Jeppe T. Pedersen

Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ1–40 using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ1–42 were compatible with the conclusions for Aβ1–40 even though the low solubility of Aβ1–42 precluded examination under the same conditions as for the Aβ1–40. Experiments with Aβ1–16 and Aβ1–28 showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ1–40 depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

Analysis of Protein Aggregation in Neurodegenerative Disease

Pathological protein and peptide aggregation are key events in a number of chronic and devastating neurodegenerative conditions including dementias such as Alzheimers and Creutzfeldt-Jakobs disease and other central nervous system diseases such as Parkinsons and Huntingtons disease and amyotrophic lateral sclerosis. Analytical methods for studying protein aggregation in these diseases are important for mapping pathophysiological events and ultimately for the development of new therapies and better diagnostic tools.

Rapid Formation of a Preoligomeric Peptide–Metal–Peptide Complex Following Copper(II) Binding to Amyloid β Peptides

Copper(II)-induced extracellular aggregation of amyloid b peptides (Ab) is implicated in the pathogenesis of Alzheimer s disease (AD). The exact role of the Cu–Ab interaction is not fully understood but it has been demonstrated that Cu–Ab oligomeric complexes may catalyze the formation of neurotoxic reactive oxygen species. Cu also induces the rapid formation of insoluble Ab oligomers that dissolve if Cu is removed. Low-order oligomers both with and without Cu are key to the neurodegeneration associated with AD. In the brain, Ab are primarily found as 40-residue (Ab1–40) and 42-residue (Ab1–42) peptides. It is known that Cu II binds to the N-terminal part of Ab and the 16-residue fragment Ab1–16 is well established as a nonfibrillating model for the Cu–Ab complex. Ab1–16 coordinates Cu II with the side chains of the amino acid residues D1, H6, H13, and H14 and presumably the N-terminal amino group; two different pHdependent coordination modes exist. The importance of the elucidation of the mechanism of Cu binding to Ab and its specific role in the aggregation process is emphasized by the fact that the Cu concentration is elevated in the amyloid plaques in brains from AD patients, and that Cu concentration transiently can reach micromolar concentrations in the extracellular space. However, detailed knowledge of the dynamics of the initial events in Cu binding to Ab and its relation to the formation of higher-order oligomers and aggregates is lacking. This information could be essential for the development of therapeutic strategies that could convert a toxic oligomerization pathway into a nontoxic pathway. Herein we address the kinetic mechanism of Cu binding to both Ab1–16 and Ab1–40, and copper(II)-induced Ab1–40 oligomerization by a combination of stopped-flow spectroscopy (fluorescence spectroscopy and light scattering), NMR relaxation, and dynamic simulations. Global fitting and dynamic simulations resulted in a unifying model for the initial steps of Cu binding to Ab, which includes a transient peptide–metal– peptide (Ab–Cu–Ab) complex that does not participate in the subsequent aggregation. Binding of Cu to Ab1–16 induces a large change in the environment of Y10 in Ab1–16 that allows characterization of the binding kinetics by stopped-flow fluorescence spectroscopy. Ab1–16 (40 mm) was mixed with solutions of Cu II with varying concentrations in HEPES buffer (0–40 mm), thus resulting in biphasic time traces (Figure 1a). The fast phase (< 30 ms) that exhibits a large decrease in signal intensity shows observed rates from approximately 35 to 1.7 10 s 1

Rapid Exchange of Metal between Zn7–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 Alzheimers 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 bodys 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.

Amyloid-β and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-β (Aβ) and α-synuclein (αS), associated with Alzheimer’s and Parkinson’s diseases, respectively, on the Cu2+-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu2+ is bound to Aβ or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aβ or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu2+ and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.

The pKa Value and Accessibility of Cysteine Residues Are Key Determinants for Protein Substrate Discrimination by Glutaredoxin

The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k1 = 1.75 × 10(5) M(-1) s(-1)) and the worst substrate (k1 = 4 × 10(2) M(-1) s(-1)). The pKa values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pKa values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pKa of the cysteine residue.

Inhibition of Cu-Amyloid-β by using Bifunctional Peptides with β-Sheet Breaker and Chelator Moieties

Breaking the mold: Inhibition of toxic amyloid-β (Aβ) aggregates and disruption of Cu-Aβ with subsequent redox-silencing of Cu have been considered promising strategies against Alzheimers disease. The design and proof of concept of simple peptides containing a Cu-chelating/redox-silencing unit and an Aβ-aggregation inhibition unit (β-sheet breaker) is described (see scheme).

Affinity capillary electrophoresis for identification and investigation of human Gc-globulin (vitamin D-binding protein) and its isoforms interacting with G-actin.

