Deposition of amyloid plaques in the parenchyma and vasculature of the brain is characteristic for AD. AP peptide is the principal constituent of these plaques and it is believed to be responsible for the neurotoxicity associated with Alzheimer's disease. The formation of peptide aggregates may be mediated by some essential metal ions.
Earlier studies have shown that metal ions like Cu2+ or Zn2+ may induce very efficient aggregation of soluble Ap. Zn2+ ions induce aggregation at pH 7.4 in vitro and this reaction is reversible with chelation [66,67], while Cu2+ ions are much more effective in conditions representing physiological acidosis (pH 6.6-6.8) . It was also shown that aggregation is negligible for the rat (mouse) peptide, which differs from the human variant by three substitutions: Arg5 ^ Gly, Tyr10 ^ Phe, and His13 ^ Arg. Preliminary data clearly indicated the role of metal interaction with the imidazole donor set of the His side chain in the aggregation process and possible multi-copper binding to Ap [66-68]. Recent studies based on EPR measurements seem to indicate that hAp binds Cu2+ in a mononuclear metal binding site with a metal: peptide ratio of 1:1 both in soluble and fibrillar form .
However, a short report has appeared suggesting that Cu2+ ions may inhibit the aggregation process in the wide pH range 5-9 . As none of these studies presented a detailed evaluation of the coordination pattern (speciation versus pH) in the considered systems, it is difficult to compare the relevance of the conclusions reached in these studies [66-70].
The analytical data concerning Alzheimer's plaque formation are extensive although there was not much analytical information about intact plaques. A recent work using Raman spectroscopy therefore provided a real progress towards the comprehension of the analytical chemistry of Alzheimer plaques . The study has confirmed the composition of isolated amyloid plaque cores, the pro-tein/peptide conformation, and possible metal ion binding sites. The Raman spectra confirmed that also in the intact plaque cores both Zn2+ and Cu2+ ions are coordinated to histidine residues and chelating agents may reverse metal ion binding leading to the loosening of the p-structure and possible solubilization of amyloid deposits.
The human variant of the Ap peptide (hAp) contains three histidine residues at positions 6, 13, and 14, which besides the N-terminal amino group can act as the potential anchoring sites for metal ions, especially Cu2+ . The interesting motif typical of hAp coordination seems to consist of the His13-His14 pair serving two adjacent imidazoles able to bind the metal ion . The protected hexapeptide fragment hAp 11-16 (Ac-EVHHQK-NH2) involves both imidazole and amide nitrogen donors to form a very stable complex with the Cu2+ ion . The human variant was found to be much more effective in copper binding than that of the mouse or rat Ap (mAp) having His13 substituted by Arg. This result indicates that both His residues could be involved in metal ion coordination in vivo affecting the peptide aggregation process. Comparison of the copper binding abilities of hAp 11-16 with that of hAp 11-28 has shown that in both cases the metal ion coordination donor set is identical but the stability of the complexes formed by the longer peptide are one to two orders of magnitude higher compared to those of hAp 11-16 . This stabilization may result from some structural organization of a peptide ligand in Cu2+ complexes.
The studies with protected amyloid peptide fragments hAc-Ap-NH2 1-16 and 1-28 have indicated the likely involvement of three His residues in binding one Cu2+ ion in the wide range of pH 5-8 . The mouse variant mAc-Ap-NH2 may use only two His imidazole side chains to bind a metal ion in the pH range 5-7, forming less stable complexes.
The unprotected Ap 1-16 and 1-28 peptides involve also the N-terminal amino group [74,75]. In hAp, the His13-His14 pair seems to be critical for metal ion binding for both protected and unprotected peptides. There is no involvement of Tyr phenolate oxygen in Cu2+ ion binding [74,75] in contrast to what was suggested earlier . The latter work suggested also the formation of the dimeric complex with two metal ions bridged by an imidazole moiety, but these findings were not supported by other studies [69,74,75].
The mechanism of induction of peptide aggregation upon metal ion coordination can be accounted for by different hypotheses. Above pH 9, when the amide nitrogen donors dominate Cu2+ ion coordination [74,75], the metal ion is unable to interact with more than one peptide molecule . However, around pH 6.6-6.8 Cu2+ coordinates to the N-terminal amino and the imidazole nitrogen atoms. Such metal ion binding could allow Cu2+ to form cross-links between different peptide molecules leading to peptide aggregation. In the case of mAp Cu2+ binding to His residues is weaker and the amide involvement occurs at lower pH making inter-peptide metal-induced cross-links ineffective. It is also possible that metal ion binding induces changes in peptide conformation and solubility leading to formation of aggregates. Cu2+ coordination will change the overall charge of the peptide molecule. Thus, copper binding will have a profound impact on the electrostatic behavior of Ap having a strongly charged N-terminal and the hydrophobic C-terminal, which may result in the aggregation process .
CD, EPR, and preliminary NMR studies on hAp and its variants in which His residues were substituted by Ala have strongly supported the critical involvement of imidazole nitrogens in metal ion coordination . Cu2+ binds to the N-terminus of Ap and three His (Figure 15). His13 seems to be the critical residue for metal ion coordination. Due to the lack of this residue in mAp, copper does not induce peptide aggregation . CD spectra suggest that binding of Cu2+ does not induce a typical p-sheet conformation found in Ap fibrils.
Although the stability  and affinity constants for the Cu2+-hAp system are known, it is still difficult to evaluate the physiological significance of metal ion binding to hAp. The reported affinities differ very much from each other  and the stability constants are difficult to be compared with the 'physiological complexes'. There is, however, general agreement that hAp has sufficient affinity to bind copper at physiological levels of Cu2+ .
The details about the binding ability of Zn2+ ions are largely understood. The major binding sites in hAp for Zn2+ seem to be three His residue .
The N-terminal region of metal-free hAp 1-28 lacks a stable structure, but its C-terminal is better defined including the helical region starting from Ala21 . The rat variant, rAp 1-28, exhibits a more extended helical structure and it is better defined (Figure 16) . Since the helical to p-strand transition of the human peptide results in amyloid formation, the more ordered conformation of rAp may protect the amyloid formation in aged rats . In the case of rAp the binding sites for Zn2+ have been suggested to be His6, His14 and Arg13 .
A good fit of the NMR titration curves was obtained for 1:1 stoichiometry which was suggested earlier for rAp 1-40 using Scatchard analysis . The affinity of Zn2+ ion binding to rAp is much weaker (4 x 102M-1)  than that found for hAp (9 x 106M-1). The binding of Zn2+ to rAp 1-28 reduces the flexibility of the N-terminal peptide region leaving the C-terminal well folded (Figure 17) . In the brain, Ap can fold into a well soluble random coil and helical structures or aggregating p-sheet structures [82,83]. Thus, the rAp 1-28 peptide with its longer helical segment than it is found in hAp 1-28 is protecting aggregation in the aged rat brain . Aggregation of hAp may be facilitated in aged brain when zinc concentration in the brain is increasing, while in rats the cerebral zinc concentration may not be high enough to induce peptide aggregation .
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