Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides
Introduction
Alzheimer's disease (AD) is a late-age neurodegenerative dementia, closely associated to protein and peptide aggregation. Two types of aberrant proteinaceous aggregates are usually found in the post-mortem analysis of brains from AD: the neurofibrillary tangles mainly composed of tau protein, and the extracellular senile plaques formed by large aggregates of amyloid peptides [1], [2], [3]. Although the origin of the disease remains obscure and controversial, the amyloid cascade hypothesis is at present the most prevalent theory conferring a framework within which to develop different approaches addressed to find a promising therapy. According to this hypothesis, the disease starts as a consequence of an excess of amyloid peptide due to an abnormal processing of the amyloid precursor protein. This accumulation results in the formation of extracellular amyloid plaques which eventually initiate a number of toxic effects resulting in neuronal death and disease [4], [5], [6], [7]. The initial molecular events by which aggregated amyloid peptides exert toxicity are not clearly established, although a number of molecular interactions and sites of action have been suggested: induction of tau protein modification [8], [9], promotion of oxidative stress [10], [11], alteration of Ca2+ permeable channels [12], [13], synapsis interaction [14], and apoptosis induction [15]. A great deal of evidence reported over the last years supports the idea that toxicity may be also related to intracellular oligomeric forms of amyloid peptides which would appear as intermediate species along the process of amyloid aggregation (see Ref. [7]). Independently of which molecular interaction is particularly relevant as the original toxic event, peptide aggregation seems to be closely related to the onset of the neurological disorder.
Heat and oxidative stress may induce the intracellular location of amyloid peptides [16]. Furthermore, the nuclear localization of these peptides has been reported and amyloid peptide aggregates have been found within the nucleus of CHO cells [17] and AD brain samples [18]. These results, together with those reports concerning the interaction of different forms of amyloid peptides with DNA [18], [20], [21], [22], [23] and those describing changes in transcription of various genes, including the apoptosis-associated p53 as a consequence of a nuclear translocation of the Aβ1–42 peptide [19], suggest that an amyloid–DNA interaction resulting in transcription deregulation may be at the origin of AD genesis [24], [25]. Similarly to amyloid peptides, some pathogenic molecules, including huntingtin [26], [27], prion protein [28], [29], α-synuclein [30], [31], and tau protein [32], [33] have been widely reported to bind DNA. Interestingly, these proteins and peptides share with amyloid peptides the tendency to aggregate as a common feature. It sounds plausible that aggregated forms of proteins and peptides related to neurodegenerative diseases display a high proneness to bind DNA. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including β-lactoglobulin, myoglobin, bovine serum albumin, histone, casein and the amyloid-β peptides related to Alzheimer's disease Aβ25–35 and Aβ1–40. Aβ25–35 is an aggregating fragment of the full-length β-amyloid 1–42, which shows high toxicity. Aβ1–40 is the most abundant amyloid peptide found as forming part of the amyloid plaques, although it aggregates more slowly than the 1–42 peptide [6]. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by the aggregated forms of proteins, or rather it is an interaction specifically related to the aggregated forms of those particular peptides or proteins related to neurodegenerative diseases.
Section snippets
Materials
H1 histone, bovine β-lactoglobulin A, myoglobin, bovine serum albumin, casein, DNA from calf thymus, poly-l-lysine, polyglutamic acid, heparin, Hepes and gold were from Sigma–Aldrich (St. Louis, USA). Aβ25–35 and Aβ1–40 from PolyPeptide (Strasbourg, France) were dissolved following the manufacturer's recommendations. Casein was dissolved in 10 mM Hepes, 100 mM NaCl, pH 7. All other proteins were dissolved in distilled water. Unless indicated, all experiments were carried out in a neutral buffer
Electron microscopy
A number of proteins including albumin, myoglobin, casein, β-lactoglobulin, histone and the amyloid peptides Aβ25–35 and Aβ1–40 were aged at room temperature over different periods of time. All of them acquired aggregated states, although displaying different forms which were easily observable under the electron microscope (Fig. 1). Small non-fibrillar aggregates, displaying an amorphous and lumpy aspect are shown by casein and albumin. Large fibrils are formed by the amyloid peptides Aβ25–35
Acknowledgment
Financial support has been provided by grants SAF2006-02424 and P2009/TIC-1476 from the Spanish Government and Comunidad de Madrid.
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