prion蛋白和prion病

ｐｒｉｏｎ蛋白是动物神经元细胞合成的通过磷酸肌醇系统锚定在细胞外表面的一种糖蛋白，该基因的突变是引起ｐｒｉｏｎ病（包括ＣＪＤ，ＧＳＳ，库鲁病等）的遗传基础。本文对该基因突变的文献资料进行综述，以引起国内医学界的注意。     

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.     

The cellular prion protein mediates neurotoxic signalling of β-sheet-rich conformers independent of prion replication

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a β-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β-sheet-rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β-peptide, (iii) yeast prion proteins or (iv) designed β-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with β-conformers. Interestingly, a secreted version of N-PrP associated with β-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various β-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.     

α-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

��-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

Copper–zinc cross-modulation in prion protein binding

In this paper we report a systematic XAS study of a set of samples in which Cu(II) was progressively added to complexes in which Zn(II) was bound to the tetra-octarepeat portion of the prion protein. This work extends previous EPR and XAS analysis in which, in contrast, the effect of adding Zn(II) to Cu(II)–tetra-octarepeat complexes was investigated. Detailed structural analysis of the XAS spectra taken at both the Cu and Zn K-edge when the two metals are present at different relative concentrations revealed that Zn(II) and Cu(II) ions compete for binding to the tetra-octarepeat peptide by cross-regulating their relative binding modes. We show that the specific metal–peptide coordination mode depends not only, as expected, on the relative metal concentrations, but also on whether Zn(II) or Cu(II) was first bound to the peptide. In particular, it seems that the Zn(II) binding mode in the absence of Cu(II) is able to promote the formation of small peptide clusters in which triplets of tetra-octarepeats are bridged by pairs of Zn ions. When Cu(II) is added, it starts competing with Zn(II) for binding, disrupting the existing peptide cluster arrangement, despite the fact that Cu(II) is unable to completely displace Zn(II). These results may have a bearing on our understanding of peptide-aggregation processes and, with the delicate cross-regulation balancing we have revealed, seem to suggest the existence of an interesting, finely tuned interplay among metal ions affecting protein binding, capable of providing a mechanism for regulation of metal concentration in cells.     

Naturally prion resistant mammals: A utopia?

Each known abnormal prion protein (PrP^Sc) is considered to have a specific range and therefore the ability to infect some species and not others. Consequently, some species have been assumed to be prion disease resistant as no successful natural or experimental challenge infections have been reported. This assumption suggested that, independent of the virulence of the PrP^Sc strain, normal prion protein (PrP^C) from these ‘resistant’ species could not be induced to misfold. Numerous in vitro and in vivo studies trying to corroborate the unique properties of PrP^Sc have been undertaken. The results presented in the article “Rabbits are not resistant to prion infection” demonstrated that normal rabbit PrP^C, which was considered to be resistant to prion disease, can be misfolded to PrP^Sc and subsequently used to infect and transmit a standard prion disease to leporids. Using the concept of species resistance to prion disease, we will discuss the mistake of attributing species specific prion disease resistance based purely on the absence of natural cases and incomplete in vivo challenges. The BSE epidemic was partially due to an underestimation of species barriers. To repeat this error would be unacceptable, especially if present knowledge and techniques can show a theoretical risk. Now that the myth of prion disease resistance has been refuted it is time to re-evaluate, using the new powerful tools available in modern prion laboratories, whether any other species could be at risk.     

Tunnelling nanotubes: A highway for prion spreading?

The discovery of tunnelling nanotubes (TNTs) and their proposed role in long intercellular transport of organelles, bacteria and viruses have led us to examine their potential role during prion spreading. We have recently shown that these membrane bridges can form between neuronal cells, as well as between dendritic cells and primary neurons and that both endogenous and exogenous PrP^Sc appear to traffic through these structures between infected and non-infected cells. Furthermore, prion infection can be efficiently transmitted from infected dendritic cells to primary neurons only in co-culture conditions permissive for TNT formation. Therefore, we propose a role for TNTs during prion spreading from the periphery to the central nervous system (CNS). Here, we discuss some of the key steps where TNTs might play a role during prion neuroinvasion.Key words: tunnelling nanotubes, TNTs, prion, PrP^Sc, prion spreading, dendritic cells, neuroinvasionPrion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that have been found in a number of species, including scrapie in sheep, bovine spongiform encephalopathy in cattle (BSE), Chronic wasting disease in deer and Creutzfeldt-Jacob, the Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia and kuru in humans (reviewed in ref. ¹). Human TSEs can be sporadic, genetic or acquired by infection. A new variant of Creutzfeldt-Jakob disease (termed vCJD) was reported from the UK in 1996.² The majority of vCJD cases diagnosed to date resulted from a peripheral exposure via the consumption of BSE-contaminated food. Pathological features of TSE diseases can include gliosis, neuronal cell loss and spongiform changes, but the common feature of all members of this group of diseases is the build-up of an aberrant form of the host cellular protein PrP^C, named PrP^Sc (from scrapie). The normal cellular isoform, PrP^C, is an endogenous glycosylphosphatidyl inositol (GPI)-anchored protein present in numerous tissues in mammals, including neurons and lymphoid cells. While the exact function of PrP^C remains unclear, evidence suggest putative roles in neuroprotection, cell adhesion and signal transduction (reviewed in refs. ³ and ⁴). According to the ‘protein-only hypothesis,’ the causative agents of prion diseases are proteinaceous infectious particles (‘prions’), which are composed essentially of misfolded PrP^C, or PrP^Sc.^5,6 Prions replicate through a molecular mechanism in which abnormally folded PrP^Sc acts as a catalyst and serves as a template to convert normal PrP^C molecules into PrP^Sc.^5,6 PrP^Sc differs from PrP^C in the conformation of its polypeptide chain, which is enriched in β-sheets and is protease resistant. Although the conversion process is believed to have a predominant role in the pathogenesis of prion diseases, the cellular and molecular basis for the pathogenic conversion of PrP are still unknown.Another important question is how PrP^Sc spreads to and within the brain. After oral exposure, PrP^Sc accumulates into lymphoid tissues, such as the spleen, lymph nodes or Peyer''s patches, prior to neuroinvasion.^7–9 The exact mechanisms and specific cells involved in the spreading from the gastrointestinal track to the lymphoid system and to the peripheral nervous system (PNS), leading to neuroinvasion of the CNS remain to be elucidated. However, a range of evidence suggests that the accumulation of PrP^Sc within lymphoid tissues is necessary for efficient neuroinvasion.^9–11 In particular it has been shown that PrP^Sc accumulates first within follicular dendritic cells (FDCs)¹² and macrophages.¹³ FDCs are stromal-differentiated cells in the germinal centres of activated lymphoid follicles. A number of studies have demonstrated that FDCs play a critical role during spreading of infection since their absence greatly impaires the neuroinvasion process.^8,11,14,15 However, because FDCs are immobile cells, it is not clear how they acquire PrP^Sc and how it spreads from the FDCs to the PNS. FDCs and nerve synapses occupy different anatomical sites^16,17 and therefore the lack of physical contact between the gut and FDCs and between FDCs and the nerve periphery imply the presence of intermediate mechanisms for the transport of PrP^Sc. Dendritic cells (DCs) have been proposed to play a critical role in the transport of PrP^Sc from the gut to FDCs.¹⁸ DCs function as sentinels for incoming pathogens. Bone-marrow dendritic cells (BMDCs) are migratory cells that are able to transport proteins within Peyer''s patches and into mesenteric lymph nodes.¹⁹ Interestingly, mucosal dendritic cells which play a role in the transport of intestinal antigen for presentation to Peyer''s patches and to mesenteric lymph nodes, can also extend trans-epithelial dendrites to directly sample bacteria in the gut.^20,21 However, the transport of PrP^Sc from FDCs to the PNS remains controversial and evidence for a direct role of DCs during this process has been debated.