-pathway assemblies (Kodali and Wetzel, 2007).
Protofilament: Intertwining strands that make up mature amyloid fibrils.
Oligomer: Macromolecular complexes of self-assembled amyloid polypeptides but not in fibril/filament form. Oligomers can be off-pathway stable structures (Gellermann et al., 2008). Other oligomers represent unstable transient nuclei that initiate fibril assembly. When visible in electron micrographs or atomic force microscopy images, stable oligomers resemble globules or ring-like assemblies (Lambert et al., 1998, Nybo et al., 1999; Goldsbury et al., 2000a, Goldsbury et al., 2000b; Lashuel et al., 2003). A complex set of terminologies exists in the literature describing oligomers – for example, for Aβ: ADDLs (Aβ-derived-diffusible-ligands; Lambert et al., 1998), globulomers (n ≈ 12 oligomers; Gellermann et al., 2008) and Aβ∗ (n ≈ 12 oligomers; Lesné et al., 2006).
Paired Helical Filaments (PHFs): Name given to amyloid fibrils formed by tau protein. PHFs have only relatively recently been widely recognized structurally as amyloid fibrils. This is due to the atypical nature of tau as the molecular subunit in the fibrils. Only a relatively small segment of this large protein contributes the β-sheet core structure that forms these filaments (Mandelkow et al., 2007). The rest of the tau protein lies peripherally and does not have β-structure. This made detection of defining cross-β diffraction patterns more challenging. PHFs also form intracellularly, in contrast to the classical extracellular amyloid deposits.
Electron microscopy (EM) of negatively stained or vitrified specimens has been applied successfully to determine the structures of many kinds of protein filaments, but has been relatively unproductive in the field of amyloid. The polymorphism of amyloid fibrils and their tendency to aggregate poses problems. In addition their smooth surface tends to generate little contrast in micrographs recorded by cryo-EM. In this situation, scanning transmission electron microscopy (STEM) has proved a powerful tool helping to delineate the assembly mechanism and structural properties of amyloids. Importantly, STEM is the only method that can directly measure the mass-per-length (MPL) of individual filaments. Working from an image, it also links the MPL to the sample appearance allowing a direct correlation to negative stain or cryo-EM images that reveal structure. If the monomer mass of the subunits that make up a fibril is known, the MPL determined by STEM imposes strong constraints on possible packing schemes within fibrils, assisting in molecular model building (Kajava et al., 2009, Petkova et al., 2005, Paravastu et al., 2008, Sen et al., 2007) and illuminating hierarchical relationships between fibrils of different kinds (Bauer et al., 1995, Goldsbury et al., 2005).
Many different filamentous protein assemblies have been examined by STEM, among them bacterial pili (Hahn et al., 2002, Köhler et al., 2004), viral filaments (Kendall et al., 2008) and neurofilaments (Leapman et al., 1997). The method can also be used to measure the masses of individual particles and, thus, define their stoichiometry. In the field of amyloid, data of this kind document the compositional heterogeneity of oligomeric particles assembled from amyloidogenic polypeptides and proteins (Lashuel et al., 2003, Goldsbury et al., 2000a, Goldsbury et al., 2000b). Further, STEM mass measurements have helped to show that polymorphic fibrils and oligomers can assemble simultaneously from a single polypeptide. Such morphological or molecular-level polymorphisms are believed to underlie the strain phenomena and species barrier in prion transmission, as well as differences in cytotoxicity in amyloid diseases.
