Prion protein fate governed by metal binding

The conversion of the normal cellular prion protein to an abnormal isoform is considered to be causal to the prion diseases or transmissible spongiform encephalopathies. The prion protein is a copper binding protein but under some conditions may bind other metals. In particular, the binding of manganese has been suggested to convert the prion protein (PrP) to a protease resistant isoform. Therefore, the differences in the way the protein binds copper and manganese might be revealing in terms of the mechanism of conversion of the protein or its normal cellular activity. We report the use of near-infrared spectroscopy for studies on aqueous solutions of prion protein binding Cu or Mn. These alloforms of the protein were analyzed by spectral data acquisition and multivariate analysis. Our results indicate that PrP binds both Mn and Cu differently. Analyses of Cu binding suggest that the PrP-Cu complex protected Cu from the water increasing protein stability. PrP-Mn does not protect Mn from water interactions. A real-time study of the protein alloforms showed that PrP-Cu remains stable in solution, but that PrP-Mn underwent highly different changes that led to fibril formation.     

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.     

Copper and zinc promote interactions between membrane-anchored peptides of the metal binding domain of the prion protein

The prion protein (PrP) has been identified as a metalloprotein capable of binding multiple copper ions and possibly zinc. Recent studies now indicate that prion self-recognition may be an important factor in both the normal function and misfunction of this protein. We have developed fluorescently labeled models of the prion protein that allow prion-prion interactions and metal binding to be investigated on the molecular level. Peptides encompassing the full metal binding region were anchored to the surface of small unilamellar vesicles, and PrP-PrP interactions were monitored by fluorescence spectroscopy as a function of added metal. Both Cu2+ and Zn2+ were found to cause an increase in the level of PrP-PrP interactions, by 117 and 300%, respectively, whereas other metals such as Ni2+, Co2+, and Ca2+ had no effect. The binding of either of these cofactors appears to act as a switch that induces PrP-PrP interactions in a reversible manner. Both glutamine and tryptophan residues, which occur frequently in the metal binding region of PrP, were found to be important in mediating PrP-PrP interactions. Experiments demonstrate that tryptophan residues are also responsible for the low level of PrP-PrP interactions observed in the absence of Cu2+ and Zn2+, and this is further supported by molecular modeling. Overall, our results indicate that PrP may be a bifunctional molecule capable of responding to fluctuations in both neuronal Cu2+ and Zn2+ levels.     

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.     

Copper has differential effect on prion protein with polymorphism of position 129

The pathology of human prion diseases is affected by polymorphism at amino acid residue 129 of the prion protein gene. Recombinant mouse prion proteins mimicking either form of the polymorphism were prepared to examine their effect on the conformation and the level of superoxide dismutase (SOD) activity of the prion protein. Following the binding of copper atoms to prion protein, antibody mapping and CD analysis detected conformational differences between the two forms of protein. However, neither the level of copper binding nor the level of SOD activity associated with this form of prion protein altered with the identity of codon 129. These results suggest that in the holo-metal binding form of the protein, prion structure but not its SOD activity is affected by polymorphism at codon 129.     

Prions and manganese: A maddening beast

The prion protein is well known because of its association with prion diseases. These diseases, which include variant CJD, are unusual because they are neurodegenerative diseases that can be transferred between individuals experimentally. The prion protein is also widely known as a copper binding protein. The binding of copper to the prion protein is possibly necessary for its normal cellular function. The prion protein has also been suggested to bind other metals, and among these, manganese. Despite over ten years of research on manganese and prion disease, this interaction has often been dismissed or at best seen as a poor cousin to the involvement of copper. However, recent data has shown that manganese could stabilise prions in the environment and that chelation therapy specifically aimed at manganese can extend the life of animals with prion disease. This article reviews the evidence for a link between prions and manganese.     

Prion protein reduces both oxidative and non-oxidative copper toxicity

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.     

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.     

