The interaction of amyloid-β (Aβ) peptide with Cu(II) appears to play an important role in the etiology of Alzheimer’s disease. At physiological pH, the Cu(II) coordination in Aβ is heterogeneous, and there exist at least two binding modes in which Cu(II) is coordinated by histidine residues. Electron spin resonance studies have revealed a picture of the Cu(II) binding at a higher or lower pH, where only one of the two binding modes is almost exclusively present. We describe a procedure to directly examine the coordination of Cu(II) to each histidine residue in the dominant binding mode at physiological pH. We use nonlabeled and residue-specifically 15N-labeled Aβ(1–16). For quantitative analysis, the intensities of three-pulse electron spin-echo envelope modulation (ESEEM) spectra are analyzed. Spectral simulations show that ESEEM intensities provide information about the contribution of each histidine residue. Indeed, the ESEEM experiments at pH 6.0 confirm the dominant contribution of His6 to the Cu(II) coordination as expected from the work of other researchers. Interestingly, however, the ESEEM data obtained at pH 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the dominant binding mode. The order indicates a significant contribution from the simultaneous coordination by His13 and His14 at physiological pH, which has been underappreciated. These findings are supported by hyperfine sublevel correlation spectroscopy experiments. The simultaneous coordination by the two adjacent residues is likely to be present in a non–β-sheet structure. The coexistence of different secondary structures is possibly the molecular origin for the formation of amorphous aggregates rather than fibrils at relatively high concentrations of Cu(II). Through our approach, precise and useful information about Cu(II) binding in Aβ(1–16) at physiological pH is obtained without any side-chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-beta(1-40) [Abeta(1-40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of Abeta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Abeta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Abeta(1-40) at a Cu(II):Abeta molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in Abeta(1-40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar beta-sheet conformation. Therefore, we propose that Abeta(1-40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) ion interferes with a conformational transition into amyloid fibrils, inducing the formation of granular amorphous aggregates.

We provide direct evidence that all three histidine residues in amyloid-β1−16 (Aβ1−16) coordinate to Cu(II). In our approach, we generate Aβ1−16 analogues, in each of which a selected histidine residue is isotopically enriched with 15N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Aβ1−16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Aβ1−16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Aβ1−40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Aβ peptide for Cu(II).

Tau protein and Cu(II) are believed to be associated with the pathogenesis of Alzheimer's disease. However, little is known about atomic-level interactions between tau protein and Cu(II). Herein, we suggest, on the basis of electron spin resonance (ESR) data, that the four pseudorepeats of tau protein in the microtubule-binding region play an important role in Cu(II) binding. We use a number of tau protein fragments in order to examine Cu(II)-binding site(s) and binding affinities. Continuous-wave (CW) ESR experiments on the four highly conserved octadecapeptides, each of which is a segment of one of the four pseudorepeats, reveal that the equimolar Cu(II) complexes of the four octadecapeptides are similar to one another in terms of the coordination environment and binding affinity. The spectra obtained with pulsed ESR techniques such as electron spinÀecho envelope modulation and hyperfine sublevel correlation provide direct evidence that a histidine residue and a backbone amide group coordinate to Cu(II) in each Cu(II)Àoctadecapeptide complex. The results of CW and pulsed ESR experiments on some chemically modified peptides indicate that the cysteine residues in the second and third pseudorepeats are unlikely to be involved in Cu(II) binding. On the other hand, similar experiments on tau fragments of the second pseudorepeat with different lengths lead to the conclusion that the affinity for Cu(II) decreases as the octadecapeptide is either truncated or elongated. The high Cu(II)-binding affinity of the octadecapeptide is presumably due to the N-terminal amino group stabilizing the Cu(II)Àoctadecapeptide complex. Finally, the ESR data for a longer tau fragment that contains two octadecapeptides suggest that the Cu(II) binding site(s) of even longer fragments of tau protein is similar to that of a single octadecapeptide.

BackgroundNephrotoxicity is the major side effect in cisplatin chemotherapy. Previously, we reported that the ginsenosides Rk3 and Rh4 reduced cisplatin toxicity on porcine renal proximal epithelial tubular cells (LLC-PK1). Here, we aimed to evaluate the protective effect of ginsenosides Rk3 and Rh4 on kidney function and elucidate their antioxidant effect using in vitro and in vivo models of cisplatin-induced acute renal failure.MethodsAn enriched mixture of ginsenosides Rk3 and Rh4 (KG-KH; 49.3% and 43.1%, respectively) was purified from sun ginseng (heat processed Panax ginseng). Cytotoxicity was induced by treatment of 20μM cisplatin to LLC-PK1 cells and rat model of acute renal failure was generated by single intraperitoneal injection of 5 mg/kg cisplatin. Protective effects were assessed by determining cell viability, reactive oxygen species generation, blood urea nitrogen, serum creatinine, antioxidant enzyme activity, and histopathological examination.ResultsThe in vitro assay demonstrated that KG-KH (50 μg/mL) significantly increased cell viability (4.6-fold), superoxide dismutase activity (2.8-fold), and glutathione reductase activity (1.5-fold), but reduced reactive oxygen species generation (56%) compared to cisplatin control cells. KG-KH (6 mg/kg, per os) also significantly inhibited renal edema (87% kidney index) and dysfunction (71.4% blood urea nitrogen, 67.4% creatinine) compared to cisplatin control rats. Of note, KG-KH significantly recovered the kidney levels of catalase (1.2-fold) and superoxide dismutase (1.5-fold).ConclusionConsidering the oxidative injury as an early trigger of cisplatin nephrotoxicity, our findings suggest that ginsenosides Rk3 and Rh4 protect the kidney from cisplatin-induced oxidative injury and help to recover renal function by restoring intrinsic antioxidant defenses.