Details of research

Formation of amyloid-type fibrils by short peptides

Proteins that form the amyloidal aggregates, such as microtubule-associated protein tau and islet amyloid polypeptide (IAPP), contain short peptide segments that play critical roles in the fibril formation. Phosphorylation/dephosphorylation of the fibrillogenic protein, human tau, is believed to play crucial roles in pathogenesis of the Alzheimer’s disease. Previous studies have demonstrated that Tyr310 is essential for the tau fibrillization and a plausible in vivo phosphorylation site. A short peptide segment VQIVY310K (PHF6) corresponding to the core part of tau fibril formation is one of such sequences capable of forming paired-helical filaments.

Fibril formation by short peptides derived from tau. PHF6 derivatives substituted at the Tyr-310 residue of PHF6 were synthesized to represent a variety of aromatic side chains. Non-natural amino acids, 4-phenylphenylalanine (PHF6FPh), 4-fluorophenylalanine (PHF6FF) and 4-methylphenylalanine (PHF6FMe) were incorporated at the Tyr-310 position of PHF6 (Figure 4).

Structures of PHF6 peptide derivatives used in this study. Tyr at the position X corresponds to the Tyr-310 residue of native tau.

TEM images of amyloid-type fibers formed from (A) PHF6 and (B) 4-phenyl derivative PHF6FPh, (C) 4-fluoro dervative PHF6FF, (D) 4-methyl derivative PHF6FMe. Samples were negatively stained with 2% uranyl acetate. Scale bar: 100 nm.

The aromatic and hydrophobic non-natural amino acids substituted at the Tyr residue of the fibril-forming core peptide of human tau play important roles in enhancing the amyloid-forming propensity of the peptide. Because the modification at the 4-position of phenyl group was accommodated in the fibrils of PHF6 derivatives, it would be possible to control the amyloid-forming propensity of short peptides by using non-natural amino acids. Such a strategy would afford novel biomaterials with defined size and shape.

This research was supported by a Grant-in-aid for Scientific Research from Ministry of Education, Science, Sports and Culture, Japan to T.M.(16651108, 17310125), and by SORST from Japan Science and Technology Corporation (JST) to T.M.

Charge-Pairing Mechanism of Phosphorylation Effect upon Amyloid Fibrillation of Human Tau Core Peptide. Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer’s disease. PHF6 (VQIVY310K) is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and its phosphorylated derivative PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.

TEM images and of amyloid-type fibers of PHF6 and PHF6pY, and hypothetical structures of amyloid-type fibrils formed by PHF6 and PHF6pY at neutral pH. Blue color and red color indicate positively charged sites of Lys, and negatively charged sites of phosphate groups, respectively.



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