Publications

Amyloid fibrils formed by the islet amyloid polypeptide cause pancreatic beta-cell damage, resulting in reduced insulin secretion and type 2 diabetes. Changes in the amino acid sequence of this peptide can influence its aggregation rate, and animals expressing variants that do not form amyloids do not develop type 2 diabetes. Conversely, specific single amino acid changes can accelerate the aggregation rate of this peptide. Here, we employ deep mutational scanning to measure the ability of 1916 islet amyloid polypeptide variants, including substitutions, insertions, truncations and deletions, to nucleate amyloids. Our results identify a continuous stretch of residues from 15 to 32 that is particularly sensitive to mutation. This region, which is likely structured in amyloids, matches the core of the early aggregated species formed by this peptide in vitro. Within this region, mutations in residues 21 to 27 have a substantial effect, suggesting tighter structural constraints. Finally, we compare the mutational atlas of the islet amyloid polypeptide to that of amyloid beta - the peptide that aggregates in Alzheimer's disease - and find that mutations that slow down nucleation correlate between the two amyloids, but mutations that accelerate nucleation in one amyloid cannot be used to predict mutational effects in the other.

JTD Keywords: Aggregation, Amylin gene, Amyloidogenicity, Atomic-resolution structure, Beta, Cryo-em structures, Diabetes-mellitus, Fibril structure, Islet, Polypeptide iapp

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

JTD Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

The role of amyloid beta (A beta) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble A beta species. Sulfated glycosaminoglycans (GAGs) promote A beta fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A beta(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A beta(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-angstrom separating A beta(42) monomers within protofibrillar beta-sheets. Incubation of A beta(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A beta(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased similar to 3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A beta(42) fibrils induced by PVS and A beta(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A beta(42) fibrils trapping soluble toxic forms.

JTD Keywords: Alzheimer's disease, Amyloid fibril structure, Fibrillogenesis enhancers and inhibitors, Polysaccharides