Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches

Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.     

The secret life of extracellular vesicles in metal homeostasis and neurodegeneration

Biologically active metals such as copper, zinc and iron are fundamental for sustaining life in different organisms with the regulation of cellular metal homeostasis tightly controlled through proteins that coordinate metal uptake, efflux and detoxification. Many of the proteins involved in either uptake or efflux of metals are localised and function on the plasma membrane, traffic between intracellular compartments depending upon the cellular metal environment and can undergo recycling via the endosomal pathway. The biogenesis of exosomes also occurs within the endosomal system, with several major neurodegenerative disease proteins shown to be released in association with these vesicles, including the amyloid‐β (Aβ) peptide in Alzheimer's disease and the infectious prion protein involved in Prion diseases. Aβ peptide and the prion protein also bind biologically active metals and are postulated to play important roles in metal homeostasis. In this review, we will discuss the role of extracellular vesicles in Alzheimer's and Prion diseases and explore their potential contribution to metal homeostasis.     

Structure and stability of the CuII complexes with tandem repeats of the chicken prion

Prion protein (PrP) misfolding is one of the pivotal issues in understanding the rudiments of neurodegenerative disorders. The conformational change of mammalian cellular PrP to scrapie PrP is caused by an unknown agent, but there is reasonable evidence supporting the key role of copper ions in this process. The structure of the avian PrP was found to be very similar to the mammalian protein, although there is only 30% homology in the secondary structure. This work shows that copper ions are very effectively bound by hexarepeat fragments of chicken prion protein, although not as effectively as it was found in the case of mammalian protein. By means of potentiometric and spectroscopic techniques (nuclear magnetic resonance, circular dichroism, UV-vis, and electronic paramagnetic resonance), it was shown that Cu(II) ions coordinate to the chicken PrP hexapeptide domain in physiological pH via imidazole nitrogen donors of His residue(s). The binding pattern changes the structure of peptide involved, indicating a possible impact of Cu(II) ions in the biology and pathology of nonmammalian PrP, which could be similar to that found for mammalian PrP. The present study shows that, similar to the human prion octapeptide repeats, chicken prion hexapeptide repeats might bind copper ions in two different ways, depending on the number of repeats and metal/ligand molar ratio: (i) an intra-repeat coordination mode in which copper ion is chelated by His imidazole and deprotonated amide nitrogen in monomeric peptide and (ii) an inter-repeat coordination mode in which a polymeric peptide ligand (dimer and trimer) forms polyimidazole complexes that are very stable at physiological pH. Two proline residues inserted into the hexapeptide unit have a critical impact on the metal-binding ability.     

Metal ions and prion diseases

Recent data demonstrate that transition metal ions such as copper not only bind the prion protein with high affinities, but also modify its biochemical properties. This has important consequences for the potential function of the protein in metal-ion transport or as an anti-oxidant molecule. In addition, this relationship between the prion protein and metal ions is likely to play a critical role in the physiopathology of prion diseases.     

Dual polarisation interferometry analysis of copper binding to the prion protein: evidence for two folding states

The prion protein is a copper binding glycoprotein expressed in neurones and other cells. Conversion of this protein to an abnormal isoform is central to the cause of prion diseases or transmissible spongiform encephalopathies. Detecting slight structural differences between different forms of the prion protein could be essential to understanding the role of the protein in health and disease. Dual polarisation interferometry (DPI) is a new method that allows detection of small structural differences. We used this technique to evaluate the effectiveness of DPI in the analysis of metal binding to recombinant mouse prion protein. DPI was able to measure mass change in the prion protein following addition of copper and could identify reproducible differences in the structure of prion protein dependent on how metal was added to the protein. These slight structural differences were confirmed by the use of circular dichroism spectroscopy and Fourier-transformed infra-red spectroscopy. These results suggest that DPI can provide important information on both transitory and stable structural difference that are induced in the prion protein. This technique could be important not only for the study of metal-protein interactions but also small structural differences that could define prion strains.     

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57-91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91-115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91-115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.     

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.     

Is there a role for copper in neurodegenerative diseases?

Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the beta-amyloid-precursor protein (beta-APP) and the prion protein (PrP(C)), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and beta-APP or PrP(C), evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of beta-APP and PrP(C), in copper metabolism and the putative role of copper in neurodegenerative diseases.     

