The prion protein and its ligands: Insights into structure-function relationships

The prion protein is a multifunctional protein that exists in at least two different folding states. It is subject to diverse proteolytic processing steps that lead to prion protein fragments some of which are membrane-bound whereas others are soluble. A multitude of ligands bind to the prion protein and besides proteinaceous binding partners, interaction with metal ions and nucleic acids occurs. Although of great importance, information on structural and functional consequences of prion protein binding to its partners is limited. Here, we will reflect on the structure-function relationship of the prion protein and its binding partners considering the different folding states and prion protein fragments.     

Prion metal interaction: Is prion pathogenesis a cause or a consequence of metal imbalance?

Functional role of cellular prion protein (PrPc) has been hypothesized to be in metal homeostasis and providing cells with a superoxide dismutase (SOD)-like activity to escape damage by reactive oxygen species (ROS). PrPc interacts with a range of divalent metal ions and undergoes Cu²⁺ as well as Zn²⁺-associated endocytosis, thereby maintaining homeostasis of these and other metal ions. Conformational change to a β-sheet rich, protease resistant entity, reminiscent of the disease-associated scrapie form called PrPsc, has been found to be induced by interaction of PrPc with metal ions like Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺. This review compiles data from various experimental studies of the interaction of metals with PrPc. The effect of metal ions on the expression and conformation of the prion protein is described in detail with emphasis on their possible physiological and pathogenic role. Further, a hypothesis is presented where attainment of altered conformation by metal-bound PrPc has been viewed as a deleterious consequence of efforts made by cells to maintain metal homeostasis. Thus, PrPc presumably sacrifices itself by converting into PrPsc form in an attempt to protect cells from the toxicity of metal imbalance. Finally, possible reasons for contradictions reported in the literature on the subject are explored and experimental approaches to resolve the same are suggested.     

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 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.     

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.     

Automated PrPres amplification using indirect sonication

Prions, which mainly consist of the scrapie isoform of the prion protein (PrP(Sc)), induce the misfolding of the physiological prion protein (PrP(C)). The Protein Misfolding Cyclic Amplification (PMCA), a process consisting of sonication and incubation, is one of the few methods thought to model autocatalytic prion replication and generation of proteinase K (PK)-resistant PrP (PrPres) in vitro. Here we show for the first time that the amplification may be achieved through direct as well as indirect sonication (water bath sonication using sealed sample containers), allowing the PMCA method to be automated. The automated method may serve as a valuable tool in high throughput screening for the diagnosis or compound identification for treatment of prion disease. The in vitro amplification process is weakly facilitated by divalent cations such as Mn, Zn and Ni, but not Cu, however, the presence of metal ions decreases the stability of PrPres against proteinase K digestion.     

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).     

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.     

Decreased brain copper due to copper deficiency has no effect on bovine prion proteins

Copper (Cu) is believed to be integral in prion biology and the lack of Cu or replacement by other metal ions on prions may be involved in prion diseases. This theory has not been evaluated in the bovine. Thus, mature cows were used to determine the effects of Cu deficiency on brain Cu concentrations and prion functional characteristics. Two Cu states were induced, Cu-adequate (n=4) and Cu-deficient (n=4). Copper deficiency resulted in decreased (44%) brain Cu concentrations but had no effect on prion concentrations. Based on Western blot analysis, the molecular weights, glycoform distributions, and elution profiles of brain prions were not affected by Cu status. Importantly, Cu status did not affect prion proteinase degradability as all prions were completely degraded by proteinase K. In conclusion, Cu status affected bovine brain Cu concentrations but had no detectable effects on brain prion protein characteristics.     

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP^C) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP^C proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP^C-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP^C in mediating copper uptake by clathrin-dependent endocytosis.     

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.     

Prion Stability and Infectivity in the Environment

The biology of normal prion protein and the property of infectivity observed in abnormal folding conformations remain thinly characterized. However, enough is known to understand that prion proteins stretch traditional views of proteins in biological systems. Numerous investigators are resolving details of the novel mechanism of infectivity, which appears to feature a protein-only, homologous replication of misfolded isoforms. Many other features of prion biology are equally extraordinary. This review focuses on the status of infectious prions in various natural and man-made environments. The picture that emerges is that prion proteins are durable under extreme conditions of environmental exposure that are uncommon in biological phenomena, and this durability offers the potential for environmental reservoirs of persistent infectivity lasting for years. A recurrent theme in prion research is a propensity for these proteins to bind to mineral and metal surfaces, and several investigators have provided evidence that the normal cellular functions of prion protein may include metalloprotein interactions. This structural propensity for binding to mineral and metal ions offers the hypothesis that prion polypeptides are intrinsically predisposed to non-physiological folding conformations that would account for their environmental durability and persistent infectivity. Similarly, the avidity of binding and potency of prion infectivity from environmental sources also offers a recent hypothesis that prion polypeptides bound to soil minerals are actually more infectious than studies with purified polypeptides would predict. Since certain of the prion diseases have a history of epidemics in economically important animal species and have the potential to transmit to humans, urgency is attached to understanding the environmental transmission of prion diseases and the development of protocols for their containment and inactivation. Special issue article in honor of Dr. George DeVries.     

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.     

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.     

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 yeast prion protein Ure2 shows glutathione peroxidase activity in both native and fibrillar forms

Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homology to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterization of the purified protein. Here we use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-butyl hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concentration and showed optima at pH 8 and 40 degrees C. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was observed. This excludes a ping-pong mechanism and implies either a rapid-equilibrium random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

Copper-dependent co-internalization of the prion protein and glypican-1

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.     

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.     

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.