The Physiological and Pathological Implications of the Formation of Hydrogels, with a Specific Focus on Amyloid Polypeptides.

Hydrogels are water-swollen and viscoelastic three-dimensional cross-linked polymeric network originating from monomer polymerisation. Hydrogel-forming polypeptides are widely found in nature and, at a cellular and organismal level, they provide a wide range of functions for the organism making them. Amyloid structures, arising from polypeptide aggregation, can be damaging or beneficial to different types of organisms. Although the best-known amyloids are those associated with human pathologies, this underlying structure is commonly used by higher eukaryotes to maintain normal cellular activities, and also by microbial communities to promote their survival and growth. Amyloidogenesis occurs by nucleation-dependent polymerisation, which includes several species (monomers, nuclei, oligomers, and fibrils). Oligomers of pathological amyloids are considered the toxic species through cellular membrane perturbation, with the fibrils thought to represent a protective sink for toxic species. However, both functional and disease-associated amyloids use fibril cross-linking to form hydrogels. The properties of amyloid hydrogels can be exploited by organisms to fulfil specific physiological functions. Non-physiological hydrogelation by pathological amyloids may provide additional toxic mechanism(s), outside of membrane toxicity by oligomers, such as physical changes to the intracellular and extracellular environments, with wide-spread consequences for many structural and dynamic processes, and overall effects on cell survival.

Rapid method for measurement of surface tension in multiwell plates.

We describe an optical method for quantifying surface tension in 96-well microtitre plates. Absorbance and fluorescence measurements in vertical beam systems as in 96-well plate photometers are complicated by the interaction of the light with the curved surface of the liquid. If the samples do not all have the same meniscus, errors in the data proportional to the degree of curvature are produced, which may confound interpretation of the data. We have harnessed this effect by modifying a 96-well plate spectrophotometer, and show that the apparent optical density measurements correlate very closely with surface tension. The method is much quicker than conventional methods for measuring multiple samples and requires considerably less technical skill. We demonstrate the applicability of the method to the study of biomolecules by investigating the surfactant properties of two peptides (Abeta and AChE(586-599)) that form amyloid fibrils in vitro, in one case replicating previously published results, and in the other case highlighting the similarity, and possibly generic nature, of the biophysical properties of these amyloidogenic peptides.

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation, but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers.

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide.

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.