Metals in ALS TDP-43 Pathology.

Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease and similar neurodegenerative disorders take their toll on patients, caregivers and society. A common denominator for these disorders is the accumulation of aggregated proteins in nerve cells, yet the triggers for these aggregation processes are currently unknown. In ALS, protein aggregation has been described for the SOD1, C9orf72, FUS and TDP-43 proteins. The latter is a nuclear protein normally binding to both DNA and RNA, contributing to gene expression and mRNA life cycle regulation. TDP-43 seems to have a specific role in ALS pathogenesis, and ubiquitinated and hyperphosphorylated cytoplasmic inclusions of aggregated TDP-43 are present in nerve cells in almost all sporadic ALS cases. ALS pathology appears to include metal imbalances, and environmental metal exposure is a known risk factor in ALS. However, studies on metal-to-TDP-43 interactions are scarce, even though this protein seems to have the capacity to bind to metals. This review discusses the possible role of metals in TDP-43 aggregation, with respect to ALS pathology.

Hereditary Transthyretin Amyloidosis (hATTR) with Polyneuropathy Clusters Are Located in Ancient Mining Districts: A Possible Geochemical Origin of the Disease.

Hereditary transthyretin amyloidosis (hATTR) with polyneuropathy (formerly known as Familial Amyloid Polyneuropathy (FAP)) is an endemic amyloidosis involving the harmful aggregation of proteins, most commonly transthyretin (TTR) but sometimes also apolipoprotein A-1 or gelsolin. hATTR appears to be transmitted as an autosomal dominant trait. Over 100 point mutations have been identified, with the Val30Met substitution being the most common. Yet, the mechanism of pathogenesis and the overall origin of hATTR remain unclear. Here, we argue that hATTR could be related to harmful metal exposure. hATTR incidence is unevenly distributed globally, and the three largest defined clusters exist in Japan, Portugal, and Sweden. All three disease regions are also ancient mining districts with associated metal contamination of the local environment. There are two main mechanisms for how harmful metals, after uptake into tissues and body fluids, could induce hATTR. First, the metals could directly influence the expression, function, and/or aggregation of the proteins involved in hATTR pathology. Such metal-protein interactions might constitute molecular targets for anti-hATTR drug design. Second, metal exposure could induce hATTR -associated genetic mutations, which may have happened several generations ago. These two mechanisms can occur in parallel. In conclusion, the possibility that hATTR could be related to metal exposure in geochemically defined regions deserves further attention.

Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

Mercury and Alzheimer's Disease: Hg(II) Ions Display Specific Binding to the Amyloid-ß Peptide and Hinder Its Fibrillization.

Brains and blood of Alzheimer's disease (AD) patients have shown elevated mercury concentrations, but potential involvement of mercury exposure in AD pathogenesis has not been studied at the molecular level. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-ß (Aß) peptides aggregated into amyloid fibrils. Aß peptide fibrillization is known to be modulated by metal ions such as Cu(II) and Zn(II). Here, we study in vitro the interactions between Aß peptides and Hg(II) ions by multiple biophysical techniques. Fluorescence spectroscopy and atomic force microscopy (AFM) show that Hg(II) ions have a concentration-dependent inhibiting effect on Aß fibrillization: at a 1:1 Aß·Hg(II) ratio only non-fibrillar Aß aggregates are formed. NMR spectroscopy shows that Hg(II) ions interact with the N-terminal region of Aß(1-40) with a micromolar affinity, likely via a binding mode similar to that for Cu(II) and Zn(II) ions, i.e., mainly via the histidine residues His6, His13, and His14. Thus, together with Cu(II), Fe(II), Mn(II), Pb(IV), and Zn(II) ions, Hg(II) belongs to a family of metal ions that display residue-specific binding interactions with Aß peptides and modulate their aggregation processes.

Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his40 and his57 ) quickly and completely, with an IC50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation.

Characterization of Mn(II) ion binding to the amyloid-ß peptide in Alzheimer's disease.

Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimer's disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-ß (Aß) peptides. Previous studies have shown that Aß displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate Aß aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the Aß(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to Aß may not have a large effect on the peptide's aggregation into amyloid fibrils. However, identification of an additional metal ion displaying Aß binding reveals more complex AD metal chemistry than has been previously considered in the literature.

The interaction of actinide and lanthanide ions with hemoglobin and its relevance to human and environmental toxicology.

Due to increasing use of lanthanides/actinides in nuclear and civil applications, understanding the impact of these metal ions on human health and environment is a growing concern. Hemoglobin (Hb), which occurs in all the kingdom of living organism, is the most abundant protein in human blood. In present study, effect of lanthanides and actinides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] on the structure and function of Hb has been investigated. Results showed that these metal ions, except Ce(IV) interacted with carbonyl and amide groups of Hb, which resulted in the loss of its alpha-helix conformation. However, beyond 75µM, these ions affected heme moiety. Metal-heme interaction was found to affect oxygen-binding of Hb, which seems to be governed by their closeness with the charge-to-ionic-radius ratio of iron(III). Consistently, Ce(IV) being closest to iron(III), exhibited a greater effect on heme. Binding constant and binding stoichiometry of Th(IV) were higher than that of U(VI). Experiments using aquatic midge Chironomus (possessing human homologous Hb) and human blood, further validated metal-Hb interaction and associated toxicity. Thus, present study provides a biochemical basis to understand the actinide/lanthanide-induced interference in heme, which may have significant implications for the medical and environmental management of lanthanides/actinides toxicity.

The affinity of yeast and bacterial SCO proteins for CU(I) and CU(II). A capture and release strategy for copper transfer.

SCO (Synthesis of Cytochrome c Oxidase) proteins are present in prokaryotic and eukaryotic cells, and are often required for efficient synthesis of the respiratory enzyme cytochrome c oxidase. The Bacillus subtilis version of SCO (i.e., BsSCO) has much greater affinity for Cu(II) than it does for Cu(I) (Davidson and Hill, 2009), and this has been contrasted to mitochondrial SCO proteins that are characterized as being specific for Cu(I) (Nittis, George and Winge, 2001). This differential affinity has been proposed to reflect the different physiological environments in which these two members of the SCO protein family reside. In this study the affinity of mitochondrial SCO1 from yeast is compared directly to that of BsSCO in vitro. We find that the yeast SCO1 protein has similar preference for Cu(II) over Cu(I), as does BsSCO. We propose a mechanism for SCO function which would involve high-affinity binding to capture Cu(II), and relatively weak binding of Cu(I) to facilitate copper transfer.

The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly.

CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na(+) at 1.7 and 1.9 Šresolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na(+) at 1.8 Šresolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type ß-bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of ß2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na(+)-TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer-trimer interfaces and the lateral clefts of the trimer. For Na(+)-PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes.