Peter Faller

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that CuII can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the CuII binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these CuII–Aβ complexes: (1) ITC showed two CuII binding sites, with an apparent Kd of 10−7 and 10−5xa0M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to CuII in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the CuII in the low-affinity binding site in Aβ28 but not in Aβ16; (5) 1H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to CuII in the high-affinity binding site; (6) the pKa of 11.3 of tyrosine in position 10 was not influenced by the binding of 2xa0equivalents of CuII.

Thioflavin Derivatives as Markers for Amyloid‐β Fibrils: Insights into Structural Features Important for High‐Affinity Binding

Alzheimer’s disease (AD) is a common neurological disease associated with chronic dementia, memory loss, and cognitive impairment. Central to the neuropathology of AD are the senile plaques (SPs) and neurofibrillar tangles (NFTs), depositions composed of amyloid-b peptide (Ab) and tau protein, respectively. Aggregated Ab in senile plaques has a b-sheet secondary structure and is arranged as fibrils. 3] Soluble Ab prior to aggregation is predominantly unstructured. Formation and accumulation of aggregates of Ab peptides in the brain are critical factors in the development and progression of AD, in which oxidative stress represents a field of current intensive studies. There is a great interest in molecules able to bind specifically to Ab aggregates. Such markers of Ab fibrils would allow their early detection and specificity would permit identification of Ab fibrils from other amyloid deposits. This is of importance from basic research to clinical application. In particular, such molecules give access to molecular imaging (by Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), or Magnetic Resonance Imaging (MRI)) therefore allowing a precise localisation and identification of the Ab aggregates in the brain. This is in contrast with the less specific information obtained either by cerebrospinal fluid analysis, or anatomic imaging by MRI. The progress of therapies that may affect Ab deposition in AD brain has added new significance to this pursuit. Three main categories of PET ligands of AD-associated aggregates are currently under investigation (Figure 1): Thioflavin T (ThT) derived compounds (for example, N-methyl-[(11)C]2-(4’-methylaminophenyl)-6-hydroxybenzothiazole (PIB)), including variations of the heteroaromatic core, styrylbenzene (SB), and compounds with an aminonaphthyl core (for example, 2-(1-{6-[(2-[F-18]fluoroethyl)ACHTUNGTRENNUNG(methyl)amino]-2-naphthyl}ethylidene)malononitrile (FDDNP)). ThT has been used for several decades to stain amyloids such as Ab. ThT is not specific for amyloids of Ab, as it reacts with many types of amyloids (for example, NFT, insulin, b2-microglobulin) but not all. Thus several derivatives of ThT have been generated with the aim of developing a biomarker of Ab fibrils with high affinity and high specificity, as exemplified by BTA-1, N-methyl-C-PIB, I-TZDM, etc. Yet not much is known about the structure–function relationships that explain the molecular nature of the interaction marker/Ab aggregate and most improvement of binding-affinity seemed to arise from empirical studies. The reported insights are scarce. It seems clear that the removal of the methyl group on the heterocyclic nitrogen of ThT and hence the removal of the positive charge increases the affinity to Ab fibrils by a factor of 40. Simultaneously, the removal of the charge increases the lipophilicity of the compounds and therefore eases crossing of the blood–brain barrier (BBB). Also removal of one of the two methyl groups on the amine nitrogen in the 4’-position did increase the affinity for a so far unknown reason. In contrast removal of the methyl group on the carbon 6 of the benzothiazole moiety did not significantly change the affinity. Better knowledge of the binding sites of the markers derived from ThT would allow a more rational ligand design. To address this question and to get a deeper insight into the nature of the interaction between uncharged ThT derivatives and in particular the role of the amine nitrogen in the 4’-position, we synthesized 18 ThT derivatives of the dimethylaminophenyl moiety and compared their binding affinity to Ab fibrils, which allowed us to propose some important features regarding the marker/Ab fibrils interaction. In the present work a highly convergent synthesis of ThT derivatives applied to 18 examples based on two similar reactions has been established (Scheme 1). Namely, the condensation of Figure 1. Structures of some known Ab aggregate markers.

The heart of photosynthesis in glorious 3D

The input of solar energy into photosynthesis, and thence into the biosphere, occurs via chlorophyll-containing proteins known as reaction centres. There are two kinds of reaction centre in oxygenic photosynthesis: photosystem I (PSI) and photosystem II (PSII). The PSII reaction centre, alias the oxygen-evolving enzyme, the water-oxidizing complex or the water-plastoquinone photo-oxidoreductase, has now been crystallized and its structure solved to a resolution of 3.8 A.

