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

Methods 53 (2011) 306–317

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Methods journal homepage: www.elsevier.com/locate/ymeth

Review Article

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion Jerson L. Silva a,⇑, Tuane C.R.G. Vieira a, Mariana P.B. Gomes a, Luciana P. Rangel a, Sandra M.N. Scapin a,b, Yraima Cordeiro c a b c

Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brazil DIPRO, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial, Brazil Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil

a r t i c l e

i n f o

Article history: Available online 8 December 2010 Keywords: Prion protein PrP Nucleic acids Glycosaminoglycans Transmissible spongiform Encephalopathy Neurodegenerative diseases PrP partners PrP structure PrP conversion

a b s t r a c t 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 (PrPC) into a misfolded, b-sheet-rich isoform (PrPSc) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrPSc, 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 PrPC 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 b-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 PrPC and PrPSc 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. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Prion diseases are classified as transmissible spongiform encephalopathies (TSEs) and have either a genetic, infectious or sporadic nature [1]. In general, these diseases are characterized by protein aggregation and neurodegeneration. They can be found in several species, such as humans (kuru and Creutzfeldt–Jakob disease), sheep (scrapie) and cattle (bovine spongiform encephalopathy, also known as ‘‘mad cow disease’’), among others [1]. Although rare, these diseases are inevitably fatal and include the symptoms of paralysis, ataxia and dementia [2]. Curiously, rabbits are the only species known to be resistant to infection with prions isolated from other species [3].

⇑ Corresponding author. Address: Universidade Federal do Rio de Janeiro, Instituto de Bioquímica, Bloco E Sala 10, Cidade Universitária, 21941-590 Rio de Janeiro, Brazil. Fax: +55 21 33814155. E-mail address: [email protected] (J.L. Silva). 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.12.002

The term ‘‘prion’’ was created as a contraction of ‘‘proteinaceous infectious particle’’, which lacks genomic nucleic acids [4]. This particle alone would be responsible for the transmission of the disease in the protein-only hypothesis [5,6]. However, several groups have suggested that a cofactor might initiate or modulate the conversion of PrPC to PrPSc [7–10]. Such a molecule would chaperone prion activity by converting PrPC into the disease-causing isoform. Among the chaperone candidates, nucleic acids or glycosaminoglycans are the most probable [10–12]. This cofactor would lower the free-energy barrier that prevents conversion between PrPC and PrPSc, thereby triggering the formation of PrPSc (reviewed in [7]). The prion particle is believed to be basically composed of an altered isoform of the cellular prion protein (PrPC), designated scrapie PrP (PrPSc). PrPC is normally found in cells and is related to many different cellular processes (for a review, see [13]), although a specific function has not yet been assigned to this protein. The expression of PrPC is required for the susceptibility to TSEs because knockout animals are unable to acquire the infection [14].

