Converting the highly amyloidogenic human calcitonin into a powerful fibril inhibitor by three-dimensional structure homology with a non-amyloidogenic analogue

Abstract

Irreversible aggregation limits bioavailability and therapeutic activity of protein-based drugs. Here we show that an aggregation-resistant mutant can be engineered by structural homology with a non-amyloidogenic analogue and that the aggregation-resistant variant may act as an inhibitor. This strategy has successfully been applied to the amyloidogenic human calcitonin (hCT). Including only five residues from the non-amyloidogenic salmon calcitonin (sCT), we obtained a variant, polar human calcitonin (phCT), whose solution structure was shown by CD, NMR, and calculations to be practically identical to that of sCT. phCT was also observed to be a potent amyloidogenesis inhibitor of hCT when mixed with it in a 1:1 ratio. Fibrillation studies of phCT and the phCT-hCT mixture mimicked the sCT behavior in the kinetics and shapes of the fibrils with a dramatic reduction with respect to hCT. Finally, the effect of phCT alone and of the mixture on the intracellular cAMP level in T47D cells confirmed for the mutant and the mixture their calcitonin-like activity, exhibiting stimulation effects identical to those of sCT, the current therapeutic form. The strategy followed appears to be suitable to develop new forms of hCT with a striking reduction of aggregation and improved activity. Finally, the inhibitory properties of the aggregation-resistant analogue, if confirmed for other amyloidogenic peptides, may favor a new strategy for controlling fibril formation in a variety of human diseases.

FIGURE 1.

Primary structures of hCT, phCT, sCT, and LAsCT. The N-terminal disulfide bridge between positions 1 and 7 is boxed, and mutation sites are boxed and shaded. In phCT and LAsCT, mutated amino acids are in bold.

FIGURE 2.

Prediction of sites putatively affecting aggregation propensities of mutated hCT sequences according to Waltz software. The histogram summarizes the analysis of ∼6000 sequences obtained by mutating the primary sequence of the amyloidogenic hCT and incorporating the corresponding residues of the aggregation-resistant sCT.

FIGURE 3.

ThT fluorescence assay time course for hCT (curve 1), LAsCT-hCT (1:1) mixture (curve 2), sCT-hCT (1:1) mixture (curve 3), phCT-hCT (1:1) mixture (curve 4), phCT (curve 5), sCT (curve 6), and LAsCT (curve 7). hCT reached its maximal fluorescence values after 42 h, whereas LAsCT-hCT (curve 2) reached its maximal fluorescence values after ∼200 h, and sCT-hCT (curve 3) and phCT-hCT (curve 4) both reached their maximal fluorescence values after ∼500 h. phCT, sCT, and LAsCT retained basal fluorescence values during the whole experiment time (860 h), indicating low propensity to form amyloid fibrils. Each point averages four independent repeats.

FIGURE 4.

Far-UV CD spectra of hCT, sCT, phCT, and phCT-hCT (1:1) mixture at three time points: after 1 h (A), after 42 h (B), and after 160 h (C). Continuous (—; 1), broken (- - -; 2), dotted (···; 3), and broken-dotted (-··-; 4) lines refer to hCT, sCT, phCT, and phCT-hCT (1:1), respectively. Signal disappearance/appearance at 205/218 nm through the time course of the experiments were assumed as an indication of a random coil → β-sheet transition. Five scans were averaged for each spectrum, and the results are reported as mean residue ellipticity. kdeg, kilodegree.

FIGURE 5.

Transmission electron micrographs of aggregates formed by CT samples at different times of incubation. A, hCT amyloid aggregates were observed at high frequency after 5 h. After 60 days, amyloid fibrils were hardly observed for sCT (B) and phCT (C), the latter showing some amorphous (i.e. non-fibrillar) structures and rare short fibrils. Amorphous structures were also observed for the phCT-hCT (1:1) mixture after 22 days (D). The scale bar corresponds to 200 nm.

FIGURE 6.

Persistent stimulation of intracellular cAMP levels after incubation of T47D cells in presence of hCT, sCT, phCT, and phCT-hCT (1:1) mixture. cAMP levels were measured after stimulation with a 100 nm concentration of each CT peptide in cells previously subjected to a pretreatment with the different peptides (restimulated) and in cells not previously exposed to such pretreatment (acute). An additional group of samples corresponded to pretreated T47D cells that had not been subjected to a second incubation with the different CT variants (recovered). Results are means ± S.E. of four independent experiments. The error bars represent the standard error.

FIGURE 7.

A, amino acid sequence of phCT and diagrammatic representation of the short and medium range NOE connectivities observed in SDS micelles at 310 and 324 K. NOE intensities are indicated by the thickness of the bars. A square below the one-letter code indicates a ³J_HNα coupling constant <5 Hz measured for that residue, whereas a circle denotes 6 < ³J_HNα < 7 Hz. A crossed square/circle indicates slow ¹H/²H amide exchange. B, backbone superposition of the 20 phCT periodically sampled structures along the 1-ns unrestrained molecular dynamics. Structures were superimposed for pairwise minimum r.m.s.d. of the N, Cα, and C atoms of residues 2–21.

FIGURE 8.

Ribbon and stick representation of phCT structure parallel (A) and perpendicular (B) to the helical axis. The color code for residue polarity is as follows: gray, hydrophobic residues; yellow, polar residues; red, acidic residues; and blue, basic residues. C and D, superposition of phCT (magenta) and sCT (green). In both panels, His¹⁷ is shown with transparent van der Waals surfaces. Views C and D are obtained from A after 45° and 90° rotations about the horizontal axis, respectively.