Human pro-islet amyloid polypeptide (ProIAPP1–48) forms amyloid fibrils and amyloid spherulites in vitro

doi:10.1016/j.jinorgbio.2010.05.012

Journal of Inorganic Biochemistry

Volume 104, Issue 10, October 2010, Pages 1125-1129

https://doi.org/10.1016/j.jinorgbio.2010.05.012 Get rights and content

Abstract

Deposition of β sheets of islet amyloid polypeptide (IAPP) in pancreatic tissue is implicated in the aetiology of type 2 diabetes mellitus (T2DM). IAPP is cleaved from its precursor protein, pro-islet amyloid polypeptide (ProIAPP) and incomplete cleavage results in ProIAPP_1–48, which is found co-deposited with IAPP. Cu(II) prevents IAPP from forming amyloid and herein we investigated if it would also prevent ProIAPP_1–48 from forming β sheets. Excess Cu(II) prevented ProIAPP_1–48 from forming amyloid and additionally reversed the formation of β sheets in pre-formed fibrils of the peptide. The latter was also true for ProIAPP_1–48 fibrils formed in the presence of Al(III). An unexpected finding was the formation of spherulites of ProIAPP_1–48 which were only observed in preparations which included Al(III). The spherulites were 40–100 μm in diameter and stained positively for Al(III) suggesting a role for this metal in their formation.

The abolition by Cu(II) of the propensity of ProIAPP_1–48 to form amyloid may have important implications for the treatment of T2DM. The immediate significance for diabetes of the equally novel observation of spherulites of ProIAPP_1–48 is unknown though, as with spherulites of Aβ₄₂ in Alzheimer's disease, there may be implications for the aetiology of the disease.

Graphical abstract

Amyloid formation by ProIAPP_1–48 is abolished by Cu(II) while Al(III) induces the formation of spherulites.

