Study on interaction between microtubule associated protein tau and prion protein

Microtubule-associated protein tau is considered to play roles in many neurodegenera-tive diseases including some transmissible spongiform encephalopathies.To address the possible molecular linkage of prion protein(PrP) and tau,a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immuno-precipitation.We found tau protein interacted with various style prion proteins such as native prion protein(PrPC) or protease-resistant isoform(PrPSc) .Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene.The domain of interaction with tau was located at N-terminal of PrP(residues 23 to 91) .The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.     

Study on interaction between microtubule associated protein tau and prion protein

Microtubule-associated protein tau is considered to play roles in many neurodegenerative diseases including some transmissible spongiform encephalopathies. To address the possible molecular linkage of prion protein (PrP) and tau, a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immunoprecipitation. We found tau protein interacted with various style prion proteins such as native prion protein (PrPC) or protease-resistant isoform (prpSc). Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene. The domain of interaction with tau was located at N-terminal of PrP (residues 23 to 91). The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.     

Prion protein interconversions

The transmissible spongiform encephalopathies (TSEs), or prion diseases, remain mysterious neurodegenerative diseases that involve perturbations in prion protein (PrP) structure. This article summarizes our use of in vitro models to describe how PrP is converted to the disease-associated, protease-resistant form. These models reflect many important biological parameters of TSE diseases and have been used to identify inhibitors of the PrP conversion as lead compounds in the development of anti-TSE drugs.     

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.     

Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.     

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.     

Dynamics of the nucleated polymerization model of prion replication

The disease process for transmissible spongiform encephalopathies (TSEs), in one way or another, involves the conversion of a predominantly alpha-helical normal host-coded prion protein (PrP(C)) to an abnormally folded (predominantly beta sheet) protease resistant isoform (PrP(Sc)). Several alternative mechanisms have been proposed for this auto-catalytic process. Here the dynamical behavior of one of these models, the nucleated polymerization model, is studied by Monte Carlo discrete-event simulation of the explicit conversion reactions. These simulations demonstrate the characteristic dynamical behavior of this model for prion replication. Using estimates for the reaction rates and concentrations, time courses are estimated for concentration of PrP(Sc), PrP(Sc) aggregates, and PrP(C) as well as size distributions for the aggregates. The implications of these dynamics on protein misfolding cyclic amplification (PMCA) is discussed.     

Identification of proteins with high affinity for refolded and native PrPC

PrPC, the cellular prion protein, is widely expressed in most tissues, including brain, muscle and the gastrointestinal tract, but its physiological role remains unclear. During propagation of transmissible spongiform encephalopathies (TSEs), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by as-yet-unknown host factors. The identification of proteins associated with PrP may provide information about the biology of prions and the pathogenesis of TSEs. In the present work, we report proteins identified from brain tissue based on their ability to bind to recombinant PrP (recPrP) or form multimolecular complexes with native PrPC in the presence of cross-linkers. Immobilized his-tagged recPrP was used as an affinity matrix to isolate PrP-interacting proteins from brain homogenates of normal individuals. In parallel, PrPC-associated proteins were characterized by cross-linking and co-immunoprecipitation assays. The unknown molecules were identified by MS and the results of LC-MS/MS analysis were subsequently verified by Western blot. Both techniques resulted in identification of proteins participating in the formation of cytoskeleton and signal transduction, further supporting the hypothesis that PrP is involved in the organization and function of receptors throughout the nervous system.     

The highways and byways of prion protein trafficking

Prions are defined as infectious agents that comprise only proteins and are responsible for transmissible spongiform encephalopathies (TSEs)--fatal neurodegenerative diseases that affect humans and other mammals and include Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. Prions have been proposed to arise from the conformational conversion of the cellular prion protein PrP(C) to a misfolded form termed PrP(Sc) that precipitates into aggregates and fibrils. The conversion process might be triggered by interaction of the infectious form with the cellular form or it might result from a mutation in the gene encoding PrP(C). Exactly how and where in the cell the interaction and the conversion of PrP(C) to PrP(Sc) occur, however, remain controversial. Recent studies have shed light on the intracellular trafficking of PrP(C), the role of protein mis-sorting and the cellular factors that are thought to be required for the conformational conversion of prion proteins.     

PrP的胞内运输与朊病毒病

朊病毒病,即传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）,是一类致死性的神经退行性疾病,存在散发性、感染性和遗传性3种形式。在朊病毒病的病理过程中,细胞正常朊蛋白PrPc（cellular PrP）转化为异常构象的PrP^Sc（scrapie PrP）是至关重要的,但是朊病毒的增殖如何导致神经元凋亡仍不清楚。PrPc的胞内运输在朊病毒病中发挥重要作用,朊病毒感染后PrP^C转化为PrP^Sc,及遗传性朊病毒病中PrP突变可能影响PrP的生物合成、亚细胞定位及转运过程,通过干扰PrP^C的正常功能或产生毒性中间体而导致神经系统病变。现对近年来关于PrP胞内运输在朊病毒病中的作用进行综述。     

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.     

Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity

Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE.     

Polymorphisms within the prion-like protein gene (Prnd) and their implications in human prion diseases, Alzheimer's disease and other neurological disorders

Only 10% of human transmissible spongiform encephalopathies (TSEs) are associated with mutations of the Prnp region encoding the prion protein (PrP). Recently, the murine PrP-like protein doppel (Dpl) was described and was shown to be overexpressed in certain strains of PrP knockout mice and to cause neurological diseases such as ataxia and Purkinje cell loss. To answer the question of whether there are any polymorphisms within the PrP-like protein gene (Prnd) that might cause or be involved in the development of TSEs, we investigated the complete open reading frame of the human Prnd gene from 58 patients who had died of genetic or sporadic Creutzfeldt-Jakob disease (CJD), Alzheimer's disease or other neurological disorders and from 111 controls. We found five new polymorphisms and one frame shift mutation. One silent polymorphism, which does not lead to an altered amino acid sequence, was also observed. Statistical analysis revealed a significant difference in the distribution of the Prnd genotype at codon 174 between sporadic CJD patients and healthy controls.     

Developmental expression of the cellular prion protein (PrP(C) ) in bovine embryos

The mammalian cellular prion protein (PrP(C) ) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc) ), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrP(Sc) conversion and its role in TSEs, the normal function of PrP(C) has not been elucidated. In adult mammals, PrP(C) is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrP(C) is expressed during neurogenesis throughout development, and it has recently been proposed that PrP(C) participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrP(C) during mammalian development, we examined PrP(C) expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrP(C) was detected in the developing central and peripheral nervous systems in Day-27, 32-, and -39 embryos. PrP(C) was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrP(C) cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrP(C) in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis.     

Tau inhibits tubulin oligomerization induced by prion protein

In previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducing tubulin oligomerization. These observations may explain the molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Here, we check whether microtubule associated proteins (MAPs) that regulate microtubule stability, influence the PrP-induced oligomerization of tubulin. We show that tubulin preparations depleted of MAPs are more prone to oligomerization by PrP than those containing traces of MAPs. Tau protein, a major neuronal member of the MAPs family, reduces the effect of PrP. Importantly, phosphorylation of Tau abolishes its ability to affect the PrP-induced oligomerization of tubulin. We propose that the binding of Tau stabilizes tubulin in a conformation less susceptible to oligomerization by PrP. Since elevated phosphorylation of Tau leading to a loss of its function is observed in Alzheimer disease and related tauopathies, our results point at a possible molecular link between these neurodegenerative disorders and TSEs.     

Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

Harnessing prions as test agents for the development of broad-range disinfectants

The development of disinfectants with broad-range efficacy against bacteria, viruses, fungi, protozoa and prions constitutes an ongoing challenge. Prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease (CJD) or its variant (vCJD) rank among the pathogens with the highest resistance to disinfection. Pilot studies have shown that different procedures devised for prion disinfection were also highly effective against microbial pathogens. This fueled the idea to systematically exploit prions as test pathogens for the identification of new potential broad-range disinfectants. Prions essentially consist of misfolded, aggregated prion protein (PrP) and putatively replicate by nucleation-dependent, or seeded PrP polymerization. Recently, we have been able to establish PrP seeding activity as a quantitative in vitro indicator for the disinfection of 263K scrapie prions on steel wires used as surrogates for medical instruments. The seeding activity on wires re-processed in different disinfectants could be (1) biochemically determined by quantitative protein misfolding cyclic amplification (qPMCA), (2) biologically detected after qPMCA in a cell assay and (3) correctly translated into residual titers of scrapie infectivity. Our approach will substantially facilitate the identification of disinfectants with efficacy against prions as promising candidates for a further microbiological validation of broad-range activity.Key words: cell assay, disinfection, prion, prion protein, protein misfolding cyclic amplification, scrapie, seeding activity, surgical instruments, transmissible spongiform encephalopathies     

The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells

The function of the prion protein (PrP(c)), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP(c) on Ca(2+) homeostasis, by analyzing local Ca(2+) fluctuations in cells transfected with PrP(c) and Ca(2+)-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrP(c) sharply increases the Ca(2+) concentration of subplasma membrane Ca(2+) domains, a feature that may explain the impairment of Ca(2+)-dependent neuronal excitability observed in TSEs. PrP(c) also limits Ca(2+) release from the endoplasmic reticulum and Ca(2+) uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP(c) paralogue, display opposite effects, which, however, are abolished by the coexpression of PrP(c). These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP(c) protects, and Doppel sensitizes, cells toward stress conditions.     

Experimental approaches to TSE prevention via inhibition of prion formation

Transmissible spongiform encepahalopathies (TSEs) are fatal diseases that damage the central nervous system. TSEs are unique in that they may be inherited, infectious or spontaneous. The central pathogenic agent is thought to be a conformationally distinct form (PrP(Sc;)) of the endogenous prion protein(PrP(c)), which is high in beta-sheet content and is resistant to proteases; infectivity is thought to involve formation of PrP(Sc) via imprinting of abnormal conformation on the normal form of the protein (PrP(c)) by seeds of PrP(Sc). A number of compounds found to inhibit the conversion of PrP(c) to PrP(Sc) have been proposed as therapeutics to halt TSEs.