Dianlu Jiang

Ternary Complexes of Iron, Amyloid-β and Nitrilotriacetic Acid: Binding Affinities, Redox Properties, and Relevance to Iron-Induced Oxidative Stress in Alzheimer’s Disease+

The interaction of amyloid-beta (Abeta) and redox-active metals, two important biomarkers present in the senile plaques of Alzheimers disease (AD) brain, has been suggested to enhance the Abeta aggregation or facilitate the generation of reactive oxygen species (ROS). This study investigates the nature of the interaction between the metal-binding domain of Abeta, viz., Abeta(1-16), and the Fe(III) or Fe(II) complex with nitrilotriacetic acid (NTA). Using electrospray ionization mass spectrometry (ESI-MS), the formation of a ternary complex of Abeta(1-16), Fe(III), and NTA with a stoichiometry of 1:1:1 was identified. MS also revealed that the NTA moiety can be detached via collision-induced dissociation. The cumulative dissociation constants of both Abeta-Fe(III)-NTA and Abeta-Fe(II)-NTA complexes were deduced to be 6.3 x 10(-21) and 5.0 x 10(-12) M(2), respectively, via measurement of the fluorescence quenching of the sole tyrosine residue on Abeta upon formation of the complex. The redox properties of these two complexes were investigated by cyclic voltammetry. The redox potential of the Abeta-Fe(III)-NTA complex was found to be 0.03 V versus Ag/AgCl, which is negatively shifted by 0.54 V when compared to the redox potential of free Fe(III)/Fe(II). Despite such a large potential modulation, the redox potential of the Abeta-Fe(III)-NTA complex is still sufficiently high for a range of redox reactions with cellular species to occur. The Abeta-Fe(II)-NTA complex electrogenerated from the Abeta-Fe(III)-NTA complex was also found to catalyze the reduction of oxygen to produce H(2)O(2). These findings provide significant insight into the role of iron and Abeta in the development of AD. The binding of iron by Abeta modulates the redox potential to a level at which its redox cycling occurs. In the presence of a biological reductant (antioxidant), redox cycling of iron could disrupt the redox balance within the cellular milieu. As a consequence, not only is ROS continuously produced, but oxygen and biological reductants can also be depleted. A cascade of biological processes can therefore be affected. In addition, the strong binding affinity of Abeta toward Fe(III) and Fe(II) indicates Abeta could compete for iron against other iron-containing proteins. In particular, its strong affinity for Fe(II), which is 8 orders of magnitude stronger than that of transferrin, would greatly interfere with iron homeostasis.

Trace Hg2+ Analysis via Quenching of the Fluorescence of a CdS-Encapsulated DNA Nanocomposite

A novel fluorescent CdS-encapsulated DNA nanocomposite was synthesized via alternate adsorption of Cd(2+) and S(2-) onto the DNA template affixed inside an agarose gel. Confining DNA molecules in the gel matrix reduces the flexibility of the DNA strand, which facilitates the formation of a uniform coating of CdS onto the DNA template. The resultant rod-shaped nanocomposite (40-90 nm in width and 200-300 nm in length) is well dispersed in solution and fluoresces at 330 nm upon excitation at either 228 or 280 nm. The fluorescence is attributed to tiny particles present in the CdS coating. It was found that the fluorescence can be significantly quenched by trace amount of Hg(2+). The high selectivity toward Hg(2+) and the apparent change in the CdS coating upon exposure to Hg(2+) indicate that Hg(2+) has reacted with the CdS coating through formation of the much more insoluble HgS and the bridging S-Hg-S bonds at the surface. The extent of quenching is dependent on the concentration of Hg(2+) in the range of 0.04-13 microM, and a remarkable detection limit (8.6 nM at 30 degrees C and 4.3 nM at 50 degrees C) can be achieved. The feasibility of the method for the analysis of Hg(2+) in a wastewater sample was demonstrated with an excellent relative standard deviation (RSD, 3.4%). The method described herein is simple, selective, and sensitive and obviates the need of extensive sample pretreatment or special instrumentation.

Copper Redox Cycling in the Prion Protein Depends Critically on Binding Mode

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches coppers inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrPs putative role in cellular signaling.

Reaction Rates and Mechanism of the Ascorbic Acid Oxidation by Molecular Oxygen Facilitated by Cu(II)-Containing Amyloid-β Complexes and Aggregates

A forefront of the research on Alzheimers disease (AD) is the interaction of amyloid beta (Abeta) peptides with redox metal ions (e.g., Cu(II), Fe(III), and Fe(II)) and the biological relevance of the Abeta-metal complexes to neuronal cell loss and homeostasis of essential metals and other cellular species. This work is concerned with the kinetic and mechanistic studies of the ascorbic acid oxidation reaction by molecular oxygen that is facilitated by Cu(II) complexes with Abeta(1-16), Abeta(1-42), and aggregates of Abeta(1-42). The reaction rate was found to linearly increase with the concentrations of Abeta-Cu(II) and dissolved oxygen and be invariant with high ascorbic acid concentrations. The rate constants were measured to be 117.2 +/- 15.4 and 15.8 +/- 2.8 M(-1) s(-1) at low (<100 muM) and high AA concentrations, respectively. Unlike free Cu(II), in the presence of AA, Abeta-Cu(II) complexes facilitate the reduction of oxygen by producing H(2)O(2) as a major product. Such a conclusion is drawn on the basis that the reaction stoichiometry between AA and O(2) is 1:1 when the Abeta concentration is kept at a much greater value than that of Cu(II). A mechanism is proposed for the AA oxidation in which the oxidation states of the copper center in the Abeta complex alternates between 2+ and 1+. The catalytic activity of Cu(II) toward O(2) reduction was found to decrease in the order of free Cu(II) > Abeta(1-16)-Cu(II) > Abeta(1-42)-Cu(II) > Cu(II) complexed by the Abeta oligomer/fibril mixture > Cu(II) in Abeta fibrils. The finding that Cu(II) in oligomeric and fibrous Abeta aggregates possesses considerable activity toward H(2)O(2) generation is particularly significant, since in senile plaques of AD patients the coexisting copper and Abeta aggregates have been suggested to inflict oxidative stress through the production of reactive oxygen species (ROS). Although Cu(II) bound to oligomeric and fibrous Abeta aggregates is less effective than free Cu(II) and the monomeric Abeta-Cu(II) complex in producing ROS, in vivo the Cu(II)-containing Abeta oligomers and fibrils might be more biologically relevant given their stronger association with cell membranes and the closer proximity of ROS to cell membranes.

The elevated copper binding strength of amyloid-β aggregates allows the sequestration of copper from albumin: a pathway to accumulation of copper in senile plaques.

Copper coexists with amyloid-β (Aβ) peptides at a high concentration in the senile plaques of Alzheimers disease (AD) patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, Aβ(1-16)(Y10W), by substituting the tyrosine residue at position 10 in the hydrophilic domain of Aβ(1-42) with tryptophan. Upon mixing Cu(II), Aβ(1-16)(Y10W), and aliquots of Aβ(1-42) taken from samples incubated for different lengths of time, we found that the Cu(II) binding strength of aggregated Aβ(1-42) has been elevated by more than 2 orders of magnitude with respect to that of monomeric Aβ(1-42). Electron paramagnetic spectroscopic measurements revealed that the Aβ(1-42) aggregates, unlike their monomeric form, can seize copper from human serum albumin, an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the Aβ(1-42) aggregates can be rationalized by a Cu(II) coordination sphere constituted by three histidines from two adjacent Aβ(1-42) molecules. Our work demonstrates that the copper binding affinity of Aβ(1-42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo.

