Investigating the inhibitory property of DM hCT on hCT fibrillization via its relevant peptide fragments

Abstract

The irreversible aggregation of proteins or peptides greatly limits their bioavailability; therefore, effective inhibition using small molecules or biocompatible materials is very difficult. Human calcitonin (hCT), a hormone polypeptide with 32 residues, is secreted by the C-cells of the thyroid gland.The biological function of this hormone is to regulate calcium and phosphate concentrations in the blood via several different pathways. One of these is to inhibit the activity of osteoclasts; thus, calcitonin could be used to treat osteoporosis and Paget's disease of the bone. However, hCT is prone to aggregation in aqueous solution and forms amyloid fibrils. Salmon and eel calcitonin are currently used as clinical substitutes for hCT. In a previous study, we found that the replacement of two residues at positions 12 and 17 of hCT with amino acids that appear in the salmon sequence can greatly suppress peptide aggregation. The double mutations of hCT (DM hCT) also act as good inhibitors by disrupting wild-type hCT fibrillization, although the inhibition mechanism is not clear. More importantly, we demonstrated that DM hCT is biologically active in interacting with the calcitonin receptor. To further understand the

Deciphering the influence of Y12L and N17H substitutions on the conformation and oligomerization of human calcitonin

“…The enhanced stability of the dynamic helix in DM-hCT compared to hCT was consistent with the CD measurements. 16,23 Overall, our analysis of residue-pairwise contacts indicated that the Y12L and N17H mutations stabilize the dynamic helical conformations, thus contributing to the suppression of the conformational transition from helix to β-sheet in the hCT monomer.…”

“…Future improvements to enhance DM-hCT's amyloid-resistance should focus on minimizing mutations to effectively reduce the remaining β-sheets. In conclusion, our simulations suggest that the experimentally determined amyloid-resistant Y12L and N17H substitutions 16,23 are achieved by stabilizing the dynamic helical conformation and suppressing its conversion to β-sheet structures. Our findings not only elucidate the molecular mechanism behind hCT aggregation and the amyloid-resistant effects of Y12L and N17H double substitutions but also offer valuable insights for developing next-generation hCT analogs for clinical applications.…”

“…In contrast, the Y12L and N17H substitutions shifted the preference towards conformations with a higher helix content and a lower β-sheet population. This evident shift towards more helices and fewer β-sheets in DM-hCT compared to hCT suggested that the double substitutions of Y12L and N17H suppressed hCT fibrillization in vivo 16,23 by enhancing helix formation and preventing β-sheet structures.…”

“…This observation aligned with prior CD measurements, which showed that DM-hCT exhibited more helical content than hCT in the monomer state. 16,23 Upon oligomerization, hCT formed β-sheet-rich aggregates and β-barrel intermediates. The β-sheet formation involved residues 8–31, with a notable presence in the phenylalanine-rich region from residues 16–25, consistent with previous NMR measurements.…”

“…20 Another hCT analog with Y12L and N17H double mutations (DM-hCT) can inhibit hCT aggregation without altering its bioactivity of binding to the hCT receptor. 16,23 Furthermore, the presence of DM-hCT can also inhibit the fibrillization of wild-type hCT. 16 With just two residue substitutions and a physiological function similar to hCT, DM-hCT could potentially provide a valuable therapeutic alternative to hCT.…”