assay protocol

Amyloid beta, APP, Amyloid fibril

Brief description:
A-beta (amyloid beta or beta-amyloid) is a peptide of 36–43 amino acids that is processed from the Amyloid precursor protein (APP), a kind of transmembrane glycoprotein with undetermined function. Aβ is well known as the main component of deposits found in the brains of patients with Alzheimer's disease. Moreover, evidence proves that A-beta is a highly multifunctional peptide with pivotal non-pathological activity.

What is amyloid precursor protein (APP)?

The amyloid precursor protein (APP) is encoded by an APP gene located on chromesome 21 and owing to alternative m-RNA splicing exists in three isoforms: APP695, APP751 and APP770. APP can be processed by α-, β- and γ-secretases and rapidly metabolized within 45-60 minutes of expression via two pathways : a non-pathogenic non-amyloidogenic pathway and a Aβ synthesizing amyloidogenic pathway (As shown in the picture on the right).
Abbreviations: AICD, amyloid intracellular domain; CTF, carboxyl terminal fragment

Beta-amyloid formation:

APP

Aβ protein is generated by series of actions of the α, β and γ secretases. Typically, the non-amyloidogenic pathway is initiated by the α-secretase-mediated cleavage of APP between residues APP687 an APP688. On the other hand, the amyloidogenic pathway is initiated by β-secrease cleavage of APP. In amyloidogenic pathway the γ secretase produces the C-terminal end of the Aβ peptide, cleaves APP within its transmembrane region and generate couples of isoforms of 36-43 amino acid length residues. (As shown in the picture on the right). The most common and intensively studied Aβisoforms are A-beta40 (beta-amyloid40) and A-beta42 (beta-amyloid42), which consist of 40 and 42 residues, respectively. Aβ40 is typically produced by cleavage that occurs in the endoplasmic reticulum, while Aβ42 is produced by cleavage in the trans-Golgi network. The A-beta40 form is more common in the two, but A-beta42 is the more fibrillogenic and is thus associated with disease states. Aβ42has been noted to be increased in early-onset Alzheimer's associated with mutations in APP. Aβ can be destroyed by several amyloid-degrading enzymes including neprilysin.

Amyloid beta structure:

Abeta cleavage

A-beta peptides are 38-43 amino acid peptides and commonly thought to be intrinsically unstructured, meaning that in solution they do not acquire a unique tertiary fold but rather populates a set of structures. Therefore amyloid beta peptides cannot be crystallized and most structural knowledge on amyloid beta comes from NMR and molecular dynamics. NMR-derived models of a 26-aminoacid polypeptide from amyloid beta (Aβ 10-35) show a collapsed coil structure devoid of significant secondary structure content. Replica exchange molecular dynamics studies suggested that amyloid beta can indeed populate multiple discrete structural states. By NMR-guided simulations, amyloid beta40 and amyloid beta 42 also seem to feature highly different conformational states, with the C-terminus of amyloid beta42 being more structured than that of amyloid beta40 fragment.

Beta-amyloid features:

A-beta peptides are amphiphilic as the first 28 aa residues are polar and the remaining residues are non-polar in nature. As a result, the peptides exhibit great differences in polarity at neutral pH and thus show a high propensity for aggregation. The unfolded A-beta monomers may self-assemble into dimmers, trimers and higher orger oligomers which may ultimately form insoluble fibrils and believed to be the main source of toxicity by causing the death of neurons.

Beta-amyloid fibrils structure:

Amyloid fibrils can be defined as fibrillar polypeptide aggregates with a cross-β structure. FTIR indicates that the β-sheet structure of A-beta40 and A-beta42 oligomers is considerably antiparallel, whereas full-length Aβ amyloid fibrils possess parallel β-sheet characteristics. NMR experiments suggest that the β-sheet packing distance and assembly could be different in Aβ42 oligomers and Aβ42 fibrils. So far, atomic structures of full-length Aβ amyloid fibrils have not been determined.

amyloid plaque

Beta-amyloid fibrils cytotoxicity:

Alzheimer disease (AD) is characterized by enhanced oxidative stress and intracellular Ca2+ concentration-both of which may serve as stimuli to induce apoptosis. As a hallmark of AD A-beta oligomers are speculated to deposit at small blood vessels, then it triggers interactions between the peptides and receptors of the endothelial cells of the blood vessel wall and activate inflammatory responses and cytokine release. These may successively lead to the destruction of the blood vessels, reducing oxygen supply to neurons and finally neuronal death.

The structure–toxicity relationship indicates that A-beta amyloid fibrils contain an intrinsic toxicity that correlates with their morphology (e.g., more regular and longer fibrils tend to be more toxic).