Metallic prions

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.     

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.     

Imbalance of antioxidant defense in mice lacking cellular prion protein

Prion diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from its normal cellular isoform, PrPC, to the infectious scrapie isoform, PrP(Sc). In spite of many studies, the physiological function of PrPC remains unknown. Recent work shows that PrPC binds Cu2+, internalizing it into the cytoplasm. Since many antioxidant enzymes depend on Cu2+ (e.g., Cu/ZnSOD), their function could be affected in prion diseases. Here we investigate a possible relationship between PrP(C) and the cellular antioxidant systems in different structures isolated from PrPC knockout and wild-type mice by determining oxidative damage in protein and lipids and activity of antioxidant enzymes (CAT, SOD) and stress-adaptive enzymes (ODC). Our results show that, in the absence of PrPC, there is an increased oxidation of lipid and protein in all structures investigated. Decreased SOD activity and changes in CAT/ODC activities were also observed. Taking into account these results, we suggest that the physiological function of PrP(C) is related to cellular antioxidant defenses. Therefore, during development of prion diseases, the whole organism becomes more sensitive to ROS injury, leading to a progressive oxidative disruption of tissues and vital organs, especially the central nervous system.     

Mapping the functional domain of the prion protein.

Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.     

Prion-associated increases in Src-family kinases

The prion diseases result from the generation and propagation of an abnormal conformer of the prion protein. It is unclear how this molecular event disrupts neuronal function and viability. Current evidence argues it is not due to loss of normal prion protein activity or direct toxic effects of the abnormal conformer. Both the normal and abnormal prion proteins are glycosylphosphatidylinositol-linked membrane proteins. Conversion to the abnormal isoform results in the formation and accumulation of prion protein aggregates. Because aggregation of glycosylphosphatidylinositol-linked proteins activates Src-family kinases, the activation status and levels of the Src-family kinases in prion disease were investigated. Elevations of Src-family kinases were found in a cell culture model and two separate animal models of prion disease. The elevations in Src kinases preceded the onset of symptoms and occurred concurrently with the appearance of detergent-insoluble prion protein. In addition, the total level of kinases phosphorylated at tyrosine residues associated with activation was increased. Similar alterations were not present in brain homogenates from presymptomatic animals early in the disease course, prion protein-ablated animals, or end-stage Tg2576 mice overexpressing mutant amyloid precursor protein. Identification of similar elevations in cell culture and animal model systems suggests the elevations are a specific response to the presence of the disease-associated conformer. Abnormal regulation of these signal transduction cascades may be a key element in the cellular pathology of the prion diseases.     

Selective oxidation of methionine residues in prion proteins.

Prion proteins are central to the pathogenesis of several neurodegenerative diseases through the postulated conversion of the endogenous cellular isoform (PrPc) into a pathogenic isoform (PrPSc). Although the cellular function of normal prion protein remains unresolved a number of studies have shown that prion proteins may be involved in the cellular response to oxidative stress. Here, using purified recombinant sources of mouse and chicken PrP refolded in the presence of copper (II) we show that the methionine residues of the protein are uniquely susceptible to oxidation. We suggest that Met residues may form an essential part of the mechanism of the antioxidant activity exhibited by normal prion protein.     

Molecular advances in understanding inherited prion diseases

The prion diseases are neurodegenerative disorders that have attracted great interest because of the possible link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (CTD) in humans. Possible transmission of these diseases has been linked to a single protein termed the prion protein. This protein is an abnormal isoform of a normal synaptic glycoprotein. The majority of prion diseases does not appear to be caused by transmission of an infectious agent but occur spontaneously with no known cause. The strongest supporting evidence that the prion protein is the causative agent in prion disease comes from specific inheritable forms of prion disease which are linked to single point mutations in the prion protein gene. Paradoxically, these point mutations, although autosomal dominant with 100% penetrance do not lead to disease until late in life. Molecular techniques are now being used extensively to determine how these point-mutations alter the prion protein’s normal structure and activity. This review deals with the latest insights into how inherited mutations in the prion protein gene lead to neurodegenerative disease.     

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.     

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.     

Prion diseases: dynamics of the infection and properties of the bistable transition

Prion diseases are thought to result from a pathogenic, conformational change in a cellular protein, the prion protein. The pathogenic isoform seems to convert the normal isoform in an autocatalytic process. In contrast to the conditions used for in vitro studies of enzyme kinetics, the concentration of the catalyst is not much lower than that of the substrate in the course of infection. This feature may endow the system with a time-hierarchy allowing the pathogenic isoform to relax very slowly in the course of infection. This may contribute to the long incubation periods observed in prion diseases. The dynamic process of prion propagation, including turnover of the cellular prion protein, displays bistable properties. Sporadic prion diseases may result from a change in one of the parameters associated with metabolism of the prion protein. The bistable transition observed in sporadic disease is reversible, whereas that observed in cases of exogenous contamination is irreversible. This model is consistent with the occurrence of rare, sporadic forms of prion diseases. It may also explain why only some individuals of a cohort develop a prion disease following transient food contamination.     

Prion diseases: copper deficiency states associated with impaired nitrogen monoxide or carbon monoxide transduction and translocation.

Literature concerning prion diseases and Cu metabolism was examined to determine merits of various suggestions concerning the relationship between these diseases and altered Cu metabolism. There are a number of recent suggestions that the normal non-pathogenic form of the prion protein (PrP(C)) contains Cu while the abnormal pathogenic form of this protein, PrP(SC), lacks Cu. Results of experiments showing oxidant sensitivity in the presence of ionically bonded Cu and millimolar concentrations of hydrogen peroxide were found to lack relevance. Demonstrating superoxide disproportionation and a correlation with cellular Cu2Zn2SOD activity is relevant and consistent with a role for PrP(C) in Cu endocytosis. There are also a number of recent suggestions that PrP(C) has a role in nerve transmission. Serum from mice that lack cellular PrP(C) was found to have an elevated Cu content consistent with a response to overcome an inflammatory disease. Attempts to induce a 'transmissible' form of prion disease requiring intracerebral injections of somewhat purified brain homogenates were found lacking in support for an etiology occurring as the result of oral ingestion of supposedly 'infected' tissues. It is suggested that PrP(C) is a normal Cu-dependent cuproglycoprotein of unknown function that may have a role in facilitating normal nitrogen monoxide- or carbon monoxide-mediated biochemistry.     

Molecular basis of cerebral neurodegeneration in prion diseases

The biochemical nature and the replication of infectious prions have been intensively studied in recent years. Much less is known about the cellular events underlying neuronal dysfunction and cell death. As the cellular function of the normal cellular isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing evidence that the normal cellular isoform of the prion protein is a key mediator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid production, and induction of neurodegeneration. Four experimental avenues will be discussed here which address scenarios of inappropriate trafficking, folding, or targeting of the prion protein.     

Prions: on the enzymatic function of PrPc from mammalian and prion "families"

This minireview deals with some approaches and results of experiments which enabled to discover SOD-like activity of the mammal PrP protein. This activity required the unchanged region of the repeated octapeptide and Cu2+ binding to the appropriate sites of the PrP molecule. It was shown that an infectious prion isoform could bind the normal isoform. This leads to the loss of PrP SOD-like function accompanied by Cu2+ release from the molecule. Also, the problem of sowing the protein seeds of prion propagation is briefly summarized and the first evidence of abnormal protein conformation induced in vivo using yeast cell and in vitro formed Sup 35 prion seed is described.     

Prion channel proteins and their role in vacuolation and neurodegenerative diseases

