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Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter., Gly-His-Lys (GHK) is a human bioactive tripeptide thought to be activated by Cu(II) binding, but little is known about the molecular aspects of its action. UV−vis, circular dichroism (CD), EPR, and NMR spectroscopies, and cyclic voltammetry were used to examine the reduction of Cu(II)GHK with glutathione (GSH), the most abundant biological thiol. A semistable GSH-Cu(II)GHK reaction intermediate was discovered, with properties suitable for delivering Cu(I) to biological transport proteins.

The metal chelator PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) acts as a terdentate ligand capable of forming binary and ternary Cu2+ complexes. It was clinically trialed as an Alzheimer’s disease (AD) therapy but failed to progress beyond phase II. The β-amyloid (Aβ) peptide associated with AD was recently concluded to form a unique Cu(Aβ) complex that is inaccessible to PBT2. Herein, it is shown that the species ascribed to this binary Cu(Aβ) complex in fact corresponds to ternary Cu(PBT2)NImAβ complexes formed by the anchoring of Cu(PBT2) on imine nitrogen (NIm) donors of His side chains. The primary site of ternary complex formation is His6, with a conditional stepwise formation constant at pH 7.4 (Kc [M−1]) of logKc = 6.4 ± 0.1, and a second site is supplied by His13 or His14 (logKc = 4.4 ± 0.1). The stability of Cu(PBT2)NImH13/14 is comparable with that of the simplest Cu(PBT2)NIm complexes involving the NIm coordination of free imidazole (logKc = 4.22 ± 0.09) and histamine (logKc = 4.00 ± 0.05). The 100-fold larger formation constant for Cu(PBT2)NImH6 indicates that outer-sphere ligand–peptide interactions strongly stabilize its structure. Despite the relatively high stability of Cu(PBT2)NImH6, PBT2 is a promiscuous chelator capable of forming a ternary Cu(PBT2)NIm complex with any ligand containing an NIm donor. These ligands include histamine, L-His, and ubiquitous His side chains of peptides and proteins in the extracellular milieu, whose combined effect should outweigh that of a single Cu(PBT2)NImH6 complex regardless of its stability. We therefore conclude that PBT2 is capable of accessing Cu(Aβ) complexes with high stability but low specificity. The results have implications for future AD therapeutic strategies and understanding the role of PBT2 in the bulk transport of transition metal ions. Given the repurposing of PBT2 as a drug for breaking antibiotic resistance, ternary Cu(PBT2)NIm and analogous Zn(PBT2)NIm complexes may be relevant to its antimicrobial properties.

The protein misfolding cyclic amplification (PMCA) technique employs repeated cycles of incubation and sonication to amplify minute amounts of misfolded protein conformers. Spontaneous (de novo) prion formation and ultrasonic power level represent two potentially interrelated sources of variation that frustrate attempts to replicate results from different laboratories. We previously established that water splitting during PMCA provides a radical-rich environment leading to oxidative damage to substrate molecules as well as the polypropylene PCR tubes used for sample containment. Here it is shown that the cross-linking agent formaldehyde is generated from buffer ions that are attacked by hydroxyl radicals. In addition, free radical damage to protein, nucleic acid, lipid, and detergent molecules produces a substantial concentration of aldehydes (hundreds of micromolar). The measurement of aldehydes using the Hantzsch reaction provides a reliable and inexpensive method for measuring the power delivered to individual PMCA samples, and for calibrating the power output characteristics of an individual sonicator. The proposed method may also be used to better account for inter-assay and inter-laboratory variation in prion replication and de novo prion generation, the latter of which may correlate with aldehyde-induced cross-linking of substrate molecules.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log c K Cu Cu ( HSA ) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4-9 production, and Cu2+ homeostasis.

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M−1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M−1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.

Neurogenesis continues in the post-developmental brain throughout life. The ability to stimulate the production of new neurones requires both quiescent and actively proliferating pools of neural stem cells (NSCs). Actively proliferating NSCs ensure that neurogenic demand can be met, whilst the quiescent pool makes certain NSC reserves do not become depleted. The processes preserving the NSC quiescent pool are only just beginning to be defined. Herein, we identify a switch between NSC proliferation and quiescence through changing intracellular redox signalling. We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life. Electronic supplementary material The online version of this article (10.1007/s00018-018-2790-3) contains supplementary material, which is available to authorized users.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

Magnetic resonance spectroscopy is one of the most important tools in chemical and bio-medical research. However, sensitivity limitations typically restrict imaging resolution to ~ 10 µm. Here we bring quantum control to the detection of chemical systems to demonstrate high-resolution electron spin imaging using the quantum properties of an array of nitrogen-vacancy centres in diamond. Our electron paramagnetic resonance microscope selectively images electronic spin species by precisely tuning a magnetic field to bring the quantum probes into resonance with the external target spins. This provides diffraction limited spatial resolution of the target spin species over a field of view of 50 × 50 µm2 with a spin sensitivity of 104 spins per voxel or ∼100 zmol. The ability to perform spectroscopy and dynamically monitor spin-dependent redox reactions at these scales enables the development of electron spin resonance and zepto-chemistry in the physical and life sciences., Electron paramagnetic resonance spectroscopy has important scientific and medical uses but improving the resolution of conventional methods requires cryogenic, vacuum environments. Simpson et al. show nitrogen vacancy centres can be used for sub-micronmetre imaging with improved sensitivity in ambient conditions.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu

Eight-hydroxyquinolines (8HQs) are a class of compounds that have been identified as potential therapeutics for a number of neurodegenerative diseases. Understanding the influence of structural modifications to the 8HQ scaffold on cellular behaviour will aid the identification of compounds that might be effective in treating dementias. In this study, we describe the action of 2-[(dimethylamino)methyl]-8-hydroxyquinoline (DMAMQ) on adult murine neural stem cells (NSCs) cultured in vitro. Treatment of NSCs with DMAMQ resulted in enhanced self-renewal and increased neurite outgrowth. Concurrent with the positive growth effects was an increase in intracellular reactive oxygen species, with the growth being inhibited by inactivation of the NADPH oxidase (Nox) enzyme family. Our results indicate that DMAMQ can stimulate neurogenesis via the Nox signalling pathway, which may provide therapeutic benefit in treating dementias of various types by replenishing neurones using the brain's own reserves. The narrow concentration range over which these effects were observed, however, suggests that there may exist only a small therapeutic window for neuro-regenerative applications.

Aβ4-42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu(II) with an affinity approximately 3000 times higher than the commonly studied Aβ1-42 and Aβ1-40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein-3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu(II) from Aβ1-40 when acting in its native Zn7 MT-3 form. This reaction is assumed to underlie the neuroprotective effect of Zn7 MT-3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1-16 and Aβ4-16 to demonstrate that the high-affinity Cu(II) complex of Aβ4-16 is resistant to Zn7 MT-3 reactivity. This indicates that the analogous complex of the full-length peptide Cu(Aβ4-42) will not yield copper to MT-3 in the brain, thus supporting the concept of a physiological role for Aβ4-42 as a Cu(II) scavenger in the synaptic cleft.

The oligomerisation of many proteins and peptides is known to be influenced by the binding of transition metal ions such as divalent copper. To investigate the oligomeric state of model peptides related to the N-terminus of α-synuclein (αSyn) in the presence of Cu(II), electron paramagnetic resonance (EPR) spectroscopy and isotopic labelling were recently used to conclude that Cu(II) occupies N-terminal bridging positions within closed-chain αSyn dimers and trimers with a Cu/peptide stoichiometry of 1:1. Herein, a statistical correction is identified and the consequences are evaluated. The analysis reveals that αSyn forms Cu-bridged antiparallel dimers and closed-chain trimers that coexist with Cu(II)-bound monomers (including a "macrochelate") and, depending on metal stoichiometry and protein environment, with open-chain Cu-bridged oligomers and heterodimers. The results demonstrate that the Cu(II) ion can be exploited as a probe of protein quaternary structure, with the potential to delineate heterogeneous oligomeric populations.

The A&beta, 4&minus, 42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer&rsquo, s Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in A&beta, 16 and A&beta, 9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)A&beta, x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-A&beta, 16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)A&beta, 16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of A&beta, 16, Pd(II)-A&beta, 16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-A&beta, complexes.

The tripeptide NH2&ndash, Gly&ndash, His&ndash, Lys&ndash, COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12- x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

X-ray absorption spectroscopy of CuII amyloid-β peptide (Aβ) under in situ electrochemical control (XAS-EC) has allowed elucidation of the redox properties of CuII bound to truncated peptide forms. The Cu binding environment is significantly different for the Aβ1–16 and the N-truncated Aβ4–9, Aβ4–12, and Aβ4–16 (Aβ4–9/12/16) peptides, where the N-truncated sequence (F4R5H6) provides the high-affinity amino-terminal copper nickel (ATCUN) binding motif. Low temperature (ca. 10 K) XAS measurements show the adoption of identical CuII ATCUN-type binding sites (CuIIATCUN) by the first three amino acids (FRH) and a longer-range interaction modeled as an oxygen donor ligand, most likely water, to give a tetragonal pyramid geometry in the Aβ4–9/12/16 peptides not previously reported. Both XAS-EC and EPR measurements show that CuII:Aβ4–16 can be reduced at mildly reducing potentials, similar to that of CuII:Aβ1–16. Reduction of peptides lacking the H13H14 residues, CuII:Aβ4–9/12, require far more forcing conditions...

The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo.

The deposition of insoluble β-amyloid (Aβ) peptide into extracellular senile plaques is a pathological hallmark of Alzheimer’s disease. Fragmented α-synuclein (αSyn) and a relatively high concentration of Cu2+ ions are found within amyloid deposits. Both Aβ and αSyn bind Cu2+ with an apparent dissociation constant in the sub-nanomolar range at physiological pH, fuelling speculation that deleterious redox chemistry and metal-mediated protein misfolding may contribute to disease progression. Binary Cu(Aβ) and Cu(αSyn) complexes have been extensively studied, although the Cu2+ coordination of heterogeneous mixtures of Aβ and αSyn has yet to be characterised. This study used synthetic N-terminal fragments Aβ1–16 and αSyn1–56 to reveal a new Cu(αSyn)(Aβ) coordination mode anchored upon a 5,6-membered chelate supplied by Met1–Asp2 of αSyn, with the fourth equatorial ligand being supplied by a His side chain of Aβ.

α-Factor-1 (WHWLQLKPGQPMY), a peptidic pheromone of Saccharomyces cerevisiae yeast, contains a XHX type copper(II) binding N-terminal site. Using a soluble analogue, WHWSKNR-amide, we demonstrated that the W(1)H(2)W(3) site alone binds copper(II) with a Kd value of 0.18 pM at pH 7.4 and also binds imidazole (Im) in a ternary complex (Kd of 1 mM at pH 7.4). This interaction boosts the ability of the peptide to sequester copper(II) depending on the Im concentration up to a subfemtomolar range, not available for any oligopeptidic system studied before. Therefore, α-factor-1 and other XHX-type peptides are likely copper(II) carriers in biological systems.

In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.

Electron paramagnetic resonance spectroscopy in conjunction with site-selective isotopic labelling provides a valuable tool for probing the local coordination environment of paramagnetic metal ions. Sometimes the metal ligands are inter-molecular in origin, such as during biological catalysis involving enzyme/metal-ion/substrate complexes or within oligomeric forms of peptides and proteins. Inter- and intra-molecular metal binding within peptide oligomers can be delineated by combining two fractions of peptide monomers containing the same amino acid sequence but having different isotopic content. Here, the principles of isotopic dilution in EPR spectroscopy are described and simulations provided to demonstrate its application to the identification of Cu2+-bridging of peptide oligomers.

In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.

The N-truncated β-amyloid (Aβ) isoform Aβ4-x is known to bind Cu(2+) via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu(2+) interactions and redox activity of Aβx-16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aβ1-16), CuL, CuL2, and ternary CuL(NIm(Aβ)) was rapid, while the corresponding equilibrium between L and Aβ4-16 occurred slowly via a metastable CuL(NIm(Aβ)) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NIm(Aβ) ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu(2+) from inert Cu(Aβ4-x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.

The beta-amyloid (Aβ) peptide and the Aβ-oligomer receptor, prion protein (PrP), both influence neurogenesis. Using in vitro murine neural stem cells (NSCs), we investigated whether Aβ and PrP interact to modify neurogenesis. Aβ imparted PrP-dependent changes on NSC self-renewal, with PrP-ablated and wild-type NSCs displaying increased and decreased cell growth, respectively. In contrast, differentiation of Aβ-treated NSCs into mature cells was unaffected by PrP expression. Such marked PrP-dependent differences in NSC growth responses to Aβ provides further evidence of biologically significant interactions between these two factors and an important new insight into regulation of NSC self-renewal in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0067-4) contains supplementary material, which is available to authorized users.

Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

The oligomerization of α-synuclein (αSyn) is one of the defining features of Parkinson's disease. Binding of divalent copper to the N terminus of αSyn has been implicated in both its function and dysfunction. Herein, the molecular details of the Cu(II) /αSyn binding interface have been revealed using a library of synthetic 56-residue αSyn peptides containing site-specific isotopic labels. Using electron paramagnetic resonance spectroscopy, αSyn is shown to coordinate Cu(II) with high affinity via two pH-dependent coordination modes between pH 6.5-8.5. Most remarkably, the data demonstrate that the dominant mode is associated with binding to oligomers (antiparallel dimers and/or cyclic trimers) in which Cu(II) ions occupy intermolecular bridging sites. The findings provide a molecular link between Cu(II) -bound αSyn and its associated quaternary oligomeric structure.

The roasting of coffee beans generates stable radicals within melanoidins produced by non-enzymatic browning. Roasting coffee beans has further been suggested to increase the antioxidant (AO) capacity of coffee brews. Herein, we have characterized the radical content and AO capacity of brews prepared from Coffea arabica beans sourced directly from an industrial roasting plant. In-tact beans exhibited electron paramagnetic resonance signals arising from Fe3+, Mn2+ and at least three distinct stable radicals as a function of roasting time, whose intensity changed upon grinding and ageing. In coffee brews, the roasting-induced radicals were harboured within the high molecular weight (> 3 kD) melanoidin-containing fraction at a concentration of 15 nM and was associated with aromatic groups within the melanoidins. The low molecular weight (< 3 kD) fraction exhibited the highest AO capacity using DPPH as an oxidant. The AO activity was not mediated by the stable radicals or by metal complexes within the brew. While other non-AO functions of the roasting-induced radical and metal complexes may be possible in vivo, we confirm that the in vitro antiradical activity of brewed coffee is dominated by low molecular weight phenolic compounds.