Juan Xiang

pH-dependent kinetics of copper ions binding to amyloid-β peptide.

Interactions of amyloid-β peptide (Aβ) with Cu(2+) are known to be pH-dependent and believed to play a crucial role in the neurotoxicity of Alzheimers disease (AD). Some research has revealed that injured brains with lowered pH have higher risks of developing AD. However, reported experiments were performed under neutral or mildly acidic conditions, and no reports about the affinity of Aβ-Cu(2+) below pH6.0. In this study, surface plasmon resonance (SPR) sensor with immobilized Aβ was used to investigate the formation of Aβ-Cu(2+) complexes under acidic pH conditions. Dissociation constants were calculated and shown to be pH-dependent, ranging from 3.5×10(-8)M to 8.7×10(-3)M in the pH range from 7.0 to 4.0. The physiological significance of K(d) was preliminarily investigated by monitoring the generation of OH() in aerobic solutions containing Aβ-Cu(2+) and Cu(2+). The results imply that acidic conditions could aggravate the oxidative stress in the presence of Cu(2+), and the weak affinities of Aβ-Cu(2+) under mildly acidic pH of 5.0-6.0 could further enhance the oxidative damage. However, the oxidative stress effect of Aβ is negligible due to the suppressed formation of Aβ-Cu(2+) below pH5.0. This work is useful for the in-depth understanding of the role of Aβ-Cu(2+) in AD neuropathology.

Amyloid-β peptide (1–42) aggregation induced by copper ions under acidic conditions

It is well known that the aggregation of amyloid-β peptide (Aβ) induced by Cu²⁺ is related to incubation time, solution pH, and temperature. In this work, the aggregation of Aβ₁₋₄₂ in the presence of Cu²⁺ under acidic conditions was studied at different incubation time and temperature (e.g. 25 and 37°C). Incubation temperature, pH, and the presence of Cu²⁺ in Aβ solution were confirmed to alter the morphology of aggregation (fibrils or amorphous aggregates), and the morphology is pivotal for Aβ neurotoxicity and Alzheimer disease (AD) development. The results of atomic force microscopy (AFM) indicated that the formation of Aβ fibrous morphology is preferred at lower pH, but Cu²⁺ induced the formation of amorphous aggregates. The aggregation rate of Aβ was increased with the elevation of temperature. These results were further confirmed by fluorescence spectroscopy and circular dichroism spectroscopy and it was found that the formation of β-sheet structure was inhibited by Cu²⁺ binding to Aβ. The result was consistent with AFM observation and the fibrillation process was restrained. We believe that the local charge state in hydrophilic domain of Aβ may play a dominant role in the aggregate morphology due to the strong steric hindrance. This research will be valuable for understanding of Aβ toxicity in AD.

Side Effect of Tris on the Interaction of Amyloid β-peptide with Cu(2+): Evidence for Tris-Aβ-Cu (2+) Ternary Complex Formation.

The interaction of amyloid β-peptide (Aβ) with Cu2+ is crucial to the development of neurotoxicity in Alzheimer’s disease (AD). Many recent studies show a variation on the dissociation constant of Aβ–Cu2+ under different solvent conditions. Among various buffers, the Tris(hydroxymethyl)aminomethane (Tris) buffer is the most reliable chelator of Cu2+. However, as a typical nucleophilic reagent capable of binding peptides, the behavior of Tris should be more complicated. In this work, the effect of Tris on the interaction of Aβ with Cu2+ was investigated. Under acidic conditions, Tris–Aβ–Cu2+ ternary complex was identified by electrospray ionization mass spectrometry and transmission electron microscopy. The results of surface plasmon resonance reveal that the formation of the ternary complex increases the dissociation constant by almost 1 order of magnitude. Consequently, the assessment of toxicity indicates that the generation of · OH induced by the Aβ–Cu2+ complex was enhanced in the presence of Tris. The work reveals the significant side effect of Tris on the interaction of Aβ with Cu2+, which will greatly improve the quantitative investigation on Aβ–Cu2+ interaction and be helpful for the in-depth understanding of the roles of Aβ and Cu2+ in AD neuropathology.

