Binding of Human Proteins to Amyloid‑β Protofibrils

The progressive neurodegeneration in Alzheimer’s disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-β (Aβ) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric Aβ may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of Aβ₄₂ (Aβ_42CC). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system, or in hemostasis. Binding of representative proteins from all of these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of Aβ enhances protein binding, as lower amounts of proteins bind monomeric Aβ. Proteins that bind to Aβ protofibrils might contribute to biological effects in which these aggregates are involved. Our results therefore suggest that an improved understanding of the mechanisms by which Aβ causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which Aβ-binding proteins are present

Hydrophobicity and Conformational Change as Mechanistic Determinants for Nonspecific Modulators of Amyloid β Self-Assembly

The link between many neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer’s disease. We show that lacmoid binds Aβ(1–40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1–40) and Aβ(1–42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide

Amyloid-β Protofibrils: Size, Morphology and Synaptotoxicity of an Engineered Mimic

<div><p>Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ₄₂cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ₄₂cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ₄₂cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ₄₂cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ₄₂cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ₄₂cc and aggregates of wild type Aβ₄₂. We suggest that Aβ₄₂cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.</p></div

Analysis of Aβ₄₂cc morphology using atomic force microscopy (AFM).

<p>(A) AFM image of Aβ₄₂cc protofibrils on dry mica surface. (B) Average <i>z</i>-heights and cross-sections of Aβ₄₂cc (black) and wild type Aβ₄₂ (red) protofibrils (grey lines represent measurements of 20 Aβ₄₂cc protofibrils). (C-F) High magnification AFM images of single protofibrils of Aβ₄₂cc (C) and wild type Aβ₄₂ (D; identified in aggregation reaction mixtures, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066101#pone.0066101.s002" target="_blank">Fig. S2</a>), and of amyloid fibrils of Aβ₄₀ (E) and Aβ₄₂ (F). Measured <i>z</i>-heights of particles are indicated in panels C-F.</p

OC serum dot blot.

<p>The fibril specific OC serum recognizes Aβ₄₂cc protofibrils and wild type Aβ₄₂ fibrils, but not monomeric Aβ₄₂cc or protofibrils that have been denatured by boiling in SDS.</p

Size distribution of Aβ₄₂cc protofibrils measured using different methods.

<p>The blue and red lines/symbols represent data from atomic force microscopy. One sample (blue) was washed briefly with deionized water, while a second sample (red) was washed extensively. The lengths of <i>ca.</i> 1500 protofibrils were measured in each case. The gray dashed line reflects an expected distribution corresponding to the analytical ultracentrifugation measurements (Fig. 3) assuming that Aβ₄₂cc protofibrils have a dehydrated diameter of 3.1 nm. The black line represents the distribution of apparent hydrodynamic radius obtained from nanoparticle tracking analysis using a NanoSight microscope.</p

Aβ₄₂cc protofibrils expose binding sites for the ANS dye.

<p>Fluorescence emission spectra of 50 µM free ANS (red) and of ANS in the presence of Aβ₄₂cc protofibrils (black) or monomeric Aβ₄₂cc (green). Peptide concentrations are in both cases 10 µM monomer units.</p

Effect of Aβ₄₂cc protofibrils (red) and wild type Aβ₄₂ oligomers (blue) on spontaneous synaptic activity in mouse primary hippocampal neurons grown on a multielectrode array (MEA) chip.

<p>Changes in firing rates are normalized to the initial electrical activity in the absence of treatment and compared to buffer-treated neurons: ** – p<0.0015, * – p<0.026 (Student's t-test); the difference between Aβ₄₂ oligomers and Aβ₄₂cc protofibrils is not significant.</p

Protein/Protein Nanocomposite Based on Whey Protein Nanofibrils in a Whey Protein Matrix

This article describes nanocomposite films with separately grown protein nanofibrils (PNFs) in a nonfibrillar protein matrix from the same protein starting material (whey). Tensile tests on the glycerol-plasticized films indicate an increased elastic modulus and a decreased extensibility with increasing content of PNFs, although the films are still ductile at the maximum PNF content (15 wt %). Infrared spectroscopy confirms that the strongly hydrogen-bonded β-sheets in the PNFs are retained in the composites. The films appear with a PNF-induced undulated upper surface. It is shown that micrometer-scale spatial variations in the glycerol distribution are not the cause of these undulations. Instead, the undulations seem to be a feature of the PNF material itself. It was also shown that, apart from plasticizing the protein film, the presence of glycerol seemed to favor to some extent exfoliation of stacked β-sheets in the proteins, as revealed by X-ray diffraction