^22,23 Several mechanisms have been proposed for the intercellular transfer of PrP^Sc, including cell-cell contact, transfer via exosomes or by GPI-painting.^24–26 For example, similar to other types of pathogens such as HIV-1, which was proposed to follow the “exosomal” pathway to be released from the cells,²⁷ it has been shown that the supernatant of prion infected cells contain large amount of PrP^Sc in membranous vesicles known as exosomes.^25,28 Thus, it was suggested that exosomes might be a way to spread prion infection in vivo.^25,28 Recently, a different type of vesicles known as plasma membrane-derived microvesicles, were also described as a potential spreading mechanism during neuroinvasion.²⁹In 2004, Rustom and colleagues discovered a new mechanism of long distance intercellular communication in mammalian cells, called tunnelling nanotubes (TNTs).³⁰ TNTs are transient, long, actin-rich projections that allow for long-distance intercellular communication (reviewed in refs. ^31–33). TNT-like structures have been described to form in vitro between numerous cell types, including neuronal and immune cells.^30,34,35 These studies demonstrated that TNT-like structures formed bridges or channels between distant cells that can be used to transfer material between cells, including Lysotracker positive or endosomal vesicles, calcium fluxes, bacteria or viruses through their cytoplasms or along the surface of the nanotubes.^31–33 Interestingly, a model GPI-anchored protein, GFP-GPI, was found to move at the surface of these tubes³⁴ and while studying the neuritic transport of prions in neuronal cells, Magalhães and colleagues noticed a strong correlation between internalized PrP-res and Lysotracker positive vesicles in neurites,³⁶ suggesting that PrP-res might also be able to transfer through TNTs during prion cell-cell spreading.The results from the studies mentioned above and random observations of TNT-like structures in neuronal model cell cultures first led us to study whether these structures could in fact provide an efficient mechanism for prion cell-cell spreading.³⁷ We initially characterized TNT-like structures in the mouse catecholaminergic neuronal cell line, Cath.a-Differentiated cells (CAD cells) a well-recognized neuronal cell model for prion infection.³⁸ Under our culturing conditions, over 40% of the CAD cells could efficiently form actin-rich TNT-like structures between differentially labelled cell populations. In CAD cells, these nanotubes were very heterogeneous, both in length and in diameters. Indeed, TNT-like structures had lengths ranging from 10 to 80 µm and while over 70% of the nanotubes had diameters smaller than 200 nm, the remaining TNT-like structures had larger diameters (200 to 800 nm). We demonstrated that vesicles of lysosomal origins, a fluorescent form of PrP (GFP-PrP), infectious Alexa-PrP^Sc, as well as both endogenous and exogenous PrP^Sc could traffic within TNTs between neuronal cells (Fig. 1). The lysosomal and GFP-PrP vesicles observed to move through TNTs had a directed movement with a speed in the range of actin-mediated motors,³⁷ consistent with previous studies suggesting the involvement of an actomyosin-dependent transport.³⁹ Interestingly, active transfer of endogenous PrP^Sc, lysosomal or GFP-PrP vesicles occurred through TNTs with larger diameters, suggesting distinct roles for the different TNT-like structures observed.³⁷ These results do not seem to be specific to CAD cells since the transfer of GFP-PrP throught TNTs was observed in different types of transfected cells, including HEK293 cells (unpublished data). Furthermore, these results were in agreement with previous observations by Onfelt and colleagues showing the presence of a fluorescent GPI model protein (GFP-GPI) in TNTs formed between EBV-transformed human B cells³⁴ suggesting that different GPI-anchored proteins can be transferred along the surface and inside vesicles within TNTs. In order to determine the relevance of this type of intercellular communication in the case of prion diseases, it was necessary to evaluate the trafficking of the pathological form of PrP (PrP^Sc) within TNTs, by analyzing the transfer of endogenous PrP^Sc between chronically infected ScCAD cells and non-infected CAD cells. By immunofluorescence after guanidium treatment, endogenous PrP^Sc was found inside TNTs and in the cytoplasm of recipient non-infected CAD cells. Similar to exogenous PrP^Sc, endogenous PrP^Sc particles were not present in non-infected CAD cells not in contact with ScCAD cells after overnight co-cultures, thus excluding exosomal transfer or protein shedding.³⁷ Similarly, no transfer was observed between cells in direct contact with one another or upon treatment with latrunculin, which inhibits TNT formation. Strikingly, the transfer of endogenous PrP^Sc was visible only when TNTs were present, demonstrating that in vitro, PrP^Sc can efficiently exploit TNTs to spread between cells of neuronal origin. These data suggested that TNTs could be a mechanism for prion spreading within the cells of the CNS.Open in a separate windowFigure 1Endogenous PrP^Sc transfer from ScCAD cells to CAD cells via TNTs. Endogenous PrP^Sc is found in punctate structures inside TNTs and in the cytoplasms of recipient cells. CAD cells were transfected with Cherry-PLAP (red) and co-cultured with ScCAD for 24 h. Cells were fixed, treated with Gnd and immunostained for PrP using SAF32 Ab (green). (A) Merge projection of Z-stacks obtained with a confocal Andor spinning-disk microscope. (B) Three-dimensional reconstruction of (A) using OsiriX software. (C) Zoom in on TNT-like structures. PrP^Sc is found in vesicular structures inside TNTs and in the cytoplasm of the recipient non-infected CAD cells (see blue arrow heads). Scale bar represents 10 µm.Interestingly, DCs were shown to form networks of TNTs both in vitro⁴⁰ and in vivo.⁴¹ In an elegant study, Watkins and Salter demonstrated that DCs could propagate calcium flux upon cell stimulation to other cells hundreds of microns away through TNTs, both between DCs and between DCs and THP-1 monocytes.⁴⁰ These data suggested the possibility that DCs could form tubular connections with neuronal cells in order to transport PrP^Sc to the PNS via TNTs. Using BMDCs in co-cultures with both cerebellar granular neurons (CGNs) and primary hippocampal neurons, we showed that BMDCs could form networks of TNTs with both types of neurons. Furthermore, these TNTs appeared to be functional, allowing for the transport of Lysotracker positive vesicles and infectious Alexa-PrP^Sc between loaded BMDCs and primary neurons, suggesting that DCs could transfer the infectious prion agent to primary neuronal cultures through TNTs. By using filters and conditions unfavorable for other mechanisms of transport, we found that moRK13 cells,²⁸ as well as CGNs (unpublished data), could be infected by co-cultures with BMDCs loaded with infectious brain homogenate.³⁷ Overall, these data indicate that TNTs could be an efficient mechanism of prion transmission between immune cells and neuronal cells, as well as between neuronal cultures. Since DCs can interact with peripheral neurons,⁴² we propose that TNTs could be involved in the process of neuroinvasion at multiple stages, from the peripheral site of entry to the PNS by neuroimmune interactions with DCs, allowing neurons to retrogradely transport prions to the CNS, and within the CNS (Fig. 2).Open in a separate windowFigure 2Transport of PrP^Sc via TNTs, an alternative spreading mechanism during neuroinvasion. Studies in our laboratory suggest that TNTs allow for the intracellular transport of PrP^Sc between dendritic cells and neurons and between neurons (see inset). The exact mechanism of transport remains to be determined. For instance, it is still not clear, whether PrP^Sc is strictly transported within endocytic vesicles, or whether it can slide along the surface or be transported as aggregosomes within the tubes. Similarly, the types of motors used, as well as the possible gated mechanisms to enter the recipient cells are not known. Because of the high propensity of DCs to form TNTs with different cell types, we propose that TNTs could play important roles in delivering PrP^Sc to the proper cell types along the neuroinvasion route. For instance, DCs could deliver PrP^Sc from the peripheral entry sites to FDCs in the secondary lymphoid tissues (2) or in a less efficient manner, they might occasionally directly transport PrP^Sc to the PNS (1). They could also bridge the immobile FDC networks and the PNS (3), since we have shown that DCs can form TNTs with nerve cells. Finally, once PrP^Sc has reached its final destination within the CNS, TNTs might play a final role in the spreading of PrP^Sc within the brain between neurons and possibly between neuronal cells and astrocytes (4).Recently, it was demonstrated that the distance between FDCs and the neighbouring PNS was critical for prion neuroinvasion.⁴³ Indeed, in the spleen of CD19^−/− mice, FDC networks were found to be 50% closer to the nerve fibers compared to wild-type mice.⁴³ The authors suggested that the increase in prion spreading efficiency in these mice was directly dependent on the reduction in the distance between the FDC networks and the PNS in these mice. These results would be consistent with a mechanism of transfer such as exosomes release. However, shortening the distance between FDCs and the PNS would also reduce the route of transport that mobile cells would have to travel and increase the chances for transfer of prions to the PNS, resulting in an increase in prion spreading efficiency. While the importance of FDCs in prion replication during the spreading to the CNS seems to be clear,^11,14,15 their specific role in the transfer of prions and their possible interactions with other mobile cells are much more debated.^22,23 In order to bridge the gap between FDCs and the PNS, a role for DCs as possible carriers of PrP^Sc has been postulated. Aucouturier and colleagues have previously shown, using RAG-1^−/− mice, which are deficient in FDCs, that CD11c⁺ DCs infected with 139A were able to carry prion infection to the CNS, without accumulation and replication in lymphoid organs,²² thus suggesting that DCs are able to transport prions from the periphery to nerve cells. Recently, however, another study using TNFR1^−/− mice, deficient for FDCs, suggested that DCs were unlikely candidates in the transport of prion to the PNS.²³ In this study, the authors showed that in TNFR1^−/− mice, ME7 or 139A infected DCs were inefficient in transferring infection to the PNS. The authors suggested that the differences between the results obtained with RAG-1^−/− mice and TNFR1^−/− mice could be due to the differences in the levels of innervation of the spleen in RAG-1^−/−mice compared to TNFR1^−/− mice. They suggested that in RAG-1^−/− mice, DCs could spread prion infection because their spleens are highly innervated, compared to TNFR1^−/− or wild-type mice, therefore increasing the propensity of DCs to encounter nerve cells and tranfer the prion agents. Because of the reduction in the levels of innervation in the spleens of wild-type mice, the authors concluded that DCs are unlikely candidate for the transport of prions directly to the PNS [see (1) in Fig. 2]. However, since these studies are using mice deficient for FDCs, it remains unclear what type of interactions might occur between FDCs and DCs, and how DCs might be able to transport prions from FDCs to the PNS in wild-type mice [see (2) in Fig. 2]. Indeed, both studies show that under the right circumstances, DCs can interact with nerve cells, similar to what was recently shown in infected mice⁴² and in agreement with our findings that DCs can form TNTs with neurons.³⁷ Within this scenario, it is clear that to determine the specific role of DCs during the spreading of prions from the gut to the PNS, the transfer mechanisms between DCs and other cell types, especially FDCs and peripheral neurons, need to be better characterized.Overall, these in vitro data strongly point toward TNTs as one possible mechanism of prion spreading. The next step will be to identify these structures in vivo and to determine whether prion spreading in vivo is the result of passive mechanisms, such as exosome release, active intercellular transport along and within TNTs or whether prions will use any means available to reach their targets. Recently, TNT-like structures were imaged in a mouse cornea,⁴¹ suggesting that while challenging, the visualization of in vivo trafficking of prions in lymphoid tissues such as in lymph nodes or in the spleen as well as in brain organotypic cultures might be possible and could be used to reveal the presence of TNTs.The discovery of the existence of nanotubular membrane bridges in vitro has opened-up a new field of research. Channels, called plasmodesmata,⁴⁴ connecting plant cells have long been known to play crucial roles in the transport of nutrients, molecules and signals during development and some of their functions were recently compared with some of the recently proposed functions of TNTs.⁴⁵ Furthermore, in vivo long, actin-rich filopodia like structures were found to be crucial during development.^46–49 For example, these structures exist in developing sea urchin embryos and were proposed to play a role in signalling and patterning during gastrulation.⁴⁷ Similar roles were proposed for thin filopodia-like structures observed during dorsal closure in drosophila.⁴⁹ In addition, TNT-like structures were observed in the mouse cornea between DCs and were shown to increase under inflammatory conditions.⁴¹ The authors postulated that these TNT-like structures could play a role in Ag-specific signalling, especially as a response to eye inflammation. Therefore, the possibility that TNTs might play numerous roles during cell development, in the immune system and as conduits for the spreading of pathogens could lead to major changes in the way we view animal cell interactions. Specifically, understanding how pathogens usurp these cellular connections to spread could allow for the screening and the identification of new therapeutic inhibitors. To this aim, characterizing the basic mechanism of TNT formation within cell model systems will be necessary to improve the knowledge of TNTs in general, to analyze the transfer of pathogens more specifically, and to identify key molecules during this process. In the case of prions, whether they hijack nanotubes to spread between cells or whether prions increase the formation of filopodia and TNT-like structures similar to some viruses^33,50 and/or the efficiency of transfer remain to be determined. Overall, in this specific field, the constant improvement of cell imaging techniques and the emergence of imaging tools to study prion spreading^{36,37,51–53} could lead to exciting new insights both in the physiology of these intercellular connections and in the pathology of these devastating diseases.     

Left handed �� helix models for mammalian prion fibrils

We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a β-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn β-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal β helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease.Key words: prion, amyloid fibril, domain swap, beta helix, computational biology     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Are Brazilian cervids at risk of prion diseases?

Prion diseases are neurodegenerative fatal disorders that affect human and non-human mammals. Chronic Wasting Disease (CWD) is a prion disease of cervids regarded as a public health problem in North America, and polymorphisms at specific codons in the PRNP gene are associated with this disease. To assess the potential CWD susceptibility of South American free-ranging deer, the presence of these polymorphisms was examined in Mazama gouazoubira, Ozotoceros bezoarticus and Blastocerus dichotomus. Despite the lack of CWD reports in Brazil, the examined codons (95, 96, 116, 132, 225, and 226) of the PRNP gene showed potential CWD susceptibility in Brazilian deer. Low abundancy of deer in Brazil possibly difficult both CWD proliferation and detection, however, CWD surveillance may not be neglected.     

Is tau ready for admission to the prion club?

Aggregation-prone proteins associated with neurodegenerative disease, such as α synuclein and β amyloid, now appear to share key prion-like features with mammalian prion protein, such as the ability to recruit normal proteins to aggregates and to translocate between neurons. These features may shed light on the genesis of stereotyped lesion development patterns in conditions such as Alzheimer disease and Lewy Body dementia. We discuss the qualifications of tau protein as a possible “prionoid” mediator of lesion spread based on recent characterizations of the secretion, uptake and transneuronal transfer of human tau isoforms in a variety of tauopathy models, and in human patients. In particular, we consider (1) the possibility that prionoid behavior of misprocessed tau in neurodegenerative disease may involve other aggregation-prone proteins, including PrP itself, and (2) whether “prionlike” tau lesion propagation might include mechanisms other than protein-protein templating.     

Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its �� isoform with high affinity

In order to obtain RNA aptamers against bovine prion protein (bPrP), we carried out in vitro selection from RNA pools containing a 55-nucleotide randomized region to target recombinant bPrP. Most of obtained aptamers contained conserved GGA tandem repeats (GGA)₄ and aptamer #1 (apt #1) showed a high affinity for both bPrP and its β isoform (bPrP-β). The sequence of apt #1 suggested that it would have a G-quadruplex structure, which was confirmed using CD spectra in titration with KCl. A mutagenic study of this conserved region, and competitive assays, showed that the conserved (GGA)₄ sequence is important for specific binding to bPrP and bPrP-β. Following 5′-biotinylation, aptamer #1 specifically detected PrP^c in bovine brain homogenate in a Northwestern blotting assay. Protein deletion mutant analysis showed that the bPrP aptamer binds within 25–131 of the bPrP sequence. Interestingly, the minimized aptamer #1 (17 nt) showed greater binding to bPrP and bPrP-β as compared to apt #1. This minimized form of aptamer #1 may therefore be useful in the detection of bPrP, diagnosis of prion disease, enrichment of bPr and ultimately in gaining a better understanding of prion diseases.Key words: RNA aptamer, prion protein, SELEX, GGA repeat, G-quadruplex     

Cross interaction of β-amyloid peptide and prion protein fragments

Summary This article presents kinetic studies of cross interaction of β-amyloid peptide and prion protein fragments. Syntheses of three peptides (β25-35, β22-35 and PrP 109–126) were performed. Those peptides were used for aggregation studies in PBS and TRIS buffers using HPLC with DAD detector. Comparison of aggregation of peptides alone and in combination with other fragments was investigated. In all cases aggregation was faster in PBS than in TRIS solution. Obtained results suggest that β-amyloid peptide and prion protein may interact to form macromolecular complexes with different ability for aggregation.