Side-Path Electron Donors: Cytochrome b559, Chlorophyll Z and β-Carotene

β-Carotene (Car), cytochrome (Cyt) b559 and a monomeric chlorophyll (Chl) designated as chlorophyll Z, all undergo oxidation in Photosystem (PS) II under some illumination conditions. These components are not part of the direct electron transfer that leads to water oxidation and plastoquinone reduction and are thus designated ‘side-path electron donors.’ Under the usual conditions of PS II function, the quantum yield for the oxidation of these components is low; however, under certain experimental conditions, particularly low temperatures, the dominant reactions can be those involving the side-path donors. Car is a branch point in the side-path electron donation, being oxidized by P+ (the kinetically competent Chl cation radical), and reduced by Cyt b559, which is itself reduced by electrons from the pool of plastoquinol, possibly through the QB site. This all occurs on the D2-side of the reaction center. When the Cyt b559 is pre-oxidized, Car+ is reduced by Chl Z. There are two candidates for Chl Z, the more obvious candidate on the D2 side and the less straightforward candidate on D1 side of the reaction center. The side-pathway is usually rationalized as a photoprotective cycle aimed at removing long-lived P+ and thus limiting oxidative damage. Based on the low quantum yields, we consider this unlikely. Instead we suggest that the side-path constitutes a photoprotective cycle in which the aim is to reduce the Car cation, rather than P+, returning the carotene cation to its unoxidized state, preventing adventitious reactions and allowing it to play its a role as a singlet O2 quencher in the heart of PS II.

Folding of the prion peptide GGGTHSQW around the copper(II) ion: identifying the oxygen donor ligand at neutral pH and probing the proximity of the tryptophan residue to the copper ion.

The GGGTHSQW sequence in the amyloidogenic part of the prion protein is a potential binding site for Cu(II). We have previously studied the binding of copper to the shorter GGGTH peptide and showed that it is highly pH dependent (Hureau et al. in J. Biol. Inorg. Chem. 11:735–744, 2006). Two predominant complexes could be characterized at pH 6.7 and 9.0 with equatorial binding modes of 3N1O and 4N for the metal ion, respectively. In this work, we have further investigated the coordination of Cu(II) to the GGGTH peptide as well as the longer GGGTHSQW peptide in order to identify the oxygen donor ligand at neutral pH and to study the proximity and redox activity of the tryptophan residue of the latter. The results for both peptides show that, at pH 6.7, Cu(II) is coordinated by a carbonyl peptide backbone. At higher pH values, the carbonyl ligand dissociates and the coordination changes to a 4N binding mode, inducing a structural rearrangement that brings the GGGTHSQW peptide’s tryptophan residue into the vicinity of the copper ion, thus affecting their respective redox properties.

The Na+/K+-ATPase and the amyloid-beta peptide aβ1-40 control the cellular distribution, abundance and activity of TRPC6 channels.

The Na(+)/K(+)-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na(+)/K(+)-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca(2+) entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca(2+) chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ(1-40), another inhibitor of the Na(+)/K(+)-ATPase, but not the shorter peptide Aβ1-16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na(+) channel), and Aβ(1-40) reduced TRPC6-mediated Ca(2+) responses whereas Aβ(1-16) was ineffective. Furthermore, when Aβ(1-40) was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na(+)/K(+)-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ(1-40) which underlines the pathophysiological significance of these processes.

Learning chemistry with multiple first-principles simulations

Huge parallel high-performance computing (HPC) architectures are today available laboratories for modelling atomic forces with high accuracy and for large samples of atoms. Modern statistical tools allow to simulate the statistics of these samples, while first-principles molecular dynamics (MD) can probe the interactions within large atomic samples, including possible chemical reactions. But a proper statistical convergence for the ensemble, represented in terms of a bundle of trajectories, is still unsatisfactory in terms of comparisons with experiments. Can we learn something by these HPC experiments? In this contribution, we show one example, where the occurrence of a chemical reaction in a disordered system is probed. The complex of the copper ion and a segment of the amyloid-β peptide, of wide interest in understanding the progress of Alzheimers disease, was modelled combining constructions based on empirical force fields with first-principles MD simulations. We simulate a bundle of 16 different structures, biasing different Cu coordination numbers and changing the charge (oxidation state) of the assembly. Even within the given approximations for forces and the poor sampling, we could identify the structures of the complex that are able to react with hydrogen peroxide. The observation explains, at a molecular level, one important linkage between Alzheimers disease and oxidative stress. This is an example of a general strategy for exploiting reactive configurations within a large set of possible reasonable candidates.

A Cu-amyloid β complex activating Fenton chemistry in Alzheimer's disease: Learning with multiple first-principles simulations

Amyloid β peptides form complexes with copper, both in vitro and in vivo, relatively soluble in water as oligomers and active as catalysts for oxidation of organic substrates by hydrogen peroxide, a species always present in cells and in their aerobic environment. All these species are present in the synapse, thus making a connection between the amyloid cascade hypothesis and the oxidative damages by reactive oxygen species in neurons, when pathological dishomeostasis of amyloid peptides and metal ions occur. In order to understand the structural features of these toxic complexes, we built several models of Cu-Aβ peptides in monomeric and dimeric forms and we found, performing multiple first-principles molecular dynamics simulations, that Cu-induced dimers are more active than monomers in converting hydrogen peroxide into aggressive hydroxyl radicals.