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PrPC is encoded by only one exon of the single-copy PRNP gene, which is located on human chromosome 20 [15]. The mature PrPC is composed of 208–209 amino acids and is attached to the outer leaflet of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor [6] in a non-, mono- or di-glycosylated form [6]. A signal peptide of 22 amino acids is cleaved from the N-terminal region, which has a flexible, random coil sequence of about 100 amino acids. This region also contains four repeats of a sequence of eight amino acids (PHGGGWGQ), named the octapeptide or octarepeat domain, which is related to copper binding [16]. This region is also related to the binding of glycosaminoglycans and nucleic acids, especially RNA [17–19]. The C-terminal region is globular, with three alpha helices at positions 144–154, 173–194 and 200– 228. A disulfide bond is formed between cysteine residues 179 and 214 [20]. In a general way, the conversion of PrPC to its altered isoform, PrPSc, leads to a refolding of alpha-helical and coil structures into a beta sheet [21,22]. These structural changes confer different physicochemical characteristics to PrPSc, such as insolubility in denaturing detergents and partial resistance to digestion by proteinases [15]. The tendency to aggregation of this isoform is related to its insolubility, and protease-resistant aggregates accumulate in the brain [6], which is one of the features of TSEs. Unlike PrPC, the three-dimensional structure of PrPSc has not yet been completely elucidated due to the heterogeneity of aggregates and the impossibility of purifying it in a soluble form. The increased beta-sheet content in PrPSc compared to PrPC has been detected through techniques such as Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichroism (CD) [21,22]; thus, a cross-beta-sheet conformation was proposed for scrapie prion rods [23]. Additionally, the overall structural organizations of fibrils from three different species (mouse, bovine and elk) have been shown to be very similar through hydrogen/deuterium exchange mass spectrometry. Moreover, two regions (24–98) and (182–212) have been observed to be highly protected, and thus, the regions between them, with a higher solvent accessibility, have been proposed to be involved in the formation of the fibrillar interface [24]. Selective antibodies recognizing epitopes in this region have been described to inhibit the formation of PrPSc and even to clear PrPSc in a cell culture model [25]. Here, we review the experimental approaches to understand the mechanism of prion conversion. We emphasize recent studies that demonstrate the participation of polyanions, especially nucleic acids and glycosaminoglycans. Biochemical, structural biology, proteomic and cell biology methodologies have been applied to identify the macromolecular partners of the prion protein conversion, which is directly related to development of TSEs.

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acids during PrP conversion [11]. We found that recombinant prion protein could bind small double-stranded DNAs with high affinities and that the PrP:nucleic acid complex could act as a catalyst, increasing the aggregation rate (Fig. 1) [11]. A nucleic acid would help to overcome the activation barriers of energy and volume changes that separate the folded from the misfolded species [31,32]. More recently, Kim et al. [33] demonstrated that the conversion of rPrP might not require any mammalian cofactor as long as a template (in this case, hamster PrPSc proteinase K-resistant core, PrP 27–30) is present. Although it has been shown that, in the presence of a template, cofactors are not necessary for the conversion, it is still unclear whether cofactors are needed for the spontaneous generation of an aberrant form of the prion protein. Another interesting feature that is not yet explained is the existence of different prion strains, which can be distinguished by different disease incubation times and clinical signs in vivo [34] and by different biochemical and immunohistological characteristics in vitro [35,36]. These many strains are related to different conformers of PrP that can be propagated. Moreover, prion strains have been shown to ‘‘accumulate’’ mutants, under selective pressure [37], with ‘‘cell-adapted’’ prions outcompeting ‘‘brainadapted’’ ones when inoculated in brains and vice versa. As mentioned before, PrPC is normally located at the outer leaflet of the cellular membrane through a GPI anchor. Despite this fact, other forms of the prion protein are present in different cell compartments. For some neurons, PrP localizes predominantly to the cytosol [38]. Transmembrane forms have also been detected in the endoplasmic reticulum (ER) membrane, and this interaction occurs through the central hydrophobic sequence [39–41]. Both the N- and C-termini can be exposed to the cytosol. These PrP forms can be found in cells comprising up to 10% of the total PrPC cellular amount. Higher levels are usually associated with TSEs [42]. It is well-established that the prion protein interacts with many different partners, and that these interactions might be related to the physiological or pathological roles of PrP. A superoxide-dismutase activity was proposed to be related to the copper-binding features of the octarepeat region in the N-terminal portion of PrPC, and the prion protein has been suggested to be involved in the homeostasis of copper ions and in the protection against oxidative stress [43]. Its partners also include glycosaminoglycans [19], nucleic acids [11,44,45] and many proteins, such as the extracellular proteins laminin and laminin receptor and even the intracellular proteins Bcl-2 and tubulin, among others (reviewed in [13]).