Complicated function of dopamine in Aβ-related neurotoxicity: Dual interactions with Tyr10 and SNK(26–28) of Aβ

With the capability to inhibit the formation of amyloid β peptides (Aβ) fibril, dopamine (DA) and other catechol derivatives have been considered for the potential treatment of Alzheimers disease (AD). Such treatment, however, remains debatable because of the diverse functions of Aβ and DA in AD pathology. Moreover, the complicated oxidation accompanying DA has caused the majority of the previous research to focus on the binding of DA oxides onto Aβ. The molecular mechanism by which Aβ interacts with the reduction state of DA, which is correlative with the brain function, should be urgently explored. By controlling rigorous anaerobic experimental conditions, this work investigated the molecular mechanism of the Aβ/DA interaction, and two binding sites were revealed. For the binding of DA, Tyrosine (Tyr10) was identified as the strong binding site, and serine-asparagine-lysing (SNK(26-28)) segment was the weak binding segment. Furthermore, the Thioflavin T (THT) fluorescence confirmed DAs positive function of inhibiting Aβ aggregation through its weakly binding with SNK(26-28) segment. Meanwhile, 7-OHCCA fluorescence exhibited DAs negative function of enhancing OH generation through inhibiting the Aβ/Cu2+ coordination. The viability tests of the neuroblastoma SH-SY5Y cells displayed that the coexistence of DA, Cu2+, and Aβ induced lower cell viability than free Cu2+, indicating the significant negative effect of excessive DA on AD progression. This research revealed the potential DA-induced damage in AD brain, which is significant for understanding the function of DA in AD neuropathology and for designing a DA-related therapeutic strategy for AD.

Electrochemical Impedance Spectroscopy for Real-Time Detection of Lipid Membrane Damage Based on a Porous Self-Assembly Monolayer Support

Layer-by-layer dissolution and permeable pore formation are two typical membrane damage pathways, which induce membrane function disorder and result in serious disease, such as Alzheimers disease, Keshan disease, Sickle-cell disease, and so on. To effectively distinguish and sensitively monitor these two typical membrane damage pathways, a facile electrochemical impedance strategy was developed on a porous self-assembly monolayer (pSAM) supported bilayer lipid membrane (BLM). The pSAM was prepared by selectively electrochemical reductive desorption of the mercaptopropionic acid in a mixed mercaptopropionic acid/11-mercaptoundecanoic acid self-assembled monolayer, which created plenty of nanopores with tens of nanometers in diameter and several nanometers in height (defined as inner-pores). The ultralow aspect ratio of the inner-pores was advantageous to the mass transfer of electrochemical probe [Fe(CN)6]3-/4-, simplifying the equivalent electric circuit for electrochemical impedance spectroscopy analysis at the electrode/membrane interface. [Fe(CN)6]3-/4- transferring from the bulk solution into the inner-pore induce significant changes of the interfacial impedance properties, improving the detection sensitivity. Based on these, the different membrane damage pathways were effectively distinguished and sensitively monitored with the normalized resistance-capacitance changes of inner-pore-related parameters including the electrolyte resistance within the pore length ( Rpore) and the metal/inner-pore interfacial capacitance ( Cpore) and the charge-transfer resistance ( Rct-in) at the metal/inner-pore interface.

Role of norepinephrine in Aβ-related neurotoxicity: dual interactions with Tyr10 and SNK(26–28) of Aβ

With their capability to inhibit the formation of amyloid-β peptide (Aβ) fibril, norepinephrine (NE), and other catechol derivatives have been considered for the potential treatment of Alzheimers disease (AD). Such treatment, however, remains debatable because of the diverse functions of Aβ and NE in AD pathology. Moreover, the complicated oxidation accompanying NE has caused the majority of the previous research to focus on the binding of NE oxides onto Aβ. The molecular mechanism by which Aβ interacts with the reduction state of NE, which is correlated with the brain function, should be urgently explored. In this work, by controlling rigorous anaerobic experimental conditions, the molecular mechanism of the Aβ/NE interaction was investigated, and two binding sites were revealed. Tyr10 was identified as the strong binding site of NE, and SNK(26-28) segment was the weak binding segment. Furthermore, thioflavin T fluorescence confirmed NEs positive function of inhibiting Aβ aggregation through its weak binding with SNK(26-28) segment. Meanwhile, 7-OHCCA fluorescence exhibited NEs negative function of enhancing ·OH generation through inhibiting the Aβ/Cu2+ coordination. The viability tests of the neuroblastoma SH-SY5Y cells displayed that the coexistence of NE, Cu2+, and Aβ induced lower cell viability than free Cu2+, indicating the significant negative effect of excessive NE on AD progression. These data revealed the possible pathway of NE-induced damage in AD brain, which is significant for understanding the function of NE in Aβ-involved AD neuropathology and for designing an NE-related therapeutic strategy for AD.

Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance

AbstractThe interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt cred), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt cox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt cred is quantitatively electro-generated from Cyt cox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt cred/Cyt cox can be evaluated accurately. In this study, the values of association/dissociation rate constants (ka, kd) for both COX/Cyt cred and COX/Cyt cox were obtained. The dissociation constants, KD, were finally calculated as 3.33 × 10–8 mol · L–1 for COX/Cyt cred and 4.25 × 10–5 mol · L–1 for COX/Cyt cox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical AbstractSchematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX