The flanking peptides issue from the maturation of the human islet amyloid polypeptide (hIAPP) slightly modulate hIAPP-fibril formation but not hIAPP-induced cell death

Highlights

• The flanking peptides of the human islet amyloid polypeptide are not amyloidogenic.

• The flanking peptides weakly reduce hIAPP fibril formation in human pancreatic islets.

• No effect of flanking peptides on the cultured cells or on human islet cells toxicity.

Abstract

Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic β cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic β-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.

Introduction

Protein misfolding and aggregation that lead to insoluble fibrils are the key factors for several human diseases, such as type 2 diabetes mellitus (T2DM), Alzheimer’s disease, Parkinson’s disease, Prion diseases as well as for many physiological processes [1,2]. In most of these misfolded protein diseases, the natively unfolded protein converts into insoluble ordered fibrillary aggregates, called as amyloid fibrils. The aggregation of amyloid proteins is a well-known process that leads to the formation of fibrils at the surface of cells, associated with cell toxicity [[2], [3], [4]]. In T2DM, the amyloid fibrils are found in the pancreatic islets of patients and are mainly composed of the human islet amyloid polypeptide (hIAPP), a 37 amino acid peptide (also known as amylin) [5,6].

hIAPP is coproduced and co-secreted along with insulin through the secretory pathway in a molar hIAPP:insulin ratio of 1:100 in healthy individuals, a ratio that can increase to 1:20 in T2DM [7,8]. During protein translation, hIAPP is processed and modified. Both hIAPP and insulin are secretory proteins initially synthesized as preprohormones that are released in response to stimuli [[9], [10], [11]]. Both hIAPP and insulin possess signal peptides that drive the targeting of nascent polypeptides from the cytosol to the endoplasmic reticulum (ER), the entry point into the secretory pathway [12,13]. The N-terminal signal peptides are removed by signal peptidase resulting in prohIAPP and proinsulin. In the oxidizing environment of the ER lumen, prohIAPP and proinsulin rapidly fold to form respectively one and three disulfide bonds, all evolutionary conserved. The resulting 67 amino acids prohIAPP and 86 amino acids proinsulin are cleaved in the trans-golgi network and then in the secretory granules by the prohormone convertases (PC1/3 and PC2) in concert with carboxypeptidase E (CPE) leading to the mature hIAPP and mature insulin. C-peptide resulting from proinsulin maturation as well as the two flanking peptides resulting from prohIAPP processing remain in the secretory granules.

The composition of the β-cell granule is extremely complex and contains many components that could influence hIAPP fibril formation and hIAPP toxicity. In fact, it is still not understood why and how hIAPP forms amyloid fibrils. However, several studies demonstrated that aggregation of amyloid proteins is easily influenced by both intrinsic features, such as mutations, expression levels, presence of peptides from the maturation, and by extrinsic factors, such as macromolecular crowding and interaction with metal ions, lipid membranes, or chaperones [[14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]]. In the case of hIAPP, it was shown that divalent metal ion, Zn(II) and Cu(II) slowed down the kinetics of fibril formation, probably through the histidine residue at position 18 as a site of interaction and/or by forming a stable non fibrillar hIAPP-metal complex [17,[27], [28], [29]]. However, the role of Cu(II) in hIAPP-induced cell death has been controversial. A few studies demonstrated that hIAPP-induced INS-1 cells toxicity increased in the presence of Cu(II) [28,30,31] while another study on INS-1 cells and also on SHSY5Y cells, reported that copper ions do not affect hIAPP-induced cell death [29]. In addition to metal ions, granule peptides such as insulin and the C-peptide could also modify hIAPP fibril formation and hIAPP-induced cell death. Westermark et al. demonstrated first that insulin, even at a low concentration, significantly inhibited hIAPP fibril formation [32]. Later, several studies confirmed this statement and proposed different mechanism of an inhibition process [[33], [34], [35]]. Importantly, C-peptide, which do not form fibril itself, could reduce the amount of hIAPP fibrils [32]. It was shown that lowering pH to mimic the acid environment of the granules protects hIAPP from fibril formation in vitro [16] and that the histidine residue 18, the only residue that titrates over the pH range, plays an important role in hIAPP aggregation and misfolding [36]. Finally, a study revealed that the prohIAPP prevents aggregation and membrane damage of mature hIAPP [37]. However, no data are currently available on a putative function of both flanking peptides neither on their influence on hIAPP fibril formation and on hIAPP toxicity. Our goal was to determine if hIAPP flanking peptides could present some physiological properties and more specifically could form fibrils and/or affect hIAPP fibrillation and toxicity. This question about a functional role of hIAPP flanking peptide was asked, as the C-peptide, which comes from the insulin maturation process, has been described as an active molecule. Indeed, for a long time, it has been thought that the C-peptide has no physiological effect and it was used as a biomarker of pancreatic β-cell activity. Since the last 20 years, the interest upon a biological action of C-peptide has grown. So far, the physiological effect of C-peptide remains unknown. In contrast, in type 1 diabetic patients, a positive effect on microvascular complications of diabetes has been demonstrated. More specifically, the C-peptide plays a neuro- and a reno-protective effects, stimulates the pancreatic β-cell antioxidant capacity and could present an anti-inflammatory action [38]. It has also been proposed that the C-peptide could promote insulin release from it aggregated storage form, enhancing the insulin biological effect [39].

To shed light on hIAPP flanking peptides function, a comprehensive set of both biophysical experiments (fluorescence, circular dichroism, transmission electron microscopy) and biological studies were performed in solution, in the presence of membrane models, on culture cells and on human pancreatic islets. In particular, we investigated the ability of the flanking peptides to form fibrils, their secondary structures, their effect on mature hIAPP fibril formation and their influence on cell toxicity. We found in solution and in the presence of model membranes that the flanking peptides are not able to completely inhibit hIAPP fibril formation and hIAPP-induced membrane leakage. In the presence of INS-1 cells, the flanking peptides appear to have no effect on the hIAPP fibril formation neither on cell toxicity. However, in human pancreatic islets both flanking peptides slightly reduce hIAPP fibril formation but not hIAPP toxicity.