The pro‐sequence of parathyroid hormone prevents premature amyloid fibril formation

Abstract

The parathyroid hormone (PTH) regulates the calcium and phosphate level in blood after secretion from parathyroid chief cells. The preand pro-sequences of precursor preproPTH get cleaved during PTH maturation. In secretory granules, PTH forms functional amyloids. Using thioflavin T fibrillation assays, circular dichroism, NMR spectroscopy, and cellular cAMP activation, we show that the pro-sequence prevents premature fibrillation by impairing primary nucleation because of Coulomb repulsion of positively charged residues. Under seeding or high salt conditions or in the presence of heparin at pH 5.5, proPTH fibril formation is delayed, but the monomer release properties are conserved. ProPTH can still activate in cellulo PTH receptor 1 but with impaired potency. These findings give some perspectives on medical applications of PTH in hormone therapy.

The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation

“…In general, PTH 84 displayed a higher R 2 / R 1 ratio for residues L7‐G38 compared to other residues (Figure 4B, lower panel). This indicated a propensity for the formation of secondary structure in this region, in good agreement with reported structures [39–42] . With increasing c 0 , R 2 / R 1 strongly increased for regimes III and IV.…”

“…In agreement with experimentally determined structures, [40,42] AlphaFold 2.0 predicted an α‐helix for residues E4‐L37, an intrinsically disordered C‐terminal part, and an additional α‐helix for residues K72‐A81 (Figure 2A). [39,41] The latter α‐helix was not experimentally observed [42] . For a detailed analysis of possible c 0 effects on the structure we used the intrinsic W23 fluorescence ratio F 350 / F 320 as an indicator for spectral changes.…”

“…Two‐dimensional NMR spectroscopy allows to derive residue specific information about which segments of PTH 84 are involved in inter‐ and intramolecular interactions. The low dispersion within the proton dimension of cross‐peaks in our c 0 dependent backbone 1 H‐ 15 N‐fHSQC spectra (Figure 4A) agrees with a predominantly disordered peptide with transient α‐helices [21,42,48] . Notably, we observed a c 0 dependent perturbation of the chemical shift (CSP) or the detected signal intensity for many cross‐peaks.…”

“…The general appearance of the circular dichroism (CD) spectra displaying a dominant minimum at λ min,1 ≈202 nm and a minor minimum at λ min,2 ≈220 nm is indicative of an intrinsically disordered peptide with additional contributions from an α‐helix (Figure S1B) [21–22,42] . In contrast, a CD spectrum calculated from the predicted structure (Figure S1B) shows the typical minima of α‐helices at λ 1 =208 nm and λ 2 =220 nm, indicating an overestimation of the α‐helix content by AlphaFold 2.0 [45] .…”

“…The hormone which acts in blood calcium and phosphate homeostasis is hypothesized to be stored in functional amyloid fibrils in secretory granules prior to its release into the blood stream [21,38] . The N‐terminal part of PTH 84 displays an α‐helical propensity while the C‐terminal part remains disordered [39–42] . PTH 84 offers a reversible fibrillation system with c crit in a μM concentration range which is easily accessible by a variety of biophysical techniques [21–23] .…”