Section snippets

Acknowledgements

M. Mold is thanked for performing the TEM. PEF acknowledges support from the Canadian Institute for Health Research.

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The influence of aluminium and copper upon the early aggregatory behaviour and size of Islet amyloid polypeptide under simulated physiological conditions
2022, Journal of Trace Elements in Medicine and Biology
Islet amyloid polypeptide/amylin deposition in the form of amyloid plaques is a common pathological feature observed in the pancreatic tissue of those with Type II Diabetes Mellitus. Its propensity to form amyloid fibrils and the resultant toxicity of this peptide in vivo is influenced by both the concentration and species of metal present in situ. Herein, we examine the influence of Al (III) and Cu (II), applied at equimolar and supra-stoichiometric concentrations on the initial aggregatory behaviour of amylin under near physiological conditions.
Dynamic light scattering measurements, which monitored the aggregation status and size of the peptide in real time, were performed during the early lag-phase of fibrillogenesis (T ≤ 30 min) in the absence or presence of metal ions.
Islet amyloid polypeptide (10 µM) rapidly aggregated when introduced into a physiological medium favouring the formation of large, agglomerated structures (> 1000 nm) after 30 min incubation. Neither the addition of equimolar or excess metals significantly influenced the size of the peptide when intensity distributions were consulted; however, number distributions indicated that both Al (III) and Cu (II) may have had, an albeit temporary, stabilising influence upon the conformations present within solution.
These results infer that small oligomeric species are likely transient entities that are rapidly incorporated into large agglomerates during the very initial stages of fibrillogenesis. While both Al (III) and Cu (II) both inhibited agglomeration to some degree, their stabilising affect upon peptide aggregation was limited over the juncture of the experiments performed herein; hence, it is difficult to say whether these metal ions play a role in enhancing the toxicity of these peptides through influencing their aggregation in the short-term.
Liquid liquid phase separation of full-length prion protein initiates conformational conversion in vitro
2021, Journal of Biological Chemistry
Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrP^Sc). Prions can convert normal cellular prion protein (PrP^C) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid–liquid phase separation (LLPS) in the absence of PrP^Sc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid–solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by β-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23–89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid–solid phase transition could be the intermediate steps in the conversion of prions.
Interaction of amylin species with transition metals and membranes
2019, Journal of Inorganic Biochemistry
Citation Excerpt :
In the case of copper(II) ions, there is controversy about their role in the aggregation of amylin. Some studies mentioned that they can inhibit amylin fibrillation as well as toxicity [24,34], while others indicate that they may contribute to the cell toxicity by forming amylin oligomers, which may contribute to cell death more than the fibrils [21,22]. Iron was shown to enhance amylin β-pleated sheet formation [35].
Islet Amyloid Polypeptide (IAPP), also known as amylin, is a 37-amino-acid peptide hormone that is secreted by pancreatic islet β-cells. Amylin is complementary to insulin in regulating and maintaining blood glucose levels in the human body. The misfolding and aggregation of amylin is primarily associated with type 2 diabetes mellitus, which is classified as an amyloid disease. Recently, the interactions between amylin and specific metal ions, e.g., copper(II), zinc(II), and iron(II), were found to impact its performance and aggregation processes. Therefore, the focus in this review will be on how the chemistry and structural properties of amylin are affected by these interactions. In addition, the impact of amylin and other amyloidogenic peptides interacting with metal ions on the cell membranes is discussed. In particular, recent studies on the interactions of amylin with copper, zinc, iron, nickel, gold, ruthenium, and vanadium are discussed.
Aggregation of the diabetes-related peptide ProIAPP<inf>1-48</inf> measured by dynamic light scattering
2019, Journal of Trace Elements in Medicine and Biology
Citation Excerpt :
It remains equivocal as to whether Al(III), Fe(III) and Zn(II) promote amyloid (β sheet) formation while it is clear that Cu(II) prevents IAPP from assembling into β sheet structures [7] as recently confirmed [9–13]. ProIAPP1-48 forms amyloid less readily than IAPP and while there are few data on its interactions with metals it is also the case that Cu(II) prevents ProIAPP1-48 from forming β sheets of amyloid [12,14,15]. It is widely believed that the cytotoxicity’s of IAPP and ProIAPP1-48 is related to their propensity to form toxic oligomers during the early stages of amyloid formation [5] and it has been suggested that metals and specifically Cu(II) potentiate toxicity through stabilisation of these oligomeric forms [11,16].
Islet amyloid polypeptide (IAPP_1-37) or amylin is implicated in the aetiology of diabetes. It is found as amyloid along with its precursor ProIAPP_1-48 in the islets of Langerhans in the pancreas. Metals have been implicated in amyloidogenesis of both IAPP and ProIAPP. Herein we have used dynamic light scattering (DLS) to investigate how Al(III) and Cu(II) influence aggregation of ProIAPP_1-48 under near-physiological conditions and in a biologically-relevant timeframe. ProIAPP_1-48 formed primarily sub-micron particles within 5 min (e.g. 470 nm at 15μM peptide) that grew to micron-sized particles (1310 nm) within a 30 min timeframe. Equimolar Al(III) had little influence upon particle size at either 5 (656 nm) or 30 min (1250 nm) while Cu(II) tended to increase particle size over the same time period (731–1300 nm). It is suggested that any effects of Al(III) and Cu(II) reflected their well known tendencies to support β-sheet or amorphous aggregates of ProIAPP_1-48 respectively.
Zn(II) - pramlintide: Stability, binding sites and unexpected aggregation
2017, Journal of Inorganic Biochemistry
Citation Excerpt :
The concentration of zinc in pancreatic β-cells is one of the highest in the body, and zinc deficiency is quite a common symptom of type 2 diabetes; [19] a link between ZnT8, an intracellular specific zinc transporter, and T2D has also been suggested [20]. Zinc has a complex effect on amylin aggregation, inhibiting fiber formation at low concentrations and accelerating it at higher concentrations [21,22]. However, according to a recent work by Ramamoorthy et al., at an early stage of aggregation (40 min) and at low concentrations of Zn(II) ions (100 μM), the Zn(II) ions promote the formation of even larger sizes of Zn(II)–amylin aggregates than those that are formed at high Zn(II) concentrations [23].
Pramlintide is an antidiabetic drug which mimics amylin – a small peptide co-secreted from pancreatic β-cells together with insulin, one of the hallmarks of type 2 diabetes. In the course of the disease, amylin misfolds into small oligomers or to an aggregated β-sheet amyloid fiber. The misfolding mechanism is not yet quite understood, but it is clear that zinc ions play an important role in the process. This work sheds new light on the role of zinc and pramlintide in the course of the disease, giving a detailed description of the Zn(II)-pramlintide complex, in which the metal ion binds to the imidazole of His18 and the amine group of Lys1, imposing a bend in the peptide between these residues. The most surprising finding is the fact that the initially well-soluble, non-aggregating Zn(II)-pramlintide complex forms fibrillar, oligomeric aggregates after a lag-time of 20 h. This raises more questions about the relationship between Zn(II) and amylin/pramlintide: could this zinc-induced change in the complex structure be a partial explanation of the formation of oligomeric aggregates of the complex, which might be much more toxic to β-cells than large fibrillar deposits and if so, is pramlintide the optimal choice of an antidiabetic drug?
Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin
2017, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
The interference of growth from the neighboring spherulites resulted in structures with high density and small size. These spherulites did not shown the typical “Maltese cross” pattern exhibited for amyloid fibrils of bovine insulin [57] or of pro-islet amyloid polypeptide (ProIAPP1–48) [58] but a pattern similar to those observed for crystallizing polymers such as polyethylene glycol (PEG) or PEG peptides derivatives able to form fibril [59]. Usually spherulites are observed on a long length scale (> 1 μm) with diameters generally between 6 and 12 nm [57].
Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264–277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264–271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile²⁶⁹ conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe²⁶⁸. Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c +.

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Short CommunicationHuman pro-islet amyloid polypeptide (ProIAPP1–48) forms amyloid fibrils and amyloid spherulites in vitro

Abstract

Graphical abstract

Section snippets

Acknowledgements

Antioxid. Redox Signal.

FEBS J.

Am. J. Pathol.

Diab. Res. Clin. Prac.

Diabetes

Biochemistry

J. Alzheimers Dis.

J. Alzheimers Dis.

J. Inorg. Biochem.

Short Communication
Human pro-islet amyloid polypeptide (ProIAPP_1–48) forms amyloid fibrils and amyloid spherulites in vitro