3. Prion protein macromolecular partners 3.1. Interaction with nucleic acids

2. Methods used in the study of the PrPC-to-PrPSc conversion The mechanism for this conversion is still unclear, but it is known that the amplification of PrPSc in a cell-free assay, called Protein Misfolding Cyclic Amplification (PMCA) [26], occurs through the incubation of PrPC with an excess of PrPSc, either from brain homogenates or from a purified source [8]. It was recently described that recombinant PrP (rPrP) could be converted to the pathogenic isoform using a protocol that involved the utilization of purified lipids and mouse liver RNA [27]. The conversion was confirmed by the observation of disease after the inoculation of these samples into mice [27]. The interaction of prion protein with nucleic acids has been characterized by different methodologies [9,11,17,28,29]. Initially, the effects of nucleic acids on polymerizing recombinant murine prion protein (rPrP) were elegantly demonstrated by Nandi’s group [30]. In 2001, our group provided the first experimental demonstration of the catalytic action of nucleic

The nucleic acids (NAs) DNA and RNA form an interesting group of PrP molecular partners and have been extensively studied by us and other groups. It has been quite a challenge to demonstrate that PrP-NA interactions, which were initially observed in vitro [11,44], are physiologically relevant. Nucleic acids were first considered to be possible PrP molecular partners by C. Weissmann, when he suggested the potential implication of a nucleic acid molecule in prion propagation [46]. Since then, a great amount of experimental evidence has been produced in order to understand the implications of those interactions (reviewed in [7,18]). Both DNA and RNA can interact with PrP [11,17,30,47,48]. Upon NA binding, PrP can form a broad variety of misfolded species with different structural and biochemical profiles (for review see [18]) (Fig. 1). Some of the aggregated species that formed upon the complexation of PrP with RNA were reported to be toxic for cultured cells (Fig. 2) and experimental animals [8,17,27]. An rPrP mutant

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Fig. 1. Evaluation of the effects of nucleic acid binding to recombinant PrP. (A) The rPrP-DNA complex increases the aggregation of the PrP (109–149) peptide but does not induce the full-length rPrP polymerization. Left, addition of increasing concentrations of rPrP(23–231) to a solution of 25 nM 18 bp-DNA labeled with rhodamine at the 30 end at pH 5.0. Right, light scattering values when PrP(109–149) (open squares) or rPrP(23–231) (filled squares) was added to the rPrP-DNA complex (adapted from Ref. [11]). (B) Scheme for the free energy diagram. Unfolding of the cellular prion protein (PrPC, in green) and its subsequent misfolding to the scrapie isoform (PrPSc, in red) has been implicated in transmissible spongiform encephalopathies. The conformational transition is separated by a large energetic barrier that is associated with unfolding and oligomerization. For these reason, it seems likely that other molecules, such as nucleic acids (purple triangle) are crucial for prion propagation acting as adjuvants by lowering the energy barrier (Adapted from Ref. [18]).

lacking residues 23–90 was not able to bind RNA, demonstrating that the amino-terminal region is crucial for RNA interactions [17,49]. Highly structured RNA molecules that bind human rPrP with high affinity were characterized by Adler and collaborators [50]. Previously, Gabus and collaborators demonstrated that the prion protein formed structures that behave similar to retroviral proteins that interact with viral RNA, therefore possessing RNAbinding chaperone characteristics [51]. We found that full-length rPrP aggregates upon RNA binding and immediately loses most of its alpha-helical content. NMR measurements with a synthetic RNA sequence (SAF9343–59) showed that the soluble portion of PrP recovered most of its original fold, but with distinct changes in the NMR HSQC spectrum [17]. The aggregates derived from the interaction of PrP with RNA extracted from neuroblastoma cells were highly cytotoxic [17] (Fig. 2). In contrast, complexes formed with synthetic RNAs were not toxic. Contradictorily, some DNA and RNA molecules also seem to prevent or stop PrP aggregation and can bind with high affinities to different prion species [52–56]. This capacity has been explored in possible therapeutic and diagnostic strategies for prion diseases, as we will discuss later in this review. Aside from disease-associated interactions, nucleic acids may be the clue to the still unknown physiological function of PrPC.

