Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23-98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23-98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23-231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.     

Melanin complex of the fungus Inonotus obliquus

The fungus Inonotus obliquus (Pers.) Pil. synthesised high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was showed that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated the melanogenesis. The production of melanin correlated with the synthesis of o- and p-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

Melanin complex of the fungusInonotus obliquus

The fungusInonotus obliquus (Pers.) Pil. synthesized high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was shown that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated melanogenesis. The production of melanin correlated with the synthesis ofo- andp-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu²⁺ or Zn²⁺ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

The ceruloplasmin and hydrogen peroxide system induces alpha-synuclein aggregation in vitro

Alpha-synuclein is a key component of Lewy bodies in the brain of patients with Parkinson's disease (PD) and recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. Since hydrogen peroxide-mediated ceruloplasmin (CP) modification can induce the formation of free radicals and release of copper ions, we investigated the role of CP in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both CP and H(2)O(2), alpha-synuclein concomitantly was induced to be aggregated. Thioflavin-S staining of alpha-synuclein aggregates showed that they displayed characteristic fibrillar structures. Hydroxyl radical scavengers and spin-trapping agent such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone significantly inhibited the aggregation of alpha-synuclein. Copper chelator, penicillamine also inhibited the CP/H(2)O(2) system-induced alpha-synuclein aggregation. This indicates that the aggregation of alpha-synuclein can be mediated by the CP/H(2)O(2) system via the generation of hydroxyl radical. The CP/H(2)O(2) system-induced alpha-synuclein aggregation resulted in the generation of protein carbonyl derivatives. Antioxidant molecules, carnosine, homocarnosine and anserine significantly inhibited the CP/H(2)O(2) system-induced aggregation of alpha-synuclein. These results suggest that the CP/H(2)O(2) system may be related to abnormal aggregation of alpha-synuclein which may be involved in the pathogenesis of PD and related disorders.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.     

Thermal aggregation of beta-lactoglobulin in presence of metal ions

In this work, we report a study of the effects of zinc and copper ions on the heat-induced aggregation of beta-lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FTIR absorption measurements show the different role played by the two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than copper. Then, at fixed temperature, formation of a large amount of aggregates, of large dimension, is observed for Zn-BLG in shorter time; on the contrary, the presence of copper in solution does not affect the aggregation process while the secondary structure changes and the formation of different stronger intermolecular H-bonds, which probably lead to build a network of bonds that takes towards gelation. Our studies show how time evolution of aggregation process of BLG is dramatically affected by the presence of metal ions in solution and structural protein modifications are induced by different divalent metal ions.     

Oxidative modification of human ceruloplasmin by methylglyoxal: an in vitro study

Methylglyoxal (MG) is an endogenous physiological metabolite which is present in increased concentrations in diabetics. MG reacts with the amino acids of proteins to form advanced glycation end products. In this in vitro study, we investigated the effect of MG on the structure and function of ceruloplasmin (CP) a serum oxidase carrier of copper ions in the human. When CP was incubated with MG, the protein showed increased electrophoretic mobility which represented the aggregates at a high concentration of MG (100 mM). MG-mediated CP aggregation led to the loss of enzymatic activity and the release of copper ions from the protein. Radical scavengers and copper ion chelators significantly prevented CP aggregation. CP is an important protein that circulates in plasma as a major copper transport protein. It is suggested that oxidative damage of CP by MG may induce perturbations of the copper transport system and subsequently lead to harmful intracellular condition. The proposed mechanism, in part, may provide an explanation for the deterioration of organs in the diabetic patient.     

Both the environment and somatic mutations govern the aggregation pathway of pathogenic immunoglobulin light chain.

Deposition of monoclonal immunoglobulin light chain (LC) aggregates in tissues is the hallmark of a class of fatal diseases with no effective treatment. In the most prevalent diseases two different types of LC aggregates are observed: fibrillar deposits in LC amyloidosis (AL) and granular aggregates in LC deposition disease (LCDD). The mechanisms by which a given LC forms either type of aggregate are not understood. Although some LCs are more aggregation-prone than others, this does not appear to be due to specific sequence determinants, but more likely from global properties that can be introduced by multiple somatic mutations. Moreover, a single LC isotype can sometimes form both fibrillar and granular aggregates within the same patient. To better understand how the different aggregation pathways arise, we developed a series of in vitro assays to analyze the formation of distinct aggregate types. The recombinant kappa IV LC (SMA) assembles into fibrils when agitated. We now show that SMA can also form granular aggregates upon exposure to copper, and that this aggregation can occur not only in vitro, but also in cells. A constellation of somatic mutations, consisting of His89/His94/Gln96, is sufficient to confer sensitivity to copper on wild-type kappa IV proteins. The formation of both types of aggregates is inhibited by synthetic peptides derived from the LC variable domain. However, the peptide that inhibits fibrillar aggregation is different from the peptide that inhibits copper-induced aggregation. Thus, distinct molecular surfaces of the LC underly each type of aggregate. We conclude that both the intrinsic properties of the sequence and extrinsic conditions govern the aggregation pathway of a LC.     

Oxidative modification of neurofilament-L by copper-catalyzed reaction

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for neuronal survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of copper and peroxide in the modification of NF-L. When disassembled NF-L was incubated with copper ion and hydrogen peroxide, then the aggregation of protein was proportional to copper and hydrogen peroxide concentrations. Dityrosine crosslink formation was obtained in copper-mediated NF-L aggregates. The copper-mediated modification of NF-L was significantly inhibited by thiol antioxidants, Nacetylcysteine, glutathione, and thiourea. A thioflavin-T binding assay was performed to determine whether the copper/H2O2 system-induced in vitro aggregation of NF-L displays amyloid-like characteristics. The aggregate of NF-L displayed thioflavin T reactivity, which was reminiscent of amyloid. This study suggests that copper-mediated NF-L modification might be closely related to oxidative reactions which may play a critical role in neurodegenerative diseases.     

Metal-ion interactions and the structural organization of Sepia eumelanin

The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra-sonicated, EDTA-washed and Fe-saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)-loaded samples. The solubilized components are found to be 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA-rich entities form distinct two-dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply-charged ions play an important role in assisting or templating the assembly of the metal-free organic components to form the three-dimensional substructure distributed along the protein scaffold within the granule.     

Effect of metal ions on de novo aggregation of full-length prion protein

It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu(2+), Mn(2+), Ni(2+), Co(2+), and Zn(2+)) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn(2+) at low micromolar concentrations that could be blocked by nanomolar concentration of Cu(2+). These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.     

Applications of affinity chromatography to the study of drug-melanin binding interactions

This short review reports on progresses in the study of drug-melanin interactions using the technique of affinity chromatography. Melanins are natural or synthetic pigments derived from the oxidation and polymerization of various precursors including L-dopa, tyrosine and cystein. Accumulation of toxic compounds, drugs, and metal ions in pigmented tissues through reversible binding to melanin has been linked to chronic toxicity. Affinity chromatography using chromatographic stationary phases based on physically adsorbed or chemically bonded melanin provides a useful tool for studying the interactions of small molecules and metal ions with melanin     

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.     

Regulation of mammalian melanogenesis. II: The role of metal cations

Melanogenesis can be divided into two phases. The first one involves two tyrosinase-catalyzed oxidations from tyrosine to dopaquinone and a very fast chemical step leading to dopachrome. The second phase, from dopachrome to melanin, can proceed spontaneously through several incompletely known reactions. However, some metal transition ions and protein factors different from tyrosinase might regulate the reaction rate and determine the structure and relative concentrations of the intermediates. The study of the effects of some divalent metal ions (Zn, Cu, Ni and Co) on some steps of the melanogenesis pathway has been approached using different radiolabeled substrates. Zn(II) inhibited tyrosine hydroxylation whereas Ni(II) and Co(II) were activators. Ni(II), Cu(II) and Co(II) accelerated chemical reactions from dopachrome but inhibited its decarboxylation. Dopachrome tautomerase also decreased decarboxylation. When metal ions and this enzyme act together, the inhibition of decarboxylation was greater than that produced by each agent separately, but amount of carboxylated units incorporated to the melanin was not higher than the amount incorporated in the presence of only cations. The amount of total melanin formed from tyrosine was increased by the presence of both agents. The action of Zn(II) was different from other ions also in the second phase of melanogenesis, and its effect on decarboxylation was less pronounced. Since tyrosine hydroxylation is the rate-limiting step in melanogenesis, Zn(II) inhibited the pathway. This ion seems to be the most abundant cation in mammalian melanocytes. Therefore, under physiological conditions, the regulatory role of metal ions and dopachrome tautomerase does not seem to be mutually exclusive, but rather complementary.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aluminum, copper, iron and zinc differentially alter amyloid-Aβ(1-42) aggregation and toxicity

Amyloid-β(1-42) (Aβ) is believed to play a crucial role in the ethiopathogenesis of Alzheimer's Disease (AD). In particular, its interactions with biologically relevant metal ions may lead to the formation of highly neurotoxic complexes. Here we describe the species that are formed upon reacting Aβ with several biometals, namely copper, zinc, iron, and with non-physiological aluminum to assess whether different metal ions are able to differently drive Aβ aggregation. The nature of the resulting Aβ-metal complexes and of the respective aggregates was ascertained through a number of biophysical techniques, including electrospray ionization mass spectrometry, dynamic light scattering, fluorescence, transmission electron microscopy and by the use of conformation-sensitive antibodies (OC, αAPF). Metal binding to Aβ is shown to confer highly different chemical properties to the resulting complexes; accordingly, their overall aggregation behaviour was deeply modified. Both aluminum(III) and iron(III) ions were found to induce peculiar aggregation properties, ultimately leading to the formation of annular protofibrils and of fibrillar oligomers. Notably, only Aβ-aluminum was characterized by the presence of a relevant percentage of aggregates with a mean radius slightly smaller than 30 nm. In contrast, both zinc(II) and copper(II) ions completely prevented the formation of soluble fibrillary aggregates. The biological effects of the various Aβ-metal complexes were studied in neuroblastoma cell cultures: Aβ-aluminum turned out to be the only species capable of triggering amyloid precursor and tau181 protein overproduction. Our results point out that Al can effectively interact with Aβ, forming "structured" aggregates with peculiar biophysical properties which are associated with a high neurotoxicity.     

Modulatory effects of pH, Cu+2 and sheet breakers on aggregation of amyloid peptides

The study explores in vitro by circular dichroism and mass spectrometry the effects of pH, Cu+2 ions and sheet-breakers on the secondary structures and self-aggregation of beta-amyloid peptides [Abeta43, Abeta42 and Abeta40] of Alzheimer's disease. Within pH 5.4-7.3, more sheet structures and aggregates containing up to 11 peptide units were observed. Cu+2 ions led to oxidative degradation or aggregation depending on its concentration and time of incubation. beta-sheet breakers can reverse the self-aggregation process, suggesting their potential therapeutic use.