Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation.

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ(42) fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ(42). Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.

Wet-spinning of amyloid protein nanofibers into multifunctional high-performance biofibers.

Amyloid nanofibers derived from hen egg white lysozyme were processed into macroscopic fibers in a wet-spinning process based on interfacial polyion complexation using a polyanionic polysaccharide as cross-linker. As a result of their amyloid nanostructure, the hierarchically self-assembled protein fibers have a stiffness of up to 14 GPa and a tensile strength of up to 326 MPa. Fine-tuning of the polyelectrolytic interactions via pH allows to trigger the release of small molecules, as demonstrated with riboflavin-5'-phophate. The amyloid fibrils, highly oriented within the gellan gum matrix, were mineralized with calcium phosphate, mimicking the fibrolamellar structure of bone. The formed mineral crystals are highly oriented along the nanofibers, thus resulting in a 9-fold increase in fiber stiffness.

Probing small molecule binding to amyloid fibrils.

Much effort has focussed in recent years on probing the interactions of small molecules with amyloid fibrils and other protein aggregates. Understanding and control of such interactions are important for the development of diagnostic and therapeutic strategies in situations where protein aggregation is associated with disease. In this perspective article we give an overview over the toolbox of biophysical methods for the study of such amyloid-small molecule interactions. We discuss in detail two recently developed techniques within this framework: linear dichroism, a promising extension of the more traditional spectroscopic techniques, and biosensing methods, where surface-bound amyloid fibrils are exposed to solutions of small molecules. Both techniques rely on the measurement of physical properties that are very directly linked to the binding of small molecules to amyloid aggregates and therefore provide an attractive route to probe these important interactions.

Aniracetam restores the effects of amyloid-beta protein or ageing on membrane fluidity and intracellular calcium concentration in mice synaptosomes

In the present study, we observed the in vitro effect of aniracetam on membrane fluidity and free calcium concentrations (Ca(2+)i) of frontal cortical (FC) and hippocampal (HP) synaptosomes of aged mice and young mice treated with amyloid-beta protein (A

The morphology of decorated amyloid fibers is controlled by the conformation and position of the displayed protein.

Self-assembled structures capable of mediating electron transfer are an attractive scientific and technological goal. Therefore, systematic variants of SH3-Cytochrome b(562) fusion proteins were designed to make amyloid fibers displaying heme-b(562) electron transfer complexes. TEM and AFM data show that fiber morphology responds systematically to placement of b(562) within the fusion proteins. UV-vis spectroscopy shows that, for the fusion proteins under test, only half the fiber-borne b(562) binds heme with high affinity. Cofactor binding also improves the AFM imaging properties and changes the fiber morphology through changes in cytochrome conformation. Systematic observations and measurements of fiber geometry suggest that longitudinal registry of subfilaments within the fiber, mediated by the interaction and conformation of the displayed proteins and their interaction with surfaces, gives rise to the observed morphologies, including defects and kinks. Of most interest is the role of small molecule modulation of fiber structure and mechanical stability. A minimum complexity model is proposed to capture and explain the fiber morphology in the light of these results. Understanding the complex interplay between these factors will enable a fiber design that supports longitudinal electron transfer.

Measuring the kinetics of amyloid fibril elongation using quartz crystal microbalances.

Kinetic measurements of amyloid growth provide insight into the free energy landscape of this supramolecular process and are crucial in the search for potent inhibitors of the main disorders with which it is associated, including Alzheimer's and Parkinson's diseases and Type II diabetes. In recent years, a new class of surface-bound biosensor assays, e.g., those based on surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) have been established as extremely valuable tools for kinetic measurements of amyloid formation. Here we describe detailed protocols of how QCM techniques can be used to monitor the elongation of amyloid fibrils in real time and to study the influence of external factors on the kinetics of amyloid growth with unprecedented accuracy.

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy.

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Rapid label-free detection of metal-induced Alzheimer's amyloid beta peptide aggregation by electrochemical method

Amyloid beta peptide plays a critical role in the pathogenesis of Alzheimer's disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate A beta conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced A beta aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced A beta aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II).

Time-dependent DNA condensation induced by amyloid beta-peptide

The major protein component of the amyloid deposition in Alzheimer's disease is a 39-43 residue peptide, amyloid beta (A beta). A beta is toxic to neurons, although the mechanism of neurodegeneration is uncertain. Evidence exists for non-B DNA conformation in the hippocampus of Alzheimer's disease brains, and A beta was reportedly able to transform DNA conformation in vitro. In this study, we found that DNA conformation was altered in the presence of A beta, and A beta induced DNA condensation in a time-dependent manner. Furthermore, A beta sheets, serving as condensation nuclei, were crucial for DNA condensation, and Cu2+ and Zn2+ ions inhibited A beta sheet-induced DNA condensation. Our results suggest DNA condensation as a mechanism of A beta toxicity.