Format

Send to

Choose Destination
J Biol Chem. 2017 Sep 8;292(36):14963-14976. doi: 10.1074/jbc.M117.791236. Epub 2017 Jul 24.

Growth-incompetent monomers of human calcitonin lead to a noncanonical direct relationship between peptide concentration and aggregation lag time.

Author information

1
From the Applied Physics Program, University of Michigan, Ann Arbor, Michigan 48109.
2
Zhou Pei-Yuan Center for Applied Mathematics, Tsinghua University, Beijing 100084, China.
3
Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan, and.
4
Department of Chemistry and Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109-1055.
5
Department of Chemistry and Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109-1055 ramamoor@umich.edu.

Abstract

The role of the peptide hormone calcitonin in skeletal protection has led to its use as a therapeutic for osteoporosis. However, calcitonin aggregation into amyloid fibrils limits its therapeutic efficacy, necessitating a modification of calcitonin's aggregation kinetics. Here, we report a direct relationship between human calcitonin (hCT) concentration and aggregation lag time. This kinetic trend was contrary to the conventional understanding of amyloid aggregation and persisted over a range of aggregation conditions, as confirmed by thioflavin-T kinetics assays, CD spectroscopy, and transmission EM. Dynamic light scattering, 1H NMR experiments, and seeded thioflavin-T assay results indicated that differences in initial peptide species contribute to this trend more than variations in the primary nucleus formation rate. On the basis of kinetics modeling results, we propose a mechanism whereby a structural conversion of hCT monomers is needed before incorporation into the fibril. Our kinetic mechanism recapitulates the experimentally observed relationship between peptide concentration and lag time and represents a novel mechanism in amyloid aggregation. Interestingly, hCT at low pH and salmon calcitonin (sCT) exhibited the canonical inverse relationship between concentration and lag time. Comparative studies of hCT and sCT with molecular dynamics simulations and CD indicated an increased α-helical structure in sCT and low-pH hCT monomers compared with neutral-pH hCT, suggesting that α-helical monomers represent a growth-competent species, whereas unstructured random coil monomers represent a growth-incompetent species. Our finding that initial monomer concentration is positively correlated with lag time in hCT aggregation could help inform future efforts for improving therapeutic applications of CT.

KEYWORDS:

amyloid; calcitonin; fiber; kinetics; oligomer; protein aggregation; protein misfolding

PMID:
28739873
PMCID:
PMC5592673
DOI:
10.1074/jbc.M117.791236
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center