Mechanistic Studies of Cu(II) Binding to Amyloid-β Peptides and the Fluorescence and Redox Behaviors of the Resulting Complexes

Due in large part to the lack of crystal structures of the amyloid-beta (Abeta) peptide and its complexes with Cu(II), Fe(II), and Zn(II), characterization of the metal-Abeta complex has been difficult. In this work, we investigated the complexation of Cu(II) by Abeta through tandem use of fluorescence and electron paramagnetic resonance (EPR) spectroscopies. EPR experiments indicate that Cu(II) bound to Abeta can be reduced to Cu(I) using sodium borohydride and that both Abeta-Cu(II) and Abeta-Cu(I) are chemically stable. Upon reduction of Cu(II) to Cu(I), the Abeta fluorescence, commonly reported to be quenched upon Abeta-Cu(II) complex formation, can be regenerated. The absence of the characteristic tyrosinate peak in the absorption spectra of Abeta-Cu(II) complexes provides evidence that the sole tyrosine residue in Abeta is not one of the four equatorial ligands bound to Cu(II), but remains close to the metal center, and its fluorescence is sensitive to the copper oxidation state and perturbations in the coordination sphere. Further analysis of the quenching and Cu(II) binding behaviors at different Cu(II) concentrations and in the presence of the competing ligand glycine offers evidence supporting the operation of two binding regimes which demonstrate different levels of fluorescence recovery upon addition of the reducing agent. We provide results that suggest the fluorescence quenching is likely caused by charge transfer processes. Thus, by using tyrosine to probe the coordination site, fluorescence spectroscopy provides valuable mechanistic insights into the oxidation state of copper ions bound to Abeta, the binding heterogeneity, and the influence of solution conditions on complex formation.

Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.

Parkinsons disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

Effective Blockage of the Interfacial Recombination Process at TiO2 Nanowire Array Electrodes in Dye-Sensitized Solar Cells

Effective blockage of recombination electron transfer of a fast electron transfer redox couple (ferrocenium/ferrocene or Fc(+)/Fc) at TiO2 nanowire array electrodes is achieved by silanization of the dye loaded TiO2 nanowire array. FT-IR clearly shows the formation of polysiloxane network at fluorine doped tin electrodes covered with TiO2 nanowire arrays and the dye molecules. Compared to the commonly used TiO2 nanoparticle film electrodes, the TiO2 nanowire array has a more spatially accessible structure, facilitating the formation of uniform polysiloxane films. Energy-dispersive X-ray spectroscopy (EDS) also reveals the presence of Si over multiple spots at the cross sections of the silanized TiO2 nanowire array electrodes. As a result, a rather high open-cell voltage Voc (0.69 V) and an enhanced efficiency (0.749 %) for DSSC with the Fc(+)/Fc couple were obtained. Contrary to the passivated TiO2 nanoparticle film electrodes at which a complex, biphasic dependence of electron lifetime on Voc was observed, we recorded a logarithm linear dependence of the lifetime on Voc after the silanization treatment. The nanowire arrays with optimal salinization treatments afford a useful surface for the study of electron recombination and photovoltaic generation in DSSCs.

Separation of intermediates of iron-catalyzed dopamine oxidation reactions using reversed-phase ion-pairing chromatography coupled in tandem with UV–visible and ESI-MS detections

Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC-ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.

t-Darpp is an elongated monomer that binds calcium and is phosphorylated by cyclin-dependent kinases 1 and 5

t‐Darpp (truncated isoform of dopamine‐ and cAMP‐regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t‐Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t‐Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t‐Darpp function, we studied its secondary structure, oligomerization status, metal‐binding properties, and phosphorylation by cyclin‐dependent kinases 1 and 5. t‐Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t‐Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin‐dependent kinase 1 and cyclin‐dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde‐mediated cross‐linking experiments demonstrate that the majority of t‐Darpp exists as a monomer, but forms low levels (< 3%) of hetero‐oligomers with its longer isoform Darpp‐32. t‐Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.