Structural characterization of amyloid fibrils from the human parathyroid hormone

Highlights

• Functional amyloid fibril formation of the parathyroid hormone hPTH(1–84)

• Fibrils exhibit the basic architecture of cross-β-sheet-rich conformations.

• Residues 25R–37L form the core of the amyloid fibrils.

• Polyphenol EGCG inhibits fibril growth by interaction with core region residues.

• Tremendous differences in the dynamics of fibrillar and non-fibrillar segments

Abstract

Amyloid deposits are common in various tissues as a consequence of misfolded proteins. However, secretory protein and peptides are often stored in membrane coated granules as functional amyloids. In this article, we present a detailed characterization of in vitro generated amyloid fibrils from human parathyroid hormone (hPTH(1–84)). Fully mature fibrils could be obtained after a short lag phase within less than one hour at 65 °C. These fibrils showed all characteristic of a cross-β structure. Protease cleavage combined with mass spectrometry identified the central region of the peptide hormone involved in the fibril core formation. EGCG, an inhibitor of amyloid fibril formation, showed binding to residues in the peptide monomers corresponding to the later fibril core and thus explaining the inhibition of the fibril growth. Conformational and dynamic studies by solid-state NMR further corroborated the cross-β core of the fibrils, but also identified highly mobile segments with a random coil structure not belonging to the rigid fibril core.

Introduction

Failure of proteins to reach a stable native structure may result in the population of misfolded or aggregated species [1], [2], [3]. Amyloid deposits are common in particular polypeptide producing tissues and typically represent a characteristic feature of diseases affecting these tissues including Alzheimer's or Parkinson's disease and type II diabetes [4]. On the other hand, because of their optimized packing properties, amyloids are found in nature for storage of proteins and peptides. Such amyloids have a non-pathological biological function and are called ‘functional amyloids’. These are present in Escherichia coli [5], silkworms [6], fungi [7], and mammalian skin [8]. Some functional amyloids of fungal prions are involved in prion replication, and the amyloid protein Pmel17 is involved in mammalian skin pigmentation. Formation of functional amyloids is also common to the secretory proteins such as hormones [9]. Hormones and secretory proteins stored in membrane coated secretory granules over long time periods at very high concentration before release into the blood stream occurs. Often, this high concentration leads to self-association of the peptide chains. It has been reported that peptide and hormones are found in the pituitary secretory granules of the endocrine system in the form of functional amyloids [9]. Few hormones including insulin and glucagon tend to form amyloid fibrils under certain in vitro conditions [10]. The basic architecture of self-assembled amyloid fibrils is the cross-β structure [11]. The cross-β motif is composed of intermolecular β-sheets arranged along the fibril axis with the β-strands aligned perpendicularly to the axis of fibrils. The amyloid-like cross-β-rich conformation was also observed for functional amyloids in secretory granules of the endocrine system [9].

Human parathyroid hormone is secreted from the parathyroid glands if the Ca^2 + level in the blood drops or the blood phosphate concentration increases [12]. Human parathyroid hormone is translated as a 115 residues comprising pre-pro protein. The 25 N-terminal residues (pre sequence) are required as signal for efficient transport to the endoplasmic reticulum [13], [14]. This signal sequence is rapidly [15] cleaved off by a signal peptidase and the resulting pro-hPTH peptide subsequently transferred to the Golgi apparatus [16]. The 6 residues of the pro sequence at the N-terminus are proteolytically removed [17] and the mature hPTH(1–84) is packaged and stored in secretory granules until the release into the blood. It has been reported that peptide and hormones found in the pituitary secretory granules of the endocrine system are stored in the form of functional amyloids [9]. Few hormones including insulin [10], glucagon [10], and PTH [18] tend to form amyloid fibrils under certain in vivo and in vitro conditions [9]. Often, mutations result in the accumulation of a conformationally defective protein in the ER which contributes to diseases such as Alzheimer's, Parkinson's, Huntington's, or type 1 diabetes [19], [20], [21]. C18R mutation in the pre sequence of pre-pro-PTH disrupts the hydrophobic core of the signal sequence leading to intracellular accumulation and causes the familial isolated hypoparathyroidism (FIH) [22].

In the present study, we test the hypothesis whether hPTH(1–84) undergoes fibril formation under in vitro conditions. Indeed, we could explore in detail amyloid fibril formation of hPTH(1–84) and elucidate its structural properties. The fibrils are curvilinear, long structures and exhibited the characteristic of repeating cross-β motif. Primarily, the N-terminal residues 25R–37L form the cross-β core structure of the fibrils. Most of the diseases related to the secreted proteins are caused by accumulation of misfolded proteins and the progressive degeneration of the associated tissues. An amyloid-like structure of peptide and protein hormones could explain most of their properties.