Amyloid Peptides
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What is the Amyloid?
The intriguing family of biopolymers known as amyloids is made up of insoluble fibrous protein aggregates that share unique structural features. These aggregates are formed by non-covalently binding misfolded polypeptides. One distinguishing feature of amyloids compared to other biopolymers like DNA and polysaccharides is that their monomers are relatively big polypeptides that may polymerize into a so-called cross-β structure. Another point is that the monomer-monomer bonds are not covalent. The individual polypeptides that make up an amyloid fibril mostly fold into a β-sheet shape due to misfolding. Protofibrils are formed when β-sheets bind strongly but not covalently. These protofibrils may then assemble into giant fibrils that are several microns long and have a diameter of several nanometers. Under certain circumstances, amyloid formations may be formed by a wide variety of peptides, both naturally occurring and engineered. Neurodegenerative disorders such as Alzheimer's, diabetes, and Parkinson's are often linked to amyloids in vivo. But there are many more uses for amyloids, both natural and man-made. For example, structuring food products.
Amyloid fibrils are biopolymers that share many characteristics with silk. Like silk, they are mostly made up of unstructured precursor proteins and are defined by their mechanical and structural properties rather than their exact chemical make-up. The basic structure of amyloid fibrils, which is a cross-β sheet, is very stiff and gives them exceptional mechanical qualities. The fibrils may have ultimate strength comparable to steel and a Young's modulus like silk. Amyloid fibrils are strong candidates for use as bio-inspired material building blocks because of this. The fibrils may be made naturally from several different types of proteins. Despite extensive chemical and biological alterations, structural and mechanical characteristics of proteins remain remarkably unaffected by changes in their amino acid sequence. Because of their unusual structure, amyloid fibrils may withstand physical stresses, proteases, detergents, denaturants, and very high and low temperatures. It is therefore not unexpected that there has been a recent uptick in study into materials derived from amyloid. In addition to their use as scaffolds for tissue engineering and effective drug delivery vehicles, amyloid fibrils have served as templates for metallic nanowires with potential applications in molecular electronics. Because of their resistance to chemical treatments, amyloid-based disorders in humans are notoriously difficult to cure medically.
Highly ordered molecular structure, exceptional stability and tensile strength, and the ability to self-replicate their shape indefinitely via seeding and self-templation are some of the unique structural and functional features of amyloids. The presence of amyloids may be determined by observing certain characteristics, such as an X-ray fiber diffraction pattern with a strong lateral reflection at about 4.7 Å, Congo red birefringence, and thioflavin T (ThT) binding. Amyloids are connected with more than fifty diseases, including Alzheimer's, Parkinson's, and Huntington's, and they are often harmful because of their stability, ability to self-replicate uncontrollably, and tendency to trigger more protein misfolding. Simultaneously, it has been shown that the ability to form amyloid is an inherent or almost inherent property of polypeptide chains, and several proteins, homopolypeptides, and non-polypeptide amphiphilic polymers that would normally not be amyloidogenic have been found to induce cross-β structure. In addition, many proteins have their most stable structural state as amyloids at physiological concentrations, indicating that the native state is often a metastable phenomena. It is not unexpected that amyloids have been used by biology for many purposes, including bacterial cell adhesion, human melanin manufacture, and, interestingly enough, memory, due to their durability, universality, and propensity for self-directed assembly. In a similar vein, amyloids are very desirable for use as catalytic scaffolds, nanoparticle templates, and new adhesives due to their advantageous mechanical characteristics and long-range molecular order. Therefore, amyloid structure studies may be a game-changer for the fields of nanotechnology and materials science, as well as for our understanding of fundamental biological processes like memory and bacterial infection.