The prion encephalopathies, which are characterized by neuropathological changes that include vacuolation, astrocytosis, the development of amyloid plaques and neuronal loss, are associated with the conversion of a normal cellular isoform of prion protein (PrP(c)) to an abnormal pathologic scrapie isoform (PrP(Sc)). The use of PrP[106-126] and its isoforms in studies of channels in lipid bilayers has revealed that it forms heterogeneous channels reflecting modifications in the peptide's structure and differences in the properties of the formed oligomeric aggregates and their intermediates. We propose that the accumulation of pathological isoforms of prion are linked to membrane abnormalities and vacuolation in prion diseases. The interlinked changes in membrane fluidity and endogenous channels induced by prion isoforms can occur independently and concurrently with channel formation, i.e. they are not mutually exclusive. We suggest that vacuolation is a cellular response triggered in order to immobilize pathological prion isoforms having the ability to form channels that compromise cellular membranes. This mechanism is similar to that of other channel-forming proteins that induce vacuolation, e.g. the well-established VacA of Helicobacter pylori, Vero cells and aerolysin, as well as melittin-induced micellization and membrane fusion. We conclude that channel formation is part of the molecular mechanisms responsible for the vacuolation associated with prion diseases. The initial vacuolation could be an adaptive cellular response to compartmentalize the increase in pathogenic prion isoforms, while an excessive accumulation of pathologic prion isoforms in later stages represents the inability of the cell to continue to compartmentalize these misfolded proteins in vacuoles.     

Selection of ovine PrP high-producer subclones from a transfected epithelial cell line

The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.     

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.     

Metals and neuroscience

Data are now rapidly accumulating to show that metallochemical reactions might be the common denominator underlying Alzheimer's disease, amyotrophic lateral sclerosis, prion diseases, cataracts, mitochondrial disorders and Parkinson's disease. In these disorders, an abnormal reaction between a protein and a redox-active metal ion (copper or iron) promotes the formation of reactive oxygen species or radicalization. It is especially intriguing how the powerful catalytic redox activity of antioxidant Cu/Zn-superoxide dismutase can convert into a pro-oxidant activity, a theme echoed in the recent proposal that Abeta and PrP, the proteins respectively involved in Alzheimer's disease and prion diseases, possess similar redox activities.     

Theoretical analysis of the implication of PrP in neuronal death during transmissible subacute spongiform encephalopathies: hypothesis of a PrP oligomeric channel

Transmissible subacute spongiform encephalopathies (TSE) are animal and human neurodegenerative diseases. The nature of the transmissible agent remains unknown. The specific molecular marker of these diseases is the abnormal isoform of the prion protein (PrP). This protein is encoded by a cellular gene and accumulates in a pathological isoform (PrPres) which is partially resistant to proteolysis. The tridimensional structure of this protein remains theoretical. F. Cohen proposed one of the most realistic models. According to this model and from molecular mechanics calculation, we suggest a PrP oligomeric ionic channel model that may be involved in TSE-induced neuronal apoptosis.     

Prions and the proteasome

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. They are unique in terms of their biology because they are caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently the precise mechanism behind prion-mediated neurodegeneration remains unclear. It is hypothesised than an unknown toxic gain of function of PrPSc, or an intermediate oligomeric form, underlies neuronal death. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. Both wild-type PrPC and disease-associated PrP isoforms accumulate in cells after proteasome inhibition leading to increased cell death, and abnormal beta-sheet-rich PrP isoforms have been shown to inhibit the catalytic activity of the proteasome. Here we review potential interactions between prions and the proteasome outlining how the UPS may be implicated in prion-mediated neurodegeneration.     

Prion processing: a double-edged sword?

The events leading to the degradation of the endogenous PrP(C) (normal cellular prion protein) have been the subject of numerous studies. Two cleavage processes, α-cleavage and β-cleavage, are responsible for the main C- and N-terminal fragments produced from PrP(C). Both cleavage processes occur within the N-terminus of PrP(C), a region that is significant in terms of function. α-Cleavage, an enzymatic event that occurs at amino acid residues 110 and 111 on PrP(C), interferes with the conversion of PrP(C) into the prion disease-associated isoform, PrP(Sc) (abnormal disease-specific conformation of prion protein). This processing is seen as a positive event in terms of disease development. The study of β-cleavage has taken some surprising turns. β-Cleavage is brought about by ROS (reactive oxygen species). The C-terminal fragment produced, C2, may provide the seed for the abnormal conversion process, as it resembles in size the fragments isolated from prion-infected brains. There is, however, strong evidence that β-cleavage provides an essential process to reduce oxidative stress. β-Cleavage may act as a double-edged sword. By β-cleavage, PrP(C) may try to balance the ROS levels produced during prion infection, but the C2 produced may provide a PrP(Sc) seed that maintains the prion conversion process.