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Binding of zinc(II) and copper(II) to the full-length Alzheimer’s amyloid-β peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta‐peptides (Αβ) may contribute to the pathogenesis of Alzheimer’s disease. Cu2+ and Zn2+ form complexes with Αβ peptides in vitro; however, the published metal‐binding affinities of Αβ vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Αβ40 under different conditions using intrinsic Αβ fluorescence and metal‐selective fluorescent dyes. We showed that Cu2+ forms a stable and soluble 1 : 1 complex with Αβ40, however, buffer compounds act as competitive copper‐binding ligands and affect the apparent KD. Buffer‐independent conditional KD for Cu(II)‐Αβ40 complex at pH 7.4 is equal to 0.035 μmol/L. Interaction of Αβ40 with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn‐Αβ40 complex (KD = 60 μmol/L) undergoes a transition to a more tight complex with KD ∼ 2 μmol/L Competition of Αβ40 with ion‐selective fluorescent dyes Phen Green and Zincon showed that the KD values determined from intrinsic fluorescence of Αβ correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Αβ peptides may occur in brain areas affected by Alzheimer’s disease and Zn2+‐induced transition in the peptide structure might contribute to amyloid plaque formation.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid‐β (Aβ) peptides is a central phenomenon in Alzheimer’s disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ42 fibrillization and initiate formation of non‐fibrillar Aβ42 aggregates, and that the inhibitory effect of Zn(II) (IC50 = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high‐affinity metal chelators including metallothioneins prevented metal‐induced Aβ42 aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ42 fibrillization. Upon prolonged incubation the metal‐induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ42. H13A and H14A mutations in Aβ42 reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross‐β core structure within region 10–23 of the amyloid fibril. Cu(II)‐Aβ42 aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)‐Aβ42 aggregates were non‐toxic. Disturbed metal homeostasis in the vicinity of zinc‐enriched neurons might pre‐dispose formation of metal‐induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal‐chelating therapies for Alzheimer’s disease is discussed in this light.

The missing link in the amyloid cascade of Alzheimer’s disease – Metal ions

Progressive deposition of amyloid beta (Aβ) peptides into amyloid plaques is the pathological hallmark of Alzheimers disease (AD). The amyloid cascade hypothesis pins this deposition as the primary cause of the disease, but the mechanisms that causes this deposition remain elusive. An increasing amount of evidence shows that biometals Zn(II) and Cu(II) can interact with Aβ, thus influencing the fibrillization and toxicity. This review focuses on the role of Zn(II) and Cu(II) in AD, and revisits the amyloid cascade hypothesis demonstrating the possible roles of Zn(II) and Cu(II) in the disease pathogenesis.

Label-Free High-Throughput Screening Assay for Inhibitors of Alzheimer’s Amyloid-β Peptide Aggregation Based on MALDI MS

Aggregation of amyloid-β (Aβ) peptides is causatively linked to Alzheimers disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of Aβ aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial Aβ derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of Aβ₄₂ fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of Aβ₄₂ fibrillization.

Zn(II) ions co-secreted with insulin suppress inherent amyloidogenic properties of monomeric insulin

Insulin, a 51-residue peptide hormone, is an intrinsically amyloidogenic peptide, forming amyloid fibrils in vitro. In the secretory granules, insulin is densely packed together with Zn(II) into crystals of Zn(2)Insulin(6) hexamer, which assures osmotic stability of vesicles and prevents fibrillation of the peptide. However, after release from the pancreatic beta-cells, insulin dissociates into active monomers, which tend to fibrillize not only at acidic, but also at physiological, pH values. The effect of co-secreted Zn(II) ions on the fibrillation of monomeric insulin is unknown, however, it might prevent insulin fibrillation. We showed that Zn(II) inhibits fibrillation of monomeric insulin at physiological pH values by forming a soluble Zn(II)-insulin complex. The inhibitory effect of Zn(II) ions is very strong at pH 7.3 (IC(50)=3.5 microM), whereas at pH 5.5 it progressively weakens, pointing towards participation of the histidine residue(s) in complex formation. The results obtained indicate that Zn(II) ions might suppress fibrillation of insulin at its release sites and in circulation. It is hypothesized that misfolded oligomeric intermediates occurring in the insulin fibrillation pathway, especially in zinc-deficient conditions, might induce autoantibodies against insulin, which leads to beta-cell damage and autoimmune Type 1 diabetes.

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid-β 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 – Alzheimers amyloid‐β (Aβ) 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 Aβ40 substantially decreased, whereas the fibrillization of Aβ42 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. Copyright

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ 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 Aβ 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 Aβ 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 Aβ 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 Aβ–Cu(II) complex.

In vitro fibrillization of Alzheimer’s amyloid-β peptide (1-42)

The amyloid deposition in the form of extracellular fibrillar aggregates of amyloid-β (Aβ) peptide is a critical pathological event in Alzheimer’s disease. Here, we report a systematic investigation of the effects of environmental factors on the kinetics of Aβ fibrillization in vitro. The effects of Aβ42 peptide concentration, temperature, pH, added solvents and the ratio of Aβ40 and Aβ42 on the peptide fibrillization under agitated conditions was studied. The analysis show that the rate of fibril growth by monomer addition is not limited by diffusion but by rearrangement in the monomer structure, which is enhanced by low concentrations of fluorinated alcohols and characterized by the activation energy of 12 kcal/mol. Fibrillization rate decreases at pH values below 7.0 where simultaneous protonation of His 13 and 14 inhibits fibril formation. The lag period for Aβ42 was only twofold shorter and the fibril growth rate twofold faster than those of Aβ40. Lag period was shortened and the fibrillization rate was increased only at 90% content of Aβ42.

Oxidation of Methionine-35 in Alzheimer’s amyloid-beta peptide and the aggregation of the oxidized peptide

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive loss of brain tissue and accumulation of amyloid-β(Aβ) and tau. Aggregation of Aβ peptides into amyloid plaques is considered to be a causative factor in AD, however the precise mechanism behind the AD onset has remained elusive. Oxidative stress (OS) is also characteristic to AD, but it is not known whether OS is a risk factor or a consequence of AD. It is assumed that Aβ aggregates generate free radicals in the presence of copper ions by participation of the Met35 residue, which can increase the OS levels. The aim of our study was to establish the role of Met35 residue in the oxidation of Aβ and peptide aggregation processes. Oxidation of Aβ was studied in the presence of two redox-active compounds: H2O2 and copper ions. In the absence of copper ions the Met35 residue was readily oxidized by H2O2 in a two electron process. The fibrillization of Aβ with Met35 oxidized to sulfoxide was threefold slower compared to that of the native peptide. TEM analysis showed that the fibrils of native and oxidized peptides are similar. The relatively small inhibitory effect of Met35 oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. In the presence of copper ions (one-electron process) the oxidation was more complex: addition of the first oxygen was still the fastest process, however, it was accompanied by multiple unspecific modifications of several amino acid residues. Addition of copper ions to the already oxidized Aβ Met35 by H2O2, resulted in a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in Aβ do not depend on the oxidation state of Met35. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ-Cu(II) complex.

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.