Milestones in Microbial Functional Amyloid Research. (Levkovich S A., et al., 2021)
Amyloid structure
The contemporary biophysical description of Amyloid is a straight protein strand with a cross-β sheet of infinite length formed by β strands that run perpendicular to the fiber axis. Amyloids are made up of several copies of a protein or peptide, often thousands. Electron microscopy (EM) makes quick work of identifying them as long, unbranched filaments of 6-12 nm in diameter. A meridional reflection at around 4.7 Å, which corresponds to the spacing between β strands, and an equatorial reflection at about 6-11 Å, which corresponds to the distance between stacked β sheets, are typical X-ray fiber diffraction patterns that are generated by the repeating cross-β sheet motif. There are a myriad of functions—some beneficial and others detrimental to the cell—given birth by this structural entity, which is unique among protein folds. With their biological actions in mind, this study aims to synthesize the newly available high-resolution structural data on amyloids.
According to the definition, every amyloid fibril has a translational symmetry component that runs perpendicular to the fibril axis. However, electron micrographs show that the majority of amyloid fibrils also include a rotating component, making them helical or screw symmetric. Therefore, diffraction patterns may be utilized to simulate the fibril structure (at low resolution) in cases when the fibrils are well-aligned. It is challenging to analyze amyloids using high-resolution structural approaches, despite their highly structured character. The fibrils aren't great candidates for three-dimensional crystallization due to their one-dimensional organization; the only crystal structures that have been found so far are of amyloidogenic peptides, which are sufficiently short to pack into a three-dimensional lattice. It may be shown from these structures that the simplest cross-β structure is really a one-dimensional crystal with a single element of translational and spinning symmetry. On the other hand, there is just one high-resolution NMR structure of an amyloid that has been made public so far, even though solid-state nuclear magnetic resonance (NMR) is an excellent technique for studying amyloids as a whole.
Hierarchical length scales relevant in amyloid research. (Schleeger M., et al., 2013)
Hierarchical organization of amyloid fibrils. (Taylor A I P., et al., 2022)
Amyloid beta (Aβ)
The much bigger precursor amyloid precursor protein (APP) is cleaved to produce the Amyloid beta (Aβ) peptides. APP is a key component in the development of Alzheimer's disease (AD). It is an integral membrane protein that is produced in several tissues, particularly in the synapses of neurons. APP is composed of a single domain that spans the cell membrane, a lengthy N-terminus that is glycosylated outside of cells, and a shorter C-terminus that is located in the cytoplasm. It belongs to a bigger family of genes in humans, of which there are three members. Apolipoprotein P1 and Apolipoprotein P2 are the other two members of this family. A number of processes, including iron export, anterograde neural transport, and synapse development and repair, have been linked to APP. Multiple isoforms, with amino acid lengths between 695 and 770, are generated during production. Unlike lengthier versions of APP, the most prevalent form in the brain (APP695) is distinguished by the absence of a Kunitz-type protease inhibitor sequence in its ectodomain. This form is primarily generated by neurons. Isoform 695 of APP is mostly found in neurons, whereas isoforms 751 and 770, which have the Kunitz-type serine protease inhibitory domain KPI, are primarily found in peripheral cells and platelets.
Oligomers, protofibrils, and amyloid fibrils are among the assemblies that may be formed when Aβ monomers assemble. Amyloid oligomers are soluble and have the potential to spread over the brain, but amyloid fibrils are bigger and insoluble, and they may further aggregate into amyloid plaques. In 1984, the main sequence of amino acids for Aβ was first found in amyloid plaques and extracellular deposits. This is the main sequence of amino acids for the 42-amino-acid Aβ isoform Aβ42. Aβ includes peptides with sizes between 37 and 49 residues. In the neocortex of Alzheimer's disease brains, amyloid plaques mostly composed of Aβ are most often seen.