A CE procedure was established for the nondenaturing separation and identification of the isoforms of the actin‐binding human plasma protein Gc‐globulin. To characterize interactions with globular actin (G‐actin), a novel method was developed for the simultaneous qualitative assessment of the binding interaction between the three major isoforms of Gc‐globulin and G‐actin using pre‐equilibrium affinity CE and UV detection. Evidence was found that some difference in binding affinity existed among the isoforms, although the quantification of this difference was not feasible by UV detection because of the high affinity nature of the binding. The difference in affinity appeared to be related to the pI of the isoforms; a high pI corresponding to a high affinity. For quantitative binding studies Gc‐globulin was fluorescently labeled with 5‐(and‐6)‐carboxyfluorescein, succinimidyl ester (CFSE). Data suggested that extensive labeling interfered with actin binding but with moderately labeled Gc‐globulin it was possible to determine a dissociation constant of Kd = 21 ± 1 nM for the binding between labeled Gc‐globulin and G‐actin using pre‐equilibrium affinity CE and LIF detection.

Aggregation-Prone Amyloid-β⋅CuII Species Formed on the Millisecond Timescale under Mildly Acidic Conditions

Metal ions and their interaction with the amyloid beta (Aβ) peptide might be key elements in the development of Alzheimers disease. In this work the effect of CuII on the aggregation of Aβ is explored on a timescale from milliseconds to days, both at physiological pH and under mildly acidic conditions, by using stopped‐flow kinetic measurements (fluorescence and light‐scattering), 1H NMR relaxation and ThT fluorescence. A minimal reaction model that relates the initial CuII binding and Aβ folding with downstream aggregation is presented. We demonstrate that a highly aggregation prone Aβ⋅CuII species is formed on the sub‐second timescale at mildly acidic pH. This observation might be central to the molecular origin of the known detrimental effect of acidosis in Alzheimers disease.

Nanosecond Dynamics at Protein Metal Sites: An Application of Perturbed Angular Correlation (PAC) of γ-Rays Spectroscopy

Metalloproteins are essential to numerous reactions in nature, and constitute approximately one-third of all known proteins. Molecular dynamics of proteins has been elucidated with great success both by experimental and theoretical methods, revealing atomic level details of function involving the organic constituents on a broad spectrum of time scales. However, the characterization of dynamics at biomolecular metal sites on nanosecond time scales is scarce in the literature. The aqua ions of many biologically relevant metal ions exhibit exchange of water molecules on the nanosecond time scale or faster, often defining their reactivity in aqueous solution, and this is presumably also a relevant time scale for the making and breaking of coordination bonds between metal ions and ligands at protein metal sites. Ligand exchange dynamics is critical for a variety of elementary steps of reactions in metallobiochemistry, for example, association and dissociation of metal bound water, association of substrate and dissociation of product in the catalytic cycle of metalloenzymes, at regulatory metal sites which require binding and dissociation of metal ions, as well as in the transport of metal ions across cell membranes or between proteins involved in metal ion homeostasis. In Perturbed Angular Correlation of γ-rays (PAC) spectroscopy, the correlation in time and space of two γ-rays emitted successively in a nuclear decay is recorded, reflecting the hyperfine interactions of the PAC probe nucleus with the surroundings. This allows for characterization of molecular and electronic structure as well as nanosecond dynamics at the PAC probe binding site. Herein, selected examples describing the application of PAC spectroscopy in probing the dynamics at protein metal sites are presented, including (1) exchange of Cd2+ bound water in de novo designed synthetic proteins, and the effect of remote mutations on metal site dynamics; (2) dynamics at the β-lactamase active site, where the metal ion appears to jump between the two adjacent sites; (3) structural relaxation in small blue copper proteins upon 111Ag+ to 111Cd2+ transformation in radioactive nuclear decay; (4) metal ion transfer between two HAH1 proteins with change in coordination number; and (5) metal ion sensor proteins with two coexisting metal site structures. With this Account, we hope to make our modest contribution to the field and perhaps spur additional interest in dynamics at protein metal sites, which we consider to be severely underexplored. Relatively little is known about detailed atomic motions at metal sites, for example, how ligand exchange processes affect protein function, and how the amino acid composition of the protein may control this facet of metal site characteristics. We also aim to provide the reader with a qualitative impression of the possibilities offered by PAC spectroscopy in bioinorganic chemistry, especially when elucidating dynamics at protein metal sites, and finally present data that may serve as benchmarks on a relevant time scale for development and tests of theoretical molecular dynamics methods applied to biomolecular metal sites.