John H. Viles

Amyloid β Protein and Alzheimer’s Disease: When Computer Simulations Complement Experimental Studies

Simulations Complement Experimental Studies Jessica Nasica-Labouze,† Phuong H. Nguyen,† Fabio Sterpone,† Olivia Berthoumieu,‡ Nicolae-Viorel Buchete, Sebastien Cote, Alfonso De Simone, Andrew J. Doig, Peter Faller,‡ Angel Garcia, Alessandro Laio, Mai Suan Li, Simone Melchionna, Normand Mousseau, Yuguang Mu, Anant Paravastu, Samuela Pasquali,† David J. Rosenman, Birgit Strodel, Bogdan Tarus,† John H. Viles, Tong Zhang,†,▲ Chunyu Wang, and Philippe Derreumaux*,†,□ †Laboratoire de Biochimie Theorique, Institut de Biologie Physico-Chimique (IBPC), UPR9080 CNRS, Universite Paris Diderot, Sorbonne Paris Cite, 13 rue Pierre et Marie Curie, 75005 Paris, France ‡LCC (Laboratoire de Chimie de Coordination), CNRS, Universite de Toulouse, Universite Paul Sabatier (UPS), Institut National Polytechnique de Toulouse (INPT), 205 route de Narbonne, BP 44099, Toulouse F-31077 Cedex 4, France School of Physics & Complex and Adaptive Systems Laboratory, University College Dublin, Belfield, Dublin 4, Ireland Deṕartement de Physique and Groupe de recherche sur les proteines membranaires (GEPROM), Universite de Montreal, C.P. 6128, succursale Centre-ville, Montreal, Quebec H3C 3T5, Canada Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom Department of Physics, Applied Physics, & Astronomy, and Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, United States The International School for Advanced Studies (SISSA), Via Bonomea 265, 34136 Trieste, Italy Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland Institute for Computational Science and Technology, SBI Building, Quang Trung Software City, Tan Chanh Hiep Ward, District 12, Ho Chi Minh City, Vietnam Instituto Processi Chimico-Fisici, CNR-IPCF, Consiglio Nazionale delle Ricerche, 00185 Roma, Italy School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore Department of Chemical and Biomedical Engineering, Florida A&M University-Florida State University (FAMU-FSU) College of Engineering, 2525 Pottsdamer Street, Tallahassee, Florida 32310, United States National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310, United States Institute of Complex Systems: Structural Biochemistry (ICS-6), Forschungszentrum Julich GmbH, 52425 Julich, Germany School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom Institut Universitaire de France, 75005 Paris, France

Copper binding to the octarepeats of the prion protein: Affinity, specificity, folding, and cooperativity: Insights from circular dichroism

The prion protein (PrP) is a Cu2+ binding cell surface glycoprotein. There is increasing evidence that PrP functions as a copper transporter. In addition, strains of prion disease have been linked with copper binding. We present here CD spectroscopic studies of Cu2+ binding to various fragments of the octarepeat region of the prion protein. We show that glycine and l-histidine will successfully compete for all Cu2+ ions bound to the PrP octapeptide region, suggesting Cu2+ coordinates with a lower affinity for PrP than the fm dissociation constant reported previously. We show that each of the octarepeats do not form an isolated Cu2+ binding motif but fold up cooperatively within multiple repeats. In addition to the coordinating histidine side chain residues, we show that the glycine residues and the proline within each octarepeat are also necessary to maintain the coordination geometry. The highly conserved octarepeat region in mammals is a hexarepeat in birds that also binds copper but with different coordination geometry. Finally, in contrast to other reports, we show that Mn2+ does not bind to the octarepeat region of PrP.

Copper(II) binding to amyloid-β fibrils of Alzheimer's disease reveals a picomolar affinity: Stoichiometry and coordination geometry are independent of Aβ oligomeric form

Cu(2+) ions are found concentrated within senile plaques of Alzheimers disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.

Substoichiometric Levels of Cu2+ Ions Accelerate the Kinetics of Fiber Formation and Promote Cell Toxicity of Amyloid-β from Alzheimer Disease

A role for Cu2+ ions in Alzheimer disease is often disputed, as it is believed that Cu2+ ions only promote nontoxic amorphous aggregates of amyloid-β (Aβ). In contrast with currently held opinion, we show that the presence of substoichiometric levels of Cu2+ ions in fact doubles the rate of production of amyloid fibers, accelerating both the nucleation and elongation of fiber formation. We suggest that binding of Cu2+ ions at a physiological pH causes Aβ to approach its isoelectric point, thus inducing self-association and fiber formation. We further show that Cu2+ ions bound to Aβ are consistently more toxic to neuronal cells than Aβ in the absence of Cu2+ ions, whereas Cu2+ ions in the absence of Aβ are not cytotoxic. The degree of Cu-Aβ cytotoxicity correlates with the levels of Cu2+ ions that accelerate fiber formation. We note the effect appears to be specific for Cu2+ ions as Zn2+ ions inhibit the formation of fibers. An active role for Cu2+ ions in accelerating fiber formation and promoting cell death suggests impaired copper homeostasis may be a risk factor in Alzheimer disease.

Deconvoluting the Cu2+ Binding Modes of Full-length Prion Protein

The prion protein (PrP) is a cell-surface Cu2+-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23–231) and constructs in which the octarepeat region has been removed, or His95 and His110 is replaced by alanine residues, have been used to elucidate the order and mode of Cu2+ coordination to PrP-(23–231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu2+ coordination to full-length PrP. At physiological pH, Cu2+ initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His95 and His110. Only subsequent Cu2+ ions bind to single histidine residues within the octarepeat region. Ni2+ ions are used to further probe metal binding and, like Cu2+, Ni2+ will bind individually to His95 and His110, involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu2+ bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu2+ binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.

Amyloid β−Cu2+ Complexes in both Monomeric and Fibrillar Forms Do Not Generate H2O2 Catalytically but Quench Hydroxyl Radicals†

Oxidative stress plays a key role in Alzheimers disease (AD). In addition, the abnormally high Cu(2+) ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid beta peptide (Abeta) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Abeta acts as a prooxidant producing H2O2. However, others have indicated Abeta acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Abeta under aerobic conditions in the presence of Cu(2+) with/without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu(2+) reduction. In contrast to previous studies, we show here that Abeta does not generate any more ROS than controls of Cu(2+) and ascorbate. Abeta does not silence the redox activity of Cu(2+/+) via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu(2+) rapidly react with Abeta; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Abeta peptide was examined using (1)H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Abeta being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Abeta in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation.

Manganese Binding to the Prion Protein

There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 μm at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called “fifth site” normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.

Human Serum Albumin Can Regulate Amyloid-β Peptide Fiber Growth in the Brain Interstitium IMPLICATIONS FOR ALZHEIMER DISEASE

Background: 95% of amyloid-β in blood plasma binds to albumin with a Kd of 5 micromolar. Results: Physiological, micromolar levels of albumin inhibit amyloid fiber formation. Conclusion: Nearly half of Aβ in the CSF will be bound to albumin and inhibited from forming fibers. Significance: Levels of albumin in CSF must represent a risk factor and therapeutic target in Alzheimer disease. Alzheimer disease is a neurodegenerative disorder characterized by extracellular accumulation of amyloid-β peptide (Aβ) in the brain interstitium. Human serum albumin (HSA) binds 95% of Aβ in blood plasma and is thought to inhibit plaque formation in peripheral tissue. However, the role of albumin in binding Aβ in the cerebrospinal fluid has been largely overlooked. Here we investigate the effect of HSA on both Aβ(1–40) and Aβ(1–42) fibril growth. We show that at micromolar cerebrospinal fluid levels, HSA inhibits the kinetics of Aβ fibrillization, significantly increasing the lag time and decreasing the total amount of fibrils produced. Furthermore, we show that the amount of amyloid fibers generated directly correlates to the proportion of Aβ not competitively bound to albumin. Our observations suggest a significant role for HSA regulating Aβ fibril growth in the brain interstitium.

Copper and the structural biology of the prion protein

PrP (prion-related protein) is a cell-surface Cu(2+)-binding glycoprotein which, when misfolded, is responsible for a number of transmissible spongiform encephalopathies. The co-ordination geometry, stoichiometry and affinity of Cu(2+) for PrP are the subject of much debate. In the present paper, we review the recent progress we have made in these areas. As many as six Cu(2+) ions bind to PrP with submicromolar affinity. Initially, two Cu(2+) ions bind to full-length PrP in the amyloidogenic region, between the octarepeats and the structured domain, at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Competitive chelators have been used to determine the affinity of the first molar equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+)-binding sites support the suggestion that PrP could act as an antioxidant by binding potentially harmful Cu(2+) ions and sacrificially quenching of free radicals generated as a result of copper redox cycling. Finally, the effect of Cu(2+) on the prion structure and misassembly into oligomers and fibres is discussed.

Fragment length influences affinity for Cu2+ and Ni2+ binding to His96 or His111 of the prion protein and spectroscopic evidence for a multiple histidine binding only at low pH

The prion protein (PrP) is a Cu2+-binding cell-surface glycoprotein. Using various PrP fragments and spectroscopic techniques, we show that two Cu2+ ions bind to a region between residues 90 and 126. This region incorporates the neurotoxic portion of PrP, vital for prion propagation in transmissible spongiform encephalopathies. Pentapeptides PrP-(92-96) and PrP-(107-111) represent the minimum motif for Cu2+ binding to the PrP-(90-126) fragment. Consequently, we were surprised that the appearance of the visible CD spectra for two fragments of PrP, residues 90-126 and 91-115, are very different. We have shown that these differences do not arise from a change in the co-ordination geometry within the two fragments; rather, there is a change in the relative preference for the two binding sites centred at His111 and His96. These preferences are metal-, pH- and chain-length dependent. CD indicates that Cu2+ initially fills the site at His111 within the PrP-(90-126) fragment. The pH-dependence of the Cu2+ co-ordination is studied using EPR, visible CD and absorption spectroscopy. We present evidence that, at low pH (5.5) and sub-stoichiometric amounts of Cu2+, a multiple histidine complex forms, but, at neutral pH, Cu2+ binds to individual histidine residues. We have shown that changes in pH and levels of extracellular Cu2+ will affect the co-ordination mode, which has implications for the affinity, folding and redox properties of Cu-PrP.