Molecular architecture of APP. (Chen G., et al., 2017)
Amyloid beta peptides
According to early models of the Aβ peptide's solution structure obtained from nuclear magnetic resonance (NMR) (1–28), the protein folds into a mostly α-helical structure with β-sheet conversion in membrane-like environments. This folding process may also happen during the first phases of amyloid production in Alzheimer's disease. The amyloid deposits in Alzheimer's disease are mostly composed of this protein, which has histidine-13 and lysine-16 side chains that are very near to each other on the same face of the helix. In aqueous sodium dodecyl sulfate (SDS) micelles, the Aβ peptide (1-40) has an α-helix conformation between residues 15 and 36, with a kink or hinge at 25-27, according to the solution structure. In contrast, the peptide is largely polar and probably solvated by water between residues 1 and 14, where it is unstructured. Prior to the aggregation of Aβ1-40, a conformational shift from a helix to a coil is facilitated by the deprotonation of two acidic amino acids inside the helix. In water, the Aβ peptide, according to models developed from solid-state NMR spectroscopy, forms a tight network of loops, strands, and twists devoid of alpha-helical or beta-sheet structure (10–35). Its conformation is stabilized by electrostatic and van der Waals interactions. There is an uninterruptedly hydrophobic surface area of about 25% and a meta-stable compact coil shape, both of which have the potential to promote fibrillization, a worldwide rearrangement of conformations, and the creation of an intermolecular beta-sheet secondary structure. Two helical sections linked by a regular type I β-turn are seen in the 3D NMR structures of the Aβ peptide (8-25) and the Aβ peptide (28-38). Synthetic Aβ peptides (25–35) are very poisonous. Its structural characteristics have been scanned by researchers using NMR and CD study of the Aβ peptide (25-35) and fluoro-alcohols. A direct mechanism of neurotoxicity may be at work here, since the peptide forms fibrillar aggregates in a lipidic environment, behaving like a normal transmembrane helix.
The NMR-guided simulations of Aβ peptides 1-40 (Aβ40) and 1-42 (AŲ42) also indicated significantly different conformational states. Aβ42 has a more structured C-terminus and a β-hairpin formed by residues 31-34 and 38-41 that decreases the C-terminal flexibility. This could explain why AŲ42 is more likely than AŲ40 to form amyloids. Multiple distinct conformations, including α-helix or β-sheet conformers, may be populated by Aβ40 and Aβ42, according to replica exchange molecular dynamics studies, and the structural states shift quickly. Recent statistical analyses have shown a multitude of distinct conformational groupings. Nonetheless, there is a notable secondary and tertiary structure in the most current nuclear magnetic resonance (NMR) structure of Aβ40. The C-terminal hydrophobic region of the Aβ protein is essential for initiating the transition from α-helical to β-sheet structure and is a vital factor in deciding how proteins aggregate in Alzheimer's disease.
Summary of Aβ structural studies (1). (Chen G., et al., 2017)
Summary of Aβ structural studies (2). (Chen G., et al., 2017)
Summary of Aβ structural studies (3). (Chen G., et al., 2017)
Amyloid beta in alzheimer's disease
The Aβ protein, which plays a crucial role in memory and cognition, builds up abnormally in some parts of the brain in Alzheimer's disease. The cellular metabolism normally produces Aβ, which originates from the APP. Arriving to the plasma membrane after completing maturation in the Golgi complex, APP is produced in the endoplasmic reticulum (ER). The β-secretase and γ-secretase break the mature APP at the plasma membrane in a sequential manner to produce Aβ. Both the extracellular space and the plasma membrane and lipid raft formations may be home to the freshly produced Aβ. In lipid rafts, Aβ aggregation is substantially promoted by its binding to ganglioside GM1. The process by which cells absorb Aβ involves ApoE attaching to it and receptor-mediated endocytosis, which is facilitated by LRP (LDL receptor-related protein). LDLR controls both the aggregation and the cellular absorption of A. The vesicular transport mechanism allows endocytosed Aβ to reach other subcellular compartments as well. Previous research suggested that Aβ fibrils were the neurotoxic substance responsible for cellular death, memory loss, and other symptoms of Alzheimer's disease. Neuronal cells are most negatively affected by oligomeric or prefibrillar forms of the Aβ peptide, according to further research conducted in the last 30 years. Aβ in its soluble form has the ability to attach to many molecules outside of cells, including as receptors on cell surfaces, metals, and cell membranes.
