Copper(II)-binding equilibria in human blood.

It has been reported that Cu(II) ions in human blood are bound mainly to serum albumin (HSA), ceruloplasmin (CP), alpha-2-macroglobulin (α2M) and His, however, data for α2M are very limited and the thermodynamics and kinetics of the copper distribution are not known. We have applied a new LC-ICP MS-based approach for direct determination of Cu(II)-binding affinities of HSA, CP and α2M in the presence of competing Cu(II)-binding reference ligands including His. The ligands affected both the rate of metal release from Cu•HSA complex and the value of KD. Slow release and KD = 0.90 pM was observed with nitrilotriacetic acid (NTA), whereas His showed fast release and substantially lower KD = 34.7 fM (50 mM HEPES, 50 mM NaCl, pH 7.4), which was explained with formation of ternary His•Cu•HSA complex. High mM concentrations of EDTA were not able to elicit metal release from metallated CP at pH 7.4 and therefore it was impossible to determine the KD value for CP. In contrast to earlier inconclusive evidence, we show that α2M does not bind Cu(II) ions. In the human blood serum ~75% of Cu(II) ions are in a nonexchangeable manner bound to CP and the rest exchangeable copper is in an equilibrium between HSA (~25%) and Cu(II)-His-Xaa ternary complexes (~0.2%).

Copper(II) partially protects three histidine residues and the N-terminus of amyloid-ß peptide from diethyl pyrocarbonate (DEPC) modification.

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue-specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid-beta (Aß) peptides and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and determined the modification sites by electrospray ionization quadrupole time-of-flight tandem MS (ESI Q-TOF MS/MS). Our results indicate that five residues in Aß1-42 are modified in the presence of 30-fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aß1-42 promotes peptide aggregation, as monitored through the loss of soluble Aß42 in a semi-quantitative MALDI-TOF MS assay. It has been previously proposed that Cu(II) ions protect Aß1-16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aß1-16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aß from DEPC modification. Sequencing of obtained Cu(II)-protected Aß1-16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aß and to speculate about the role of His residues in these processes.

Mercury and Alzheimer's Disease: Hg(II) Ions Display Specific Binding to the Amyloid-ß Peptide and Hinder Its Fibrillization.

Brains and blood of Alzheimer's disease (AD) patients have shown elevated mercury concentrations, but potential involvement of mercury exposure in AD pathogenesis has not been studied at the molecular level. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils. Aß peptide fibrillization is known to be modulated by metal ions such as Cu(II) and Zn(II). Here, we study in vitro the interactions between Aß peptides and Hg(II) ions by multiple biophysical techniques. Fluorescence spectroscopy and atomic force microscopy (AFM) show that Hg(II) ions have a concentration-dependent inhibiting effect on Aß fibrillization: at a 1:1 Aß·Hg(II) ratio only non-fibrillar Aß aggregates are formed. NMR spectroscopy shows that Hg(II) ions interact with the N-terminal region of Aß(1-40) with a micromolar affinity, likely via a binding mode similar to that for Cu(II) and Zn(II) ions, i.e., mainly via the histidine residues His6, His13, and His14. Thus, together with Cu(II), Fe(II), Mn(II), Pb(IV), and Zn(II) ions, Hg(II) belongs to a family of metal ions that display residue-specific binding interactions with Aß peptides and modulate their aggregation processes.

Effect of methionine-35 oxidation on the aggregation of amyloid-ß peptide.

Aggregation of Aß peptides into amyloid plaques is considered to trigger the Alzheimer's disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aß aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H2O2. The fibrillization of Aß with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aß with oxidized Met35 in the presence of H2O2, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aß-Cu(II) complex.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid-ß peptide 1-42 but not amylin and insulin fibrils can grow under quiescent conditions.

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides--Alzheimer's amyloid-ß (Aß) 1-40 and 1-42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aß40 substantially decreased, whereas the fibrillization of Aß42 peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive.