Curcumin binds to the alpha-helical intermediate and to the amyloid form of prion protein - a new mechanism for the inhibition of PrP(Sc) accumulation

Conversion of the native, predominantly α-helical conformation of prion protein (PrP) into the β-stranded conformation is characteristic for the transmissible spongiform encephalopathies such as Creutzfeld–Jakob disease. Curcumin, an extended planar molecule and a dietary polyphenol, inhibits in vitro conversion of PrP and formation of protease resistant PrP in neuroblastoma cell lines. Curcumin recognizes the converted β-form of the PrP both as oligomers and fibrils but not the native form. Curcumin binds to the prion fibrils in the left-handed chiral arrangement as determined by circular dichroism. We show that curcumin labels the plaques of the brain sections of variant Creutzfeld–Jakob disease cases and stains the same structures as antibodies against the PrP. In contrast to thioflavin T, curcumin also binds to the α-helical intermediate of PrP present at acidic pH at stoichiometry of 1 : 1. Congo red competes with curcumin for binding to the α-intermediate as well as to the β-form of PrP but is toxic and binds also to the native form of PrP. We therefore show that the partially unfolded structural intermediate of the PrP can be targeted by non-toxic compound of natural origin.     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.     

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

ABSTRACT

A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP^Sc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP^Sc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP^Sc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP^Sc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.     

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.     

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

Abstract

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.     

In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features

The "protein only" hypothesis of prion propagation postulates that the abnormal isoform of the prion protein, PrP(Sc), acts as a causative and transmissible agent of prion disease. In attempt to reconstitute prion infectivity in vitro, we previously developed a cell-free conversion protocol for generating amyloid fibrils from a recombinant prion protein encompassing residues 89-231 (rPrP 89-230) [Baskakov et al. (2002) J. Biol. Chem. 277, 21140]. When inoculated into transgenic mice, these amyloid fibrils induced prion disease, which can be efficiently transmitted to both wild-type and transgenic mice [Legname et al. (2004) Science 305, 673]. Here we show that the polymerization of rPrPs into the fibrils displays a number of distinctive kinetic features that are not typical for polymerization by other amyloidogenic polypeptides. Specifically, the lag phase of polymerization showed only modest dependence on protein concentration, and the conversion reaction displayed a dramatic volume-dependent threshold effect. To explain these unique kinetic features, we proposed that the conversion reaction is regulated by the dynamics between the rates of multiplication and deactivation of self-propagating fibrillar isoforms. Our further studies demonstrated that surface-dependent sorption of fibrillar isoforms is responsible for their deactivation in vitro, while fibril fragmentation seems to account for the multiplication of the active centers of polymerization. Our findings support the hypothesis that development of prion disease is controlled by a fine dynamic balance between self-propagation and clearance/deactivation of PrP(Sc).     

Nucleic acid and prion protein interaction produces spherical amyloids which can function in vivo as coats of spongiform encephalopathy agent

The infectious agent of transmissible spongiform encephalopathies (TSE) has been considered to be PrP(SC), a structural isoform of cellular prion protein PrP(C). PrP(SC) can exist as oligomers and/or as amyloid polymers. Nucleic acids induce structural conversion of recombinant prion protein PrP and PrP(C) to PrP(SC) form in solution and in vitro. Here, we report that nucleic acids, by interacting with PrP in solution, produce amyloid fibril and fibres of different morphologies, similar to those identified in the diseased brains. In addition, the same interaction produces polymer lattices and spherical amyloids of different dimensions (15-150 nm in diameters). The polymer lattices show apparent morphological similarity to the two-dimensional amyloid crystals obtained from linear amyloids isolated in vivo. The spherical amyloids structurally resemble "spherical particles" observed in natural spongiform encephalopathy (SE) and in scrapie-infected brains (TSE). We suggest that spherical amyloids, PrP(SC)-amylospheroids, are probable constituents of the coat of the spherical particles found in vivo and the latter can act as protective coats of the SE and TSE agents in vivo.     

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.     

Cloning and expression of a prion protein (PrP) gene from Korean bovine (Bos taurus coreanae) and production of rabbit anti-bovine PrP antibody

A PrP gene, from a Korean bovine, exhibiting a nonsense and a missense polymorphism respectively at nucleotides 576 and 652 has been cloned. The latter resulted in Glu to Lys substitution at amino acid residue 218. After expression and purification of the recombinant bovine PrP (recBoPrP) with Glu218Lys substitution, a polyclonal antibody against this protein was raised. ELISA and Western blot analysis suggested that the recBoPrP obtained in this study had a unique conformation not presented in native PrP(C), and the polyclonal antibody recognized PrP in a conformation dependent manner. These reagents will be valuable tools for studying PrP conformation.     

Reversible aggregation of mouse prion protein derivatives with PrPSC-like structural properties

Three carbamylated derivatives of reduced mouse prion protein (mPrP) were isolated during the aborted oxidative folding in the presence of urea. These three prion protein derivatives (mPrP-a, mPrP-b, and mPrP-c) exist as monomer in the acidic solution (pH < 2.0) and exhibit prevalent random coil structure. However, they undergo rapid aggregation and transformation to a predominant -sheet structure upon exposure to ionic buffer with pH greater than 3.0. The stability of aggregates of mPrP conformers is in part dependent upon the time that they were allowed to develop. The nascent aggregates comprise a significant fraction of loosely packed mPrP monomers that can be dissociated by treatment with strong acidic solution. Matured aggregates acquired through prolonged sample incubation contain more tightly packed mPrP monomers that cannot be dissociated by strong acid but can be disaggregated by denaturant. The properties of reversible aggregation of mPrP-a, mPrP-b, and mPrP-c bear a striking resemblance to that observed with aggregates of hamster PrP^SC.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP(Sc) formation

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.     

Mouse prion protein (PrP) segment 100 to 104 regulates conversion of PrP(C) to PrP(Sc) in prion-infected neuroblastoma cells

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).     

Efficient inhibition of prion replication by PrP-Fc(2) suggests that the prion is a PrP(Sc) oligomer

Soluble dimeric prion protein (PrP-Fc(2)) binds to the disease-associated prion protein PrP(Sc), and inhibits prion replication when expressed in transgenic mice. Prion inhibition is effective even if PrP-Fc(2) is expressed at low levels, suggesting that its affinity for PrP(Sc) is higher than that of monomeric PrP(C). Here, we model prion accumulation as an exponential replication cycle of prion elongation and breakage. The exponential growth rate corresponding to this cycle is reflected in the incubation period of the disease. We use a mathematical model to calculate the exponential growth rate, and fit the model to in vivo data on prion incubation times corresponding to different levels of PrP(C) and PrP-Fc(2). We find an excellent fit of the model to the data. Surprisingly, targeting of PrP(Sc) can be effective at concentrations of PrP-Fc(2) lower than that of PrP(C), even if PrP-Fc(2) and PrP(C) have the same affinity for PrP(Sc). The best fit of our model to data predicts that the replicative prion consists of PrP(Sc) oligomers with a mean size of four to 15 units.     

Semiautomated cell-free conversion of prion protein: applications for high-throughput screening of potential antiprion drugs

Transmissible spongiform encephalitis (TSE) is a lethal illness with no known treatment. Conversion of the cellular prion protein (PrP(C)) into the infectious isoform (PrP(Sc)) is believed to be the central event in the development of this disease. Recombinant PrP (rPrP) protein folded into the amyloid conformation was shown to cause the transmissible form of prion disease in transgenic mice and can be used as a surrogate model for PrP(Sc). Here, we introduced a semiautomated assay of in vitro conversion of rPrP protein to the amyloid conformation. We have examined the effect of known inhibitors of prion propagation on this conversion and found good correlation between their activity in this assay and that in other in vitro assays. We thus propose that the conversion of rPrP to the amyloid isoform can serve as a high-throughput screen for possible inhibitors of PrP(Sc) formation and potential anti-TSE drugs.