Influence of divalent copper, manganese and zinc ions on fibril nucleation and elongation of the amyloid-like yeast prion determinant Sup35p-NM

There is a large body of evidence that divalent metal ions, particularly copper, might play a role in several protein folding pathologies like Alzheimer’s disease, Parkinson’s disease or the prion diseases. However, contribution of metal ions on pathogenesis and their molecular influence on the formation of amyloid structures is not clear. Therefore, the general influence of metals on the formation of amyloids is still controversially discussed. We have utilized the well established system of yeast Sup35p-NM to investigate the role of three different metal ions, Cu²⁺, Mn²⁺ and Zn²⁺, on amyloidogenesis. Recently, it has been shown that the prion determining region NM of the Saccharomyces cerevisiae prion protein Sup35p, which is responsible for the yeast prion phenotype [PSI⁺], specifically binds Cu²⁺ ions. We further characterized the affinity of NM for Cu²⁺, which were found to be comparable to that of other amyloidogenic proteins like the mammalian prion protein PrP. The specific binding sites could be located in the aminoterminal N-region which is known to initiate formation of amyloidogenic nuclei. In the presence of Cu²⁺, fibril nucleation was significantly delayed, probably due to influences of copper on the oligomeric ensemble of soluble Sup35p-NM, since Cu²⁺ altered the tertiary structure of soluble Sup35p-NM, while no influences on fibril elongation could be detected. The secondary structure of soluble or fibrous protein and the morphology of the fibrils were apparently not altered when assembled in presence of Cu²⁺. In contrast, Mn²⁺ and Zn²⁺ did not bind to Sup35p-NM and did not exhibit significant effects on the formation of NM amyloid fibrils.     

Effect of copper and manganese on the de novo generation of protease-resistant prion protein in yeast cells

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to bind manganese and copper, the latter being involved in the normal function of the protein. Indeed, upon expression in yeast we noted a major increase in intracellular copper and a decrease in manganese. Interestingly, protease-resistant PrP(Sc)-like protein (PrP(res)) formation was induced when PrP-expressing yeast cells were grown in copper- and/or manganese-supplemented media. The pattern of PrP banding in SDS-PAGE was dominantly determined by manganese. This conformational transition was stable against EDTA treatment but not in the presence of the copper chelators bathocuproinedisulfonic acid or clioquinol. Conclusively, PrP itself influences manganese and copper metabolism, and a replacement of copper in PrP complexes with manganese is highly likely under the condition of copper depletion or if excess amounts of copper and manganese are present. Taken together, our present study demonstrates the involvement of PrP in the regulation of intracellular metal ion homeostasis and uncovers copper and, more severely, manganese ions as in vivo risk factors for the conversion into PrP(Sc).     

Prion infection impairs copper binding of cultured cells

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies (TSEs) remains unclear. Using radioactive copper ((64)Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSE.     

Copper-induced structural propensities of the amyloidogenic region of human prion protein

Transmissible spongiform encephalopathies are associated with the misfolding of the cellular Prion Protein (PrP^C) to an abnormal protein isoform, called scrapie prion protein (PrP^Sc). The structural rearrangement of the fragment of N-terminal domain of the protein spanning residues 91–127 is critical for the observed structural transition. The amyloidogenic domain of the protein encloses two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for metal ion binding. Previous studies have shown that Cu(II) sequestration by both sites may modulate the peptide’s tendency to aggregation as it inflicts the hairpin-like structure that stabilizes the transition states leading to β-sheet formation. On the other hand, since both His sites differ in their ability to Cu(II) sequestration, with His-111 as a preferred binding site, we found it interesting to test the role of Cu(II) coordination to this single site on the structural properties of amyloidogenic domain. The obtained results reveal that copper binding to His-111 site imposes precise backbone bending and weakens the natural tendency of apo peptide to β-sheet formation.     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

Prion protein binds copper within the physiological concentration range

The prion protein is known to be a copper-binding protein, but affinity and stoichiometry data for the full-length protein at a physiological pH of 7 were lacking. Furthermore, it was unknown whether only the highly flexible N-terminal segment with its octarepeat region is involved in copper binding or whether the structured C-terminal domain is also involved. Therefore we systematically investigated the stoichiometry and affinity of copper binding to full-length prion protein PrP(23-231) and to different N- and C-terminal fragments using electrospray ionization mass spectrometry and fluorescence spectroscopy. Our data indicate that the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at approximately 2 microm. This argues strongly for a direct role of the prion protein in copper metabolism, since it is almost saturated at about 5 microm, and the exchangeable copper pool concentration in blood is about 8 microm.     

Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.     

The prion protein and neuronal zinc homeostasis

Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive. Octapeptide repeats in the N terminus of PrP bind metal ions and are required for the endocytosis of PrP upon exposure of cells to copper or zinc. As the concentration of zinc in the extracellular spaces of the brain is higher than that for copper, we put forward the hypothesis that PrP is involved in neuronal zinc homeostasis; PrP might be involved in transport of zinc into the cell or might act as a zinc sensor. In prion disease, when the protein undergoes a conformational change to the infectious form, this function of PrP in zinc homeostasis might be compromised.     

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein–membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.     

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimer's disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.     

Prion,amyloid beta-derived Cu(II) ions,or free Zn(II) ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate

Copper are generally bound to proteins, e.g. the prion and the amyloid beta proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid beta peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.