The extracellular deposition of Aβ in neuritic plaques and its interaction with various receptors are defining features of Alzheimer's disease. The interaction of Aβ with many receptors has been suggested as a contributor to neuronal toxicity: Aβ oligomers are believed to provoke mitochondrial malfunction and oxidative stress in Alzheimer's disease neurons, leading to significant calcium influx and neuronal toxicity. Moreover, soluble oligomeric Aβ has been implicated as toxic due to its interaction with various receptors, including lipids, proteoglycans, and proteins. The interactions between Aβ and its receptors are hypothesized to produce and convey neurotoxic signals to neurons, resulting in cellular impairments such as mitochondrial malfunction and endoplasmic reticulum stress responses. Furthermore, some Aβ receptors are expected to import Aβ into neurons, resulting in specific cellular abnormalities.
The many types of Aβ including soluble Aβ, Aβ oligomers, and Aβ found in amyloid plaques. Moreover, a dynamic compartmentalization of various Aβ types may occur between plaques and soluble Aβ, with distinct Aβ forms potentially contributing to neurodegeneration at different disease phases. Aβ has been shown to form aggregates via two primary kinds of reactions: non-metal-dependent association and metal-dependent association. Non-metal Aβ aggregates produce soluble oligomers and amyloid fibrils, while metal Aβ aggregates generate ionically bridged aggregates, covalently crosslinked oligomers, and seeds for non-metal-dependent Aβ fibrillization. The accumulation of Aβ first results in the formation of Aβ oligomers, which progressively deposit as fibrils and senile plaques. Tau protein undergoes hyperphosphorylation due to alterations in kinase/phosphatase activity induced by Aβ aggregation, resulting in the development of neurofibrillary tangles (NFTs), neuronal and synaptic dysfunction, ultimately culminating in Alzheimer's disease. The self-aggregation process on neuronal membranes produces a hazardous aldehyde known as 4-hydroxynonenal, resulting in lipid peroxidation that may impair the functionality of ion-motive ATPases, glucose transporters, and glutamate transporters. Aβ induces depolarization of the synaptic membrane, excessive calcium influx, and mitochondrial damage, hence impairing cellular capacity to perform normal physiological functions.
Extracellular deposits of fibrils or amorphous aggregates of Aβ peptide create plaques and diffuse deposits, while intracellular fibrillar aggregates of hyperphosphorylated and oxidized tau may develop neurofibrillary tangles. These plaques and neurofibrillary tangles are mostly formed in brain areas, including the hippocampus, amygdala, entorhinal cortex, and basal forebrain, which affect memory, learning, and emotional responses. Aβ may impair synapses and neurites, whereas plaque accumulation in cerebral areas diminishes synapse quantity. Aβ selectively impairs neurons that synthesize serotonin and norepinephrine or use glutamate or acetylcholine as neurotransmitters. Following the discovery that synthetic Aβ fragments induce the death of cultured neurons, a succession of investigations elucidated the chemical and cellular biology mechanisms behind synaptic failure and neuronal death in Alzheimer's disease. Aβ, especially in its aggregated forms, may disrupt synaptic ion and glucose transporters, and electrophysiological research has shown that Aβ hinders synaptic plasticity. Reduced sAPPα levels may enhance neuronal resilience to oxidative and metabolic stress, aligning with the notion that sAPPα contributes to neuronal degeneration, which coincides with elevated Aβ formation in Alzheimer's disease. Memory problems are associated with the emergence of Aβ oligomers, which manifest early in the Aβ deposition process in APP mutant mice. The vaccination of APP mutant mice with human Aβ42 led to the elimination of Aβ deposits in the brain and the restoration of cognitive function, reinforcing the notion that Aβ deposition is a critical factor in Alzheimer's disease.
Biological functions of Aβ. (Chen G., et al., 2017)
References
- Levkovich S A., et al., Two decades of studying functional amyloids in microorganisms, Trends in Microbiology, 2021, 29(3): 251-265.
- Schleeger M., et al., Amyloids: from molecular structure to mechanical properties, Polymer, 2013, 54(10): 2473-2488.
- Taylor A I P., et al., General principles underpinning amyloid structure, Frontiers in Neuroscience, 2022, 16: 878869.
- Greenwald J., et al., Biology of amyloid: structure, function, and regulation, Structure, 2010, 18(10): 1244-1260.
- Chen G., et al., Amyloid beta: structure, biology and structure-based therapeutic development, Acta pharmacologica sinica, 2017, 38(9): 1205-1235.