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Ordering in a glycine-rich peptide conjugate: microscopic, fluorescence, and metalation studies

Glycine residues play an intriguing role in peptide/protein structure where they can act as tightly packing amino acids with flexible bond angles. For example, structural role of glycines is highlighted in natural silk fibers where different structural polymorphs have been reported. This study deals with a glycine-rich segment from the conserved octarepeat (PHGGGWGQ) in prion protein. We have synthesized a bis-conjugate 3, containing a truncated pentapeptide segment (GGGWG), to study its time-dependent solution phase aggregation by a combination of microscopic methods and fluorescence. This discontinuous peptide conjugate 3 exhibited interesting photophysical properties upon self-assembly allowing us to propose a possible model of peptide filament formation. Taking note of the fact that prion octarepeats bind copper, we also demonstrate the ability of this conjugate to bind copper and the growth and ultrastructure of metallized fibers formed upon incubation. Enforcing peptide fiber formation in metal binding motifs offers an entry into metal impregnated fibers for possible nanobiotechnological applications.     

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrP(Sc)). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5-7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.     

A mechanism for copper inhibition of infectious prion conversion

We employ ab initio electronic structure calculations to obtain two structural models for copper bound in the strongest binding site of the noninfectious form of the prion protein. The models are compatible with available experimental constraints from electron spin resonance data. The bending of the peptide backbone attendant with the copper binding is not compatible with the requisite straight beta-strand backbone structure for the same sequence contained in two recently proposed models of the prion protein structure in its infectious form. We hypothesize that copper binding at this site is protective against conversion to the infectious form, discuss experimental data that appear to support and conflict with our hypothesis, and propose tests using recombinant prion protein, genetically modified cultured neurons, and transgenic mice.     

Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Effects of the Pathological Q212P Mutation on Human Prion Protein Non-Octarepeat Copper-Binding Site

Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrP(C). PrP(C) interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dyshomeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP-HuPrP(90-231), both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.     

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.     

Prion protein expression modulates neuronal copper content

The prion protein is a copper (Cu)-binding protein. The abnormal isoform of this protein is associated with the transmissible spongiform encephalopathies or prion diseases. In prion diseases, the prion protein loses its Cu binding capacity. The effect of prion protein expression on the Cu content of the brain was investigated. Transgenic mice, either overexpressing the prion protein or expressing a mutant form lacking the Cu-binding region of the protein, were compared with wild-type mice and mice in which expression of the protein was knocked out. Age-dependent differences in Cu content of the brain were detected. Also, synaptosomal fractions from the brains of the mice showed different Cu content depending on the expression of the prion protein. Mice expressing prion protein, but without the Cu-binding domain showed reduced Cu content. Mice overexpressing the prion protein showed little difference in Cu in the brain compared with wild type but also the prion protein expressed by the mice showed a reduction in the level of Cu bound. These results confirm that prion protein expression modulates the Cu level found at the synapse and this effect is dependent on its Cu binding capacity. Loss of normal Cu binding by the prion protein altered age-related increases in metals in the brain. This may explain why many forms of human prion disease do not develop until late in life.     

Copper converts the cellular prion protein into a protease-resistant species that is distinct from the scrapie isoform

Several lines of evidence have suggested that copper ions play a role in the biology of both PrP(C) and PrP(Sc), the normal and pathologic forms of the prion protein. To further investigate this intriguing connection, we have analyzed how copper ions affect the biochemical properties of PrP(C) extracted from the brains of transgenic mice and from transfected cells. We report that the metal rapidly and reversibly induces PrP(C) to become protease-resistant and detergent-insoluble. Although these two properties are commonly associated with PrP(Sc), we demonstrate using a conformation-dependent immunoassay that copper-treated PrP is structurally distinct from PrP(Sc). The effect of copper requires the presence of at least one of the five octapeptide repeats normally present in the N-terminal half of the protein, consistent with the idea that the metal alters the biochemical properties of PrP by directly binding to this region. These results suggest potential roles for copper in prion diseases, as well as in the physiological function of PrP(C).     

Manganese binding to the prion protein

There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 mum at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called "fifth site" normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.     

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.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.