Inhibition Of Fibril Assembly Research Articles

Fine Mapping of the Amyloid β-Protein Binding Site on Myelin Basic Protein

The assembly and deposition of amyloid β-protein (Aβ) in brain is a key pathological feature of Alzheimer's disease and related disorders. Factors have been identified that can either promote or inhibit Aβ assembly in brain. We previously reported that myelin basic protein (MBP) is a potent inhibitor of Aβ fibrillar assembly [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., et al. (2009) Biochemistry 48, 4720-4727]. Moreover, the region on MBP responsible for this activity was localized to the 64 N-terminal amino acids (MBP1-64) [Liao, M. C., et al. (2010) J. Biol. Chem. 285, 35590-35598]. In the study presented here, we sought to further define the site on MBP1-64 involved in this activity. Deletion mapping studies showed that the C-terminal region (residues 54-64) is required for the ability of MBP1-64 to bind Aβ and inhibit fibril assembly. Alanine scanning mutagenesis revealed that amino acids K54, R55, G56, and K59 within MBP1-64 are important for both Aβ binding and inhibition of fibril assembly as assessed by solid phase binding, thioflavin T binding and fluorescence, and transmission electron microscopy studies. Strong spectral shifts are observed by solution nuclear magnetic resonance spectroscopy of specific N-terminal residues (E3, R5, D7, E11, and Q15) of Aβ42 upon the interaction with MBP1-64. Although the C-terminal region of MBP1-64 is required for interactions with Aβ, a synthetic MBP50-64 peptide was itself devoid of activity. These studies identify key residues in MBP and Aβ involved in their interactions and provide structural insight into how MBP regulates Aβ fibrillar assembly.

Influence of phosphorus dendrimers on the aggregation of the prion peptide PrP 185–208

Inhibition of fibril assembly is a potential therapeutic strategy in prion diseases. The effect of cationic phosphorous dendrimers on the aggregation process of the prion peptide PrP 185–208 was studied using a spectrofluorometric assay with thioflavin T (ThT) and Fourier transformed infrared spectroscopy in order to monitor the kinetics of the process and the changes in the peptide secondary structure. The results show that phosphorous dendrimers are able to clearly interfere with PrP 185–208 aggregation process by both slowing down the formation of aggregates (by causing a decrease of the nucleation rate) and by lowering the final amount of amyloid fibrils, a common hallmark of conformational diseases. The dendrimers effect on the aggregation process would imply their interaction with peptide monomers and oligomers during the nucleation phase.

Influence of dendrimer’s structure on its activity against amyloid fibril formation

Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer’s diseases. Highly branched, globular polymers—dendrimers—are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185–208 and the Alzheimer’s peptide Aβ 1–28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer–peptide interaction mechanism is presented based on the dendrimers’ molecular structure.

Self-Assembly of β-Amyloid 42 Is Retarded by Small Molecular Ligands at the Stage of Structural Intermediates

Assemblyof the amyloid-β peptide (Aβ) into fibrils and its deposition in distinct brain areas is considered responsible for the pathogenesis of Alzheimer's disease (AD). Thus, inhibition of fibril assembly is a potential strategy for therapeutic intervention. Electron cryomicroscopy was used to monitor the initial, native assembly structure of Aβ42. In addition to the known fibrillar intermediates, a nonfibrillar, polymeric sheet-like structure was identified. A temporary sequence of supramolecular structures was revealed with (i) polymeric Aβ42 sheets during the onset of assembly, inversely related to the appearance of (ii) fibril intermediates, which again are time-dependently replaced by (iii) mature fibrils. A cell-based primary screening assay was used to identify compounds that decrease Aβ42-induced toxicity. Hit compounds were further assayed for binding to Aβ42, radical scavenger activity, and their influence on the assembly structure of Aβ42. One compound, Ro 90-7501, was found to efficiently retard mature fibril formation, while extended polymeric Aβ42 sheets and fibrillar intermediates are accumulated. Ro 90-7501 may serve as a prototypic inhibitor for Aβ42 fibril formation and as a tool for studying the molecular mechanism of fibril assembly.

Inhibition of the Self-assembly of Collagen I into Fibrils with Synthetic Peptides: DEMONSTRATION THAT ASSEMBLY IS DRIVEN BY SPECIFIC BINDING SITES ON THE MONOMERS

A series of experiments were carried out to test the hypothesis that the self-assembly of collagen I monomers into fibrils depends on the interactions of specific binding sites in different regions of the monomer. Six synthetic peptides were prepared with sequences found either in the collagen triple helix or in the N- or C-telopeptides of collagen I. The four peptides with sequences found in the telopeptides were found to inhibit self-assembly of collagen I in a purified in vitro system. At concentrations of 2.5 mM, peptides with sequences in the C-telopeptides of the alpha1(I) and alpha2(I) chain inhibited assembly at about 95%. The addition of the peptide with the alpha2-telopeptide sequence was effective in inhibiting assembly if added during the lag phase and early propagation phase but not later in the assembly process. Experiments with biotinylated peptides indicated that both the N- and C-telopeptides bound to a region between amino acid 776 and 822 of the alpha(I) chain. A fragment of nine amino acids with sequences in the alpha2-telopeptide was effective in inhibiting fibril assembly. Mutating two aspartates in the 9-mer peptide to serine had no effect on inhibition of fibril assembly, but mutating two tyrosine residues and one phenylalanine residue abolished the inhibitory action. Molecular modeling of the binding sites demonstrated favorable hydrophobic and electrostatic interactions between the alpha2telopeptide and residues 781-794 of the alpha(I) chain.