Direct Competition of ATCUN Peptides with Human Serum Albumin for Copper(II) Ions Determined by LC-ICP MS.

Copper is an indispensable biometal, primarily serving as a redox-competent cofactor in numerous proteins. Apart from preformed copper-binding sites within the protein structures, small peptide motifs exist called ATCUN, which are composed of an N-terminal tripeptide XZH, able to bind Cu(II) ions in exchangeable form. These motifs are common for serum albumin, but they are also present in a wide range of proteins and peptides. These proteins and peptides can be involved in copper metabolism, and copper ions can affect their biological role. The distribution of copper between the ATCUN peptides, including truncated amyloid-ß (Aß) peptides Aß4-42 and Aß11-42, which may be involved in Alzheimer's disease pathogenesis, is mainly determined by their concentrations and relative Cu(II)-binding affinities. The Cu(II)-binding affinity (log Kd) of several ATCUN peptides, determined by different methods and authors, varies by more than three orders of magnitude. This variation may be attributed to the chemical properties of peptides but can also be influenced by the differences in methods and experimental conditions used for the determination of Kd. In the current study, we performed direct competition experiments between selected ATCUN peptides and HSA by using an LC-ICP MS-based approach. We demonstrated that ATCUN and truncated Aß peptides Aß4-16 and Aß11-15 bind Cu(II) ions with an affinity similar to that for HSA. Our results demonstrate that ATCUN motifs cannot compete with excess HSA for the binding of Cu(II) ions in the blood and cerebrospinal fluid.

Characterization of Uranyl (UO22+) Ion Binding to Amyloid Beta (Aß) Peptides: Effects on Aß Structure and Aggregation.

Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer's disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aß aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aß production, and these metals bind to Aß peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aß peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to Aß peptides with affinities in the micromolar range, induce structural changes in Aß monomers and oligomers, and inhibit Aß fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

Mercury Ion Binding to Apolipoprotein E Variants ApoE2, ApoE3, and ApoE4: Similar Binding Affinities but Different Structure Induction Effects.

Mercury intoxication typically produces more severe outcomes in people with the APOE-ε4 gene, which codes for the ApoE4 variant of apolipoprotein E, compared to individuals with the APOE-ε2 and APOE-ε3 genes. Why the APOE-ε4 allele is a risk factor in mercury exposure remains unknown. One proposed possibility is that the ApoE protein could be involved in clearing of heavy metals, where the ApoE4 protein might perform this task worse than the ApoE2 and ApoE3 variants. Here, we used fluorescence and circular dichroism spectroscopies to characterize the in vitro interactions of the three different ApoE variants with Hg(I) and Hg(II) ions. Hg(I) ions displayed weak binding to all ApoE variants and induced virtually no structural changes. Thus, Hg(I) ions appear to have no biologically relevant interactions with the ApoE protein. Hg(II) ions displayed stronger and very similar binding affinities for all three ApoE isoforms, with K D values of 4.6 µM for ApoE2, 4.9 µM for ApoE3, and 4.3 µM for ApoE4. Binding of Hg(II) ions also induced changes in ApoE superhelicity, that is, altered coil-coil interactions, which might modify the protein function. As these structural changes were most pronounced in the ApoE4 protein, they could be related to the APOE-ε4 gene being a risk factor in mercury toxicity.

Evaluation of Zn2+- and Cu2+-Binding Affinities of Native Cu,Zn-SOD1 and Its G93A Mutant by LC-ICP MS.

The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn2+ and Cu2+ were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the KD values. From the Arrhenius plots of the demetallation in the absence of chelators ΔH‡ = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high ΔH values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar.

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.

Insulin Fibrillization at Acidic and Physiological pH Values is Controlled by Different Molecular Mechanisms.

Formation of amyloid-like fibrils by insulin was studied at different insulin concentrations, pH and temperatures. At low pH (pH 2.5) the insulin fibrillization occurred only at high ([10 lM) peptide concentrations, whereas at physiological pH values the fibril formation is inhibited at higher insulin concentrations. The enthalpy of activation Ea of the fibril growth at pH 2.5 equals to 33 kJ/mol, which is considerably lower than 84 kJ/mol at physiological pH. The fibrillization rate of insulin decreases with increasing pH at high, 250 lM concentration, which was opposite to the pH effect observed in 2.5 lM insulin solutions. The latter effect indicates that protonation of histidine residues seems to be important for the fibrillization of monomeric insulin, whereas the pH effect at high concentration may result from off-pathway oligomerization propensity. Together, the different effect of environmental factors on the insulin fibrillization suggest that the reaction rate is controlled by different molecular events in acidic conditions and at physiological pH values.

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.

Interference of low-molecular substances with the thioflavin-T fluorescence assay of amyloid fibrils.

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type-II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low-molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid-ß fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag-period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes.

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.