Interactions between PrP and NAs can alter both the nucleic acid and the prion protein structures (for review see [18]). Moreover, PrP shares similarities with nucleic acid binding proteins [51,57] and has a mapped DNA-binding region on its globular domain [47,58]. It has also been found through a microarray assay that PrP interacts with nucleic acid binding proteins [59]. Therefore, aside from the implication of nucleic acids in prion pathology, several findings point to a physiological role for PrP as an NA-binding protein. In the last decade, a large number of studies on non-coding RNAs (ncRNA) have found that ncRNAs can act in post-translational regulation and cell morphogenesis, with crucial participation in embryonic and neural development [60]. The interactions of PrP with nucleic acids that have potential NA chaperone activities elicit the possibility that PrP might have some effects on ncRNA processing. In fact, recent studies demonstrated that cytoplasmic PrP induced large ribonucleoprotein particles, which had curious similarities to the chromatoid body, an RNA granule that appears to function in post-transcriptional gene regulation [61]. 3.2. Interaction with glycosaminoglycans Glycosaminoglycans (GAGs) are the main group of sulfated polysaccharides that are found in animal tissues. These molecules

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Fig. 2. RNA effects on PrP. Interaction with RNA molecules occurs through the N-terminal unstructured domain of rPrP leading to aggregation and loss of secondary structure. Aggregates formed upon RNA binding have different characteristics, depending on RNA origin. Some of these aggregates are also toxic to cultured cells The graph A shows secondary structure content of rPrP constructions in the absence (solid lines) or the presence of N2aRNA (dashed lines) measured by Circular Dichroism (CD). rPrP23–231 (black); rPrPD32–121 (blue); and rPrPD51–90 (red). The inset represents the CD spectrum of free N2aRNA free in solution. rPrP (N-terminal) deletion mutants do not seem to interact with RNA leading to the conclusion that RNA-induced aggregation of PrP depends on the N-terminal region of this protein, indicating that at least part of the 51 to 90 PrP region is responsible for this process. The graph B represents statistics of cell viability measured by Live/Dead assay. For simplicity, results are expressed as the percentage of living cells relative to the value for control cells (cells in culture medium only). The aggregates formed upon PrP incubation with total RNA extract from N2a cells (N2aRNA) were significantly cytotoxic, leading to 60 % loss in cell viability. In contrast, significant toxicity was not observed when rPrP aggregates were induced by small synthetic oligonucleotides (SAF9343–59). Pannels C and D represent aggregates of rPrP23–231 formed upon incubation with N2aRNA (C) or SAF9343–59 (D) observed by Transmission Electronic Microscopy. Scale bars: 0.2 lM. (Adapted from Ref. [17]).

have a negative charge due to the presence of carboxylic acid and sulfate residues [62]. GAGs, mainly heparan sulfate (HS), are the focus of many prion disease studies. Snow et al. [63] were the first to demonstrate the presence of HS in prion amyloid plaques. The direct interaction between GAGs and PrPC was also disclosed [64,65]. The decrease of cellular HS leads to a reduction in PrPSc formation, thereby regulating the metabolism of prions [66]. These observations indicate that the interaction between HS and PrPC/PrPSc is essential for prion pathogenesis. After identification of GAGs as part of amyloid aggregates, many studies showed that these molecules are able to inhibit the polymerization of synthetic prion peptides into amyloid fibrils [67] and PrPres (resistant to proteinase treatment) accumulation in scrapie-infected cells [68]. This last effect was suggested to occur due to the increase in the PrPC endocytosis rate that is stimulated by these compounds [69]. Conversely, Wong et al. [10] suggested that sulfate carbohydrates induce conformational changes in PrPsen (susceptible to proteinase treatment) and stimulate its conversion to a PrP-res isoform. Moreover, GAGs stimulate the amplification of PrP-res in vitro [12] and induce the formation of amyloid aggregates of a prion fragment (PrP 185–208) in neuronal cells [70]. All these results point to a paradoxical effect of sulfated polysaccharides, either stimulating or preventing PrP conversion, which lead us to conclude that more mechanistic studies on PrP– GAG interactions are needed. Recently, we described the chemical and physical properties of the murine recombinant PrP 23–231 interaction with low-molecular-weight heparin (LMWHep) [71,72] LMWHep interacts with rPrP 23–231, leading to transient aggregation, which evolves into

