Metabolism of copper and possibilities for its regulation

Copper is an indispensable biometal participating as a redox catalyst in many important biochemical processes. However, if uncontrolled, copper ions induce the formation of reactive oxygen species and become toxic. For this reason, cellular copper metabolism is tightly regulated and specific proteins â copper chaperones â participate in the metalation of cellular copper transporters and enzymes. The thermodynamic background for cellular copper distribution is known, and copper is driven to cellular destinations according to shallow affinity gradients. Copper metabolism is disturbed in the case of Wilsonâs, Menkes, and Alzheimerâs disease (AD), characterized by copper overload, deficiency, and misdistribution, respectively. Wilsonâs and Menkes disease could be treated by copper chelators and supplements, respectively; however, with AD, a search for effective molecular tools for the correction of copper metabolism is ongoing. One natural copper­binding ligand â α­-lipoic acid â has shown positive results in cellular and fruit fly models of AD and serves as a promising candidate for the regulation of copper metabolism in the case of AD

A structural-dynamical characterization of human Cox17.

Human Cox17 is a key mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy transducing respiratory chain. A structural and dynamical characterization of human Cox17 in its various functional metallated and redox states is presented here. The NMR solution structure of the partially oxidized Cox17 (Cox17(2S-S)) consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds involving Cys(25)-Cys(54) and Cys(35)-Cys(44), preceded by a flexible and completely unstructured N-terminal tail. In human Cu(I)Cox17(2S-S) the copper(I) ion is coordinated by the sulfurs of Cys(22) and Cys(23), and this is the first example of a Cys-Cys binding motif in copper proteins. Copper(I) binding as well as the formation of a third disulfide involving Cys(22) and Cys(23) cause structural and dynamical changes only restricted to the metal-binding region. Redox properties of the disulfides of human Cox17, here investigated, strongly support the current hypothesis that the unstructured fully reduced Cox17 protein is present in the cytoplasm and enters the intermembrane space (IMS) where is then oxidized by Mia40 to Cox17(2S-S), thus becoming partially structured and trapped into the IMS. Cox17(2S-S) is the functional species in the IMS, it can bind only one copper(I) ion and is then ready to enter the pathway of copper delivery to cytochrome c oxidase. The copper(I) form of Cox17(2S-S) has features specific for copper chaperones

The Native Copper- and Zinc- Binding Protein Metallothionein Blocks Copper-Mediated Aβ Aggregation and Toxicity in Rat Cortical Neurons

Background: A major pathological hallmark of AD is the deposition of insoluble extracellular b-amyloid (Ab) plaques. There are compelling data suggesting that Ab aggregation is catalysed by reaction with the metals zinc and copper. Methodology/Principal Findings: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Ab1–40 and Ab1–42. This action of MT-2A appears to involve a metal-swap between Zn 7MT-2A and Cu(II)-Ab, since neither Cu 10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Ab aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Ab induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. Conclusions/Significance: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Ab aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Ab leaving a metal-free Ab that can readily bind metals again, we believe that MT-2A might represent a different therapeuti

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10-23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.12 page(s

In situ fibrillizing amyloid-beta 1-42 induces neurite degeneration and apoptosis of differentiated SH-SY5Y cells

<div><p>The progression of Alzheimer’s disease is causatively linked to the accumulation of amyloid-β aggregates in the brain, however, it is not clear how the amyloid aggregates initiate the death of neuronal cells. The <i>in vitro</i> toxic effects of amyloid peptides are most commonly examined using the human neuroblastoma derived SH-SY5Y cell line and here we show that differentiated neuron-like SH-SY5Y cells are more sensitive to amyloid peptides than non-differentiated cells, because the latter lack long neurites. Exogenous soluble amyloid-β 1–42 covered cell bodies and whole neurites in differentiated cells with dense fibrils, causing neurite beading and fragmentation, whereas preformed amyloid-β 1–42 fibrils had no toxic effects. Importantly, spontaneously fibrillizing amyloid-β 1–42 peptide exhibited substantially higher cellular toxicity than amyloid-β 1–40, which did not form fibrils under the experimental conditions. These results support the hypothesis that peptide toxicity is related to the active fibrillization process in the incubation mixture.</p></div

RA/BDNF differentiated cells establish a neuron-like phenotype with long neurites.

<p>(A) Immunocytochemistry of non-differentiated and RA/BDNF differentiated SH-SY5Y cells for DAPI (blue; left), anti-TUJ-1 (green; middle). Scale bar 50 μm.</p

Aβ40 and Aβ42 induce pathological changes in neurite morphology after 72 h.

<p>(A) RA/BDNF differentiated live cell imaging for differential interference contrast (right), CalceinAM fluorescence (green; middle). Red arrows indicate fragmented neurites. Scale bar 20μm. (B) Greater magnification of a neurite with beads <i>versus</i> a fragmented neurite. Scale bar 5 μm. (C) Quantification of pathological changes in RA/BDNF differentiated cell culture. The figure displays the mean± SEM; at least <i>n = 3</i> independent experiments; **p≤0.005; *p≤0.05, One-way ANOVA followed by a Bonferroni's multiple comparisons test at the 0.05 level was used to determine differences between the conditions.</p

Evaluation of Zn2+- and Cu2+-Binding Affinities of Native Cu,Zn-SOD1 and Its G93A Mutant by LC-ICP MS

The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn2+ and Cu2+ were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the KD values. From the Arrhenius plots of the demetallation in the absence of chelators &Delta;H&Dagger; = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high &Delta;H values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar