Binding of Zn(II) ions to α-lactalbumin

The binding of Zn(II) ions to human and bovine -lactalbumin has been studied by fluorescence, scanning microcalorimetry, and proteolytic digestion. The intrinsic tryptophan fluorescence spectrum of Ca(II)-loaded -lactalbumin is insensitive to Zn(II) binding to the strong cation binding sites (Zn:protein ratios up to 20), yet the thermal denaturation transition, as detected by intrinsic fluorescence, is shifted toward lower temperatures. On the other hand, low concentrations of Zn(II) ([Zn]:[protein]<1) shift heat sorption curves toward lower temperatures. It was concluded that -lactalbumin possesses several relatively strong Zn(II) binding sites, which are filled sequentially, the process being accompanied by protein aggregation. The strongest Zn(II) binding (5×10⁵ M^–1) increases its susceptibility to tryptic and chymotryptic digestion, slightly decreases its affinity for the fluorescent probe, bis-ANS, and alters its interactions with UDP-galactose. Zn(II) binding to aggregated forms of -lactalbumin increases its affinity to bis-ANS.     

Mitochondrial import and degradation of amyloid-β peptide

Mitochondrial dysfunctions associated with amyloid-β peptide (Aβ) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. Aβ is produced by sequential action of β- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aβ production, from which Aβ is further transported into the mitochondria. In vitro, Aβ was shown to be imported into the mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (PreP) is responsible for Aβ degradation reducing toxic effects of Aβ on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of Aβ production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aβ clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells

Background  

Self-assembly of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting Aβ self-assembly could serve as therapeutic agents and endogenous molecules that modulate Aβ self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Aβ aggregates warns that inhibition at intermediate stages of Aβ self-assembly may prove detrimental. Here, we explore the inhibition of Aβ_1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Aβ_1–40 activation of endothelial cells for monocyte adhesion.     

Rapid exchange of metal between Zn(7)-metallothionein-3 and amyloid-β peptide promotes amyloid-related structural changes

Metal ions, especially Zn(2+) and Cu(2+), are implemented in the neuropathogenesis of Alzheimer's disease (AD) by modulating the aggregation of amyloid-β peptides (Aβ). Also, Cu(2+) may promote AD neurotoxicity through production of reactive oxygen species (ROS). Impaired metal ion homeostasis is most likely the underlying cause of aberrant metal-Aβ interaction. Thus, focusing on the body's natural protective mechanisms is an attractive therapeutic strategy for AD. The metalloprotein metallothionein-3 (MT-3) prevents Cu-Aβ-mediated cytotoxicity by a Zn-Cu exchange that terminates ROS production. Key questions about the metal exchange mechanisms remain unanswered, e.g., whether an Aβ-metal-MT-3 complex is formed. We studied the exchange of metal between Aβ and Zn(7)-MT-3 by a combination of spectroscopy (absorption, fluorescence, thioflavin T assay, and nuclear magnetic resonance) and transmission electron microscopy. We found that the metal exchange occurs via free Cu(2+) and that an Aβ-metal-MT-3 complex is not formed. This means that the metal exchange does not require specific recognition between Aβ and Zn(7)-MT-3. Also, we found that the metal exchange caused amyloid-related structural and morphological changes in the resulting Zn-Aβ aggregates. A detailed model of the metal exchange mechanism is presented. This model could potentially be important in developing therapeutics with metal-protein attenuating properties in AD.     

Membrane-mimetic systems for biophysical studies of the amyloid-β peptide

The interplay between the amyloid-β (Aβ) peptide and cellular membranes have been proposed as an important mechanism for toxicity in Alzheimer's disease (AD). Membrane environments appear to influence Aβ aggregation and may stabilize intermediate Aβ oligomeric states that are considered to be neurotoxic. One important role for molecular biophysics within the field of Aβ studies is to characterize the structure and dynamics of the Aβ peptide in various states, as well as the kinetics of transfer between these states. Because biological cell membranes are very complex, simplified membrane models are needed to facilitate studies of Aβ and other amyloid proteins in lipid environments. In this review, we examine different membrane-mimetic systems available for molecular studies of Aβ. An introduction to each system is given, and examples of important findings are presented for each system. The benefits and drawbacks of each system are discussed from methodical and biological perspectives.     

On the origin of the stronger binding of PIB over thioflavin T to protofibrils of the Alzheimer amyloid-β peptide: a molecular dynamics study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ(9-40) protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ(17-42) revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ(17-42) cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.     

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.     

Staphylococcal display for combinatorial protein engineering of a head-to-tail affibody dimer binding the Alzheimer amyloid-β peptide

We have previously generated an affibody molecule for the disease-associated amyloid beta (Aβ) peptide, which has been shown to inhibit the formation of various Aβ aggregates and revert the neurotoxicity of Aβ in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the Aβ-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and Aβ binding. Overall, the detailed engineering and characterization of this promising Aβ-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.     

Minimal Zn(2+) binding site of amyloid-β

Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aβ.     

Carbohydrate adducts with zinc-group-metal ions. Interactions of β-d-fructose with Zn(II), Cd(II), and Hg(II) cations,and the effects of metal-ion coordination on the sugar isomer binding

Interaction of β-d-fructose with hydrated salts of zinc-group-metal has been studied in aqueous solution and solid adducts of the type M(d-fructose)X₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl⁻ or Br⁻, and n = 0–2, have been isolated, and characterized by means of F.t.-i.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. The marked spectral similarities observed with the Mg(d-fructose)X₂·4 H₂O (X = Cl⁻ or Br⁻) compounds indicated that the Zn(II) and Cd(II) ions are six-coordinated, binding to two d-fructose molecules through O-2, O-3 of the first d-fructose, and O-4, O-5 of the second, as well as to two H₂O. The Hg(II) ion binds to two sugar moieties in the same fashion as do the Zn(II) and Cd(II) ions, resulting in four-coordination geometry around the mercury ion. The crystalline sugar is in the β-d-fructopyranose form, and the coordination of the of the Ca(II) ion takes place through the β-d-fructopyranose isomer, whereas the binding of the Mg(II), Zn(II), Cd(II), Hg(II), and UO²⁺₂ cations could be via the β-d-fructopyranose and the β-d-fructofuranose structures.     

Modulation of aggregate size- and shape-distributions of the amyloid-β peptide by a designed β-sheet breaker

A peptide with 42 amino acid residues (Aβ42) plays a key role in the pathogenesis of the Alzheimer’s disease. It is highly prone to self aggregation leading to the formation of fibrils which are deposited in amyloid plaques in the brain of diseased individuals. In our study we established a method to analyze the aggregation behavior of the Aβ peptide with a combination of sedimentation velocity centrifugation and enhanced data evaluation software as implemented in the software package UltraScan. Important information which becomes accessible by this methodology is the s-value distribution and concomitantly also the shape-distribution of the Aβ peptide aggregates generated by self-association. With this method we characterized the aggregation modifying effect of a designed β-sheet breaker molecule. This compound is built from three head-to-tail connected aminopyrazole moieties and represents a derivative of the already described Tripyrazole. By addition of this compound to a solution of the Aβ42 peptide the maximum of the s-value distribution was clearly shifted to smaller s-values as compared to solutions where only the vehicle DMSO was added. This shift to smaller s-values was stable for at least 7 days. The information about size- and shape-distributions present in aggregated Aβ42 solutions was confirmed by transmission electron microscopy and by measurement of amyloid formation by thioflavin T fluorescence.     

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein-ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.     

Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal–Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal–protein interactions, including those in metal–Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu²⁺ binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10⁹ and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu²⁺-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu²⁺ binding to Aβ and other intrinsically disordered peptides.     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

Benchmarking of copper(II) LFMM parameters for studying amyloid-β peptides

Ligand field molecular mechanics (LFMM) parameters have been benchmarked for copper (II) bound to the amyloid-β_1–16 peptide fragment. Several density functional theory (DFT) optimised small test models, representative of different possible copper coordination modes, have been used to test the accuracy of the LFMM copper bond lengths and angles, resulting in errors typically less than 0.1 Å and 5°. Ligand field molecular dynamics (LFMD) simulations have been carried out on the copper bound amyloid-β_1–16 peptide and snapshots extracted from the subsequent trajectory. Snapshots have been optimised using DFT and the semi-empirical PM7 method resulting in good agreement against the LFMM calculated geometry. Analysis of substructures within snapshots shows that the larger contribution of geometrical difference, as measured by RMSD, lies within the peptide backbone, arising from differences in DFT and AMBER, and the copper coordination sphere is reproduced well by LFMM. PM7 performs excellently against LFMM with an average RMSD of 0.2 Å over 21 tested snapshots. Further analysis of the LFMD trajectory shows that copper bond lengths and angles have only small deviations from average values, with the exception of a carbonyl moiety from the N-terminus, which can act as a weakly bound fifth ligand.     

Synthesis, Characterization and Antitumor Studies of Mn(II), Ni(II), Cu(II) and Zn(II) Complexes of N-Nicotinoyl-N′-o-Hydroxythiobenzhydrazide

A new ligand N-Nicotinoyl-N-o-hydroxythiobenzhydrazide (H₂Notbh) forms complexes [Mn(Notbh)(H₂O)], [M(Notbh)] [M=Ni(II) Cu(II) and Zn(II)] which were characterized by various physico-chemical techniques. All the metal complexes were observed to inhibit the growth of tumor in vitro, whereas, ligand did not. In vivo administration of these complexes resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with metal complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H₂Notbh and its metal complexes in tumor regression and tumor growth associated immunosuppression.     

The clathrin assembly protein AP180 regulates the generation of amyloid-β peptide

The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer’s disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar clathrin assembly proteins, AP180 and CALM. We show that RNA interference-mediated knockdown of AP180 reduces the generation of Aβ1-40 and Aβ1-42, whereas CALM knockdown has no effect on Aβ generation. Thus AP180 is among the traffic controllers that oversee and regulate amyloid precursor protein processing pathways. Our results also suggest that AP180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.     

Probing the efficacy of peptide-based inhibitors against acid- and zinc-promoted oligomerization of amyloid-β peptide via single-oligomer spectroscopy

One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-β peptide (Aβ). Given the deleterious effects reported for Aβ dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled Aβ peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2-5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of Aβ species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments.     

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Extended X-ray absorption fine structure studies of the metallo-β-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn₁ site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal–metal interaction at 3.42 Å. Reaction with the β-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn–Zn interaction to 3.62 Å.     

Computational study of the binding of CuII to Alzheimer’s amyloid-β peptide: Do Aβ42 and Aβ40 bind copper in identical fashion?

One of the many hypotheses on the pathogenesis of Alzheimer’s disease is that the amyloid-β peptide (Aβ) binds Cu^II and can catalytically generate H₂O₂, leading to oxidative damage in brain tissues. For a molecular level understanding of such catalysis it is critical to know the structure of the Aβ–Cu^II complex precisely. Unfortunately, no high-resolution structure is available to date and there is considerable debate over the copper coordination environment with no clear consensus on which residues are directly bound to Cu^II. Considering all plausible isomers of the copper-bound Aβ42 and Aβ40 using a combination of density functional theory and classical molecular dynamics methods, we report an atomic resolution structure for each possible complex. We evaluated the relative energies of these isomeric structures and surprisingly found that Aβ42 and Aβ40 display very different binding modes, suggesting that shorter peptides that are truncated at the C-terminus may not be realistic models for understanding the chemistry of the most neurotoxic peptide, Aβ42. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.