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.     

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.     

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.     

Copper brain homeostasis: role of amyloid precursor protein and prion protein

The main proteins associated with Alzheimer's and prion diseases (amyloid precursor protein (APP) and prion protein (PrP(C)), respectively, have binding sites for copper and it has therefore been suggested that they play a role in copper metabolism. Here, we review evidence indicating that the copper binding domains (CuBD) of APP and PrP(C) are able to modulate the oxidation state of copper, and prevent neurotoxic effects and memory impairments induced by copper. Results with transgenic and other animal models have established the relation between these pathogenic proteins and copper. In particular, APP transgenic models, suggest a beneficial effect for copper in AD.     

Aberrant metal binding by prion protein in human prion disease

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.     

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.     

Tau融合蛋白及其缺失突变体与朊蛋白的体外作用分析

在部分朊病毒病（prion diseases）中,高度磷酸化的微管相关蛋白tau与朊蛋白(prion protein,PrP)发生共定位,tau蛋白可能在朊病毒病的病理机制中有重要作用. 本室已经证明二者可以发生分子间相互作用,本文进一步分析了tau蛋白与prion的体外相互作用及作用位点. 利用RT-PCR方法从人源细胞系SHSY5Y cDNA中扩增出微管相关蛋白tau全长cDNA序列,克隆至质粒pGEX-2T载体,在大肠杆菌中诱导表达融合蛋白GST-tau. 利用GST pull-down及免疫共沉淀方法检测全长tau蛋白与PrP23-231的分子间相互作用. 进一步表达tau 蛋白的各种缺失突变体,确定tau蛋白与PrP蛋白的相互作用位点. 结果表明,所表达的全长tau蛋白及各种缺失突变体均为可溶性蛋白,Western印迹结果显示,各种蛋白均能很好的被tau蛋白单抗识别. GST pull-down和免疫共沉淀实验均显示,原核表达的全长tau蛋白可与全长的PrP蛋白在体外发生相互作用,并确定相互作用位点位于tau蛋白的N端序列及中段的重复区. 上述结果为研究tau蛋白与PrP的相互作用在朊病毒病的发病机制中的意义提供了一定的理论基础.     

Fragment molecular orbital calculations reveal that the E200K mutation markedly alters local structural stability in the human prion protein

The E200K mutation of the human prion protein (PrP) is known to cause familial Creutzfeldt–Jakob disease. In order to elucidate the effects of the mutation on the local structural stability of PrP, we performed ab initio fragment molecular orbital calculations for the wild-type human PrP and the E200K variant modeled under neutral and mild acidic conditions. The calculations revealed that this substitution markedly altered the intramolecular interactions in the PrP, suggesting that the local structural instabilities induced by the E200K mutation might cause initial denaturation of the PrP and its subsequent conversion to a pathogenic form. This work presents a new approach for quantitatively elucidating structural instabilities in proteins that cause misfolding diseases.     

Copper(II) binding to the human Doppel protein may mark its functional diversity from the prion protein

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP(C)) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP(C) seems to be involved in Cu(2+) recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP(C) and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5-6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP(C), addition of Cu(2+) to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP(C) and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.     

High affinity binding between copper and full-length prion protein identified by two different techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.     

Difference in redox behaviors between copper-binding octarepeat and nonoctarepeat sites in prion protein

We studied the redox behavior of copper-binding sites in prion protein (PrP) to clarify copper’s role in the pathological mechanism underlying prion diseases. We investigated the coordination structures, binding affinities, and redox potentials of copper-binding peptide fragments derived from the N-terminal domain of PrP by density functional theory calculations. We used four models for copper-binding moieties in PrP(60–96): two were derived from the PHGGGWGQ octapeptide repeat region of PrP(60–91), and the others were tripeptide Gly-Thr-His fragments derived from the copper-binding nonoctarepeat site around His96. We found that such PrP-derived copper-binding complexes exhibit conformationally dependent redox behavior; for example, the copper-binding complex derived from the octarepeat region tends to possess high reduction potential for the Cu(II)/Cu(I) couple, exceeding 0 V versus the standard hydrogen electrode, whereas the copper-binding nonoctarepeat model around His96 tends to possess high oxidation potential for the Cu(II)/Cu(III) couple and stabilize the higher-valent Cu(III) state. It is possible that such distinct redox activities of a copper-binding PrP are involved in the mechanism underlying prion diseases.     

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).     

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.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

Copper binding to the octarepeats of the prion protein. Affinity,specificity, folding,and cooperativity: insights from circular dichroism

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein. There is increasing evidence that PrP functions as a copper transporter. In addition, strains of prion disease have been linked with copper binding. We present here CD spectroscopic studies of Cu(2+) binding to various fragments of the octarepeat region of the prion protein. We show that glycine and l-histidine will successfully compete for all Cu(2+) ions bound to the PrP octapeptide region, suggesting Cu(2+) coordinates with a lower affinity for PrP than the fm dissociation constant reported previously. We show that each of the octarepeats do not form an isolated Cu(2+) binding motif but fold up cooperatively within multiple repeats. In addition to the coordinating histidine side chain residues, we show that the glycine residues and the proline within each octarepeat are also necessary to maintain the coordination geometry. The highly conserved octarepeat region in mammals is a hexarepeat in birds that also binds copper but with different coordination geometry. Finally, in contrast to other reports, we show that Mn(2+) does not bind to the octarepeat region of PrP.     

Context dependent neuroprotective properties of prion protein (PrP)

Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure—independent of infectious prion conformation—to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-β, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer’s disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP’s neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson’s disease or tauopathy. Deletion of PrP in one of two Huntington’s disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington’s motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.     

The role of PrP in health and disease

Transmissible spongiform encephalopathies (TSEs) such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jacob disease (CJD) and Gerstmann-Str?ussler-Scheinker syndrome (GSS) in humans, are caused by an infectious agent designated prion. The "protein only" hypothesis states that the prion consists partly or entirely of a conformational isoform of the normal host protein PrPc and that the abnormal conformer, when introduced into the organism, causes the conversion of PrPc into a likeness of itself. Since the proposal of the "protein only" hypothesis more than three decades ago, cloning of the PrP gene, studies on PrP knockout mice and on mice transgenic for mutant PrP genes allowed deep insights into prion biology. Reverse genetics on PrP knockout mice containing modified PrP transgenes was used to address a variety of problems: mapping PrP regions required for prion replication, studying PrP mutations affecting the species barrier, modeling familial forms of human prion disease, analysing the cell specificity of prion propagation and investigating the physiological role of PrP by structure-function studies. Many questions regarding the role of PrP in susceptibility to prions have been elucidated, however the physiological role of PrP and the pathological mechanisms of neurodegeneration in prion diseases are still elusive.     

The role of prion peptide structure and aggregation in toxicity and membrane binding

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.     

Single particle analysis of manganese-induced prion protein aggregates

Prion diseases are characterized by the conversion of the cellular prion protein (PrP(C)) to a disease-specific aggregated isoform (PrP(Sc)). We have shown that Mn(2+) ions amplify aggregation, whereas Cu(2+) has an inhibitory effect. To characterize Mn(2+)-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn(2+) was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn(2+) induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu(2+) required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrP(C) which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo.