Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP)

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Abstract

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer’s disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of β amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AlATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to β amyloid and amylin may be substrates or receptors for ATP in vivo.

Introduction

The precipitation in the brain of β amyloid (Aβ) is a pathological hallmark of Alzheimer’s disease [1]. The process of precipitation is neurotoxic [2] though the identity of the toxic form of the peptide has not yet been resolved [3], [4], [5], [6], [7]. The mechanism by which Aβ will form fibrils has been the subject of a great deal of research [8], [9]. Fibril formation involves a number of well defined stages and will occur spontaneously at super-physiological concentrations of peptide. However, the cause of fibril formation in vivo in which an undersaturated concentration of peptide will be induced to precipitate is still unknown.

The hypothesis that has the precipitation of Aβ as a seminal event in the aetiology of Alzheimer’s disease [10] has stimulated an enormous amount of research into factors which both promote and inhibit Aβ deposition [11], [12], [13], [14], [15], [16], [17], [18]. We are interested in the role of aluminium in the initiation of amyloid fibril formation in Alzheimer’s disease [19]. We showed that aluminium abolished the α-helical conformation of Aβ(1-40) in a membrane-mimicking environment and promoted the conformational transition of the peptide to β-sheet via random coil [20]. The binding of aluminium by Aβ has subsequently been confirmed by a number of independent groups [21], [22], [23], [24], [25], [26]. Recently we have investigated how small molecules, such as glucose [27] and ATP [28], [29], which are present in the body in high concentrations, influence the interaction of aluminium with Aβ. Glucose increased the biological availability of aluminium and, coincidentally, the nature of the fibrils of Aβ formed in the presence of aluminium [27], whilst ATP promoted the formation of thioflavin T-reactive Aβ fibrils and aluminium potentiated this effect [29].

The possibility that ATP has a role in amyloidosis in vivo was investigated further in this research. We have used thioflavin T (ThT) fluorescence [30] to demonstrate that AlATP promoted the formation of anti-parallel β-sheet fibrils of both Aβ (Aβ(25-35); Aβ(1-40); Aβ(1-42)) and amylin. Strong evidence was provided by reverse phase high performance liquid chromatography (RP-HPLC) that this effect was due to AlATP being bound by one or more monomers of the amyloidogenic peptide.

Section snippets

Materials

Synthetic Aβ(25-35), Aβ(1-40) and Aβ(1-42) and synthetic human amylin were obtained from Bachem (Saffron Walden, UK). ATP, ADP and AMP were purchased from Boehringer Mannheim (Lewes, UK). Suramin hexasodium was bought from RBI-Sigma (Poole, UK). All other chemicals were obtained from either Sigma (Poole, UK) or BDH (Poole, UK) and were of the highest quality available.

Stock solutions

Stock solutions of Aβ(25-35) (0.840 mM), Aβ(1-40) (0.100 mM) and amylin (0.100 mM) were prepared in ultrapure water

ThT fluorescence of Aβ(25-35) in assays made from either freshly prepared or aged peptide stock solutions

Measurements of A405 of Aβ(25-35) stock solutions (0.840 mM) showed time-dependent changes which suggested that the aggregation states of freshly prepared (<6 h old) and aged (>24 h old) stocks might be different from one another (Table 1). Upon dissolution of the peptide in ultrapure water the stock absorbance increased steadily during the first 6–9 h before achieving a relatively constant level for up to 72 h. This raised the possibility that the age of peptide stocks could, upon their

Discussion

The preparation and storage of stock solutions of amyloidogenic peptides can be critical to the propensity of the peptides to form amyloid fibrils in biological milieu. As an aid to the interpretation of fibril formation it is important that the conditions under which peptides are used are accurately defined. We experimented with a number of different procedures which included additions, such as TFA, to the solvent and the storage of stocks as frozen aliquots. Our objective was to identify a

Acknowledgments

This research was supported by The Wellcome Trust, The North Staffordshire Heart Committee and The Royal Society.

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