Amyloid Fibril Formation by Pentapeptide and Tetrapeptide Fragments of Human Calcitonin

Abstract

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH 2 -DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH 2 -DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH 2 -FNKF-COOH tetrapeptide, the NH 2 -DFN-COOH tripeptide, or the NH 2 -DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.The process of amyloid fibril formation is associated with a large number of diseases of unrelated origin. A partial list includes Alzheimer's disease, Parkinson's disease, Type II diabetes, Prion diseases, Familial British dementia, and several familial amyloidosises (1-7). All of these diseases are characterized by the transformation of soluble proteins into aggregated fibrillar deposits in different organs and tissues. The pathological significance of amyloid fibril formation is not completely understood in all cases. One such example is aortic medial amyloid deposits that occur virtually in all individuals older than 60 years, but its medical consequence is not known yet (8).Although different amyloid-forming proteins do not share clear sequence homology, the fibrillar structures that are being formed have very similar physicochemical and ultrastructural characteristics as determined by transmission electron microscopy x-ray fiber diffraction, and other biophysical techniques (1-7). Furthermore, amyloid fibrils are being formed in vitro also by disease-unrelated proteins (9 -13). This finding suggests that amyloid fibrils may serve as a generic structural form of aggregated proteins (1-7). Nevertheless, despite the significant medical importance of the amyloid fibril formation process and its consideration as a universal structural form, the exact mechanism that leads to the self-assembly of polypeptides into ordered fibrils is not fully understood.Human calcitonin (hCT) 1 is a 32-amino acid polypeptide hor...

Modulating Calcitonin Fibrillogenesis

“…Therefore, aggregation of hCT does not rely upon a specific secondary structure but on characteristic local, chemical properties. This is in line with the finding that short hCT peptides based upon the 15-19 region are able to form fibrils if Phe 16 and Phe 19 are preserved (5).…”

“…It is not known which structural features cause specific proteins to form amyloid fibrils in vivo; however, evidence is accumulating that aggregation is initiated from specific regions within a polypeptide chain (2)(3)(4)(5)(6).…”

A systematic screen of β 2 -microglobulin and insulin for amyloid-like segments

“…In fact, there are numerous examples of small peptides (5-9), some as short as three or four residues, that form fibrils (10,11). How such short peptides can form fibrils was illuminated by the crystal structures of the amyloidlike peptides of sequence NNQQNY and GNNQQNY (12).…”

Molecular basis for insulin fibril assembly

“…Peptide segments, as short as three to four residues, can form fibrils by themselves in vitro (26)(27)(28)(29)(30). Furthermore, these segments from the spine of the fibril often act as inhibitors of fibrillation of their parent proteins.…”