a soluble LMWHep–PrP complex. Nuclear magnetic resonance (NMR) results show that prion protein that is complexed with LMWHep has the same general fold pattern as the free protein, indicating that, in the non-aggregated complex, this interaction does not form a scrapie-like isoform. Notably, the Hep–rPrP complex is immune to RNA-induced aggregation. The transient aggregation of PrP may explain why some GAGs have been reported to induce the conversion into the misfolded, scrapie conformation, whereas others are thought to protect against conversion. We also showed that LMWHep binds the octapeptide repeat region at pH 7.4; however, at acidic pH values, rPrP 23–231 exhibits a second binding region, where histidine residues also mediate binding. These results show that, depending on the cellular milieu, PrP may expose different regions that can bind GAGs [71,72]. The ability of PrP to interact with GAGs appears to be involved not only with the pathogenic functions, but also the physiological functions of both molecules. PrPC is found mainly at the cell membrane [15], where it probably interacts with proteoglycans that are attached to the cell surface, syndecan (Sdc) and glypicans (Gpc), or in the extracellular matrix. PrPC is involved in neurite outgrowth and neuronal survival through signal transduction pathways, including p59 (Fyn, a Src-related family member), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase [13]. N-syndecans are transmembrane proteoglycans that interact with Src kinases to mediate neurite outgrowth [73]. Therefore, we hypothesize that the cited PrP effect could be related to its interaction with HS proteoglycans. GAGs were shown to mediate the interaction of PrP with other proteins, such as 37-kDa/67-kDa (LRP/LR), and to act as cofactors

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for PrP in t-PA-mediated plasminogen activation in vitro to regulate pericellular proteolysis (review in [13]). Another association occurs during cellular trafficking: glypican-1 (Gpc-1) and PrPC are cointernalized, stimulated by Cu+2, and PrPC controls the internalization and self-pruning of Gpc-1 [74]. Sulfated polysaccharides also stimulate PrPC endocytosis [69]. 3.3. PrP interactome studies As mentioned before, the prion protein is able to interact with many partners, including proteins, nucleic acids and glycosaminoglycans [42]. Several studies have been dedicated to the description of the PrP interaction with various macromolecules and the investigation of their role in prion biology. Many different tools have been applied to these studies, such as yeast two-hybrid screens and cross-linking experiments. Classical PrPC partners have been discovered in this way, such as the 37-kDa/67-kDa laminin receptor precursor [75] and the neural cell adhesion molecule (Ncam1) [76]. However, the ‘‘omics’’ era has brought vast amounts of information, and new approaches are being developed for their processing. The PrP-interacting proteome, or interactome studies of the prion protein, may help clarify the elusive function of this protein and the paths leading to neurodegeneration. Different techniques have been applied in this new approach. They include classical proteomics tools, like 2D gel electrophoresis and mass spectrometry [77], the use of protein tags for affinity chromatography [78] and specific antibody binding that allows for both the elution [79] and the precipitation of supramolecular complexes [80] with the subsequent identification of these molecules. In general, these studies provide a list of interactors, and authors try to find a connection among the proteins identified, such as protein families or groups, in order to find a general function for PrPC. Interestingly, Schmitt-Ulms et al. described not only the interaction of PrPC with metal ion transporters of the ZIP family, but also an evolutionary descent of the prion protein gene from a ZIP-like ancestor gene [81]. Rutishauser et al. [80] developed a fully functional PrP tagged with a C-terminal myc epitope. The immunopurification of 96, which specifically released proteins that formed complexes with

PrP, led to the isolation of seven proteins that co-eluted in an equimolar ratio with PrPC; these proteins are thought to be components of multiprotein complexes. Many of these proteins were related to axomyelinic maintenance [80]. A protein microarray has been described by Satoh et al. [59], in which a nitrocellulose-coated glass slide containing a protein library was incubated with recombinant PrP 23–231, and the interacting proteins were located using a western blotting analysis with PrP-specific antibodies. This work has led to the identification of 47 new partners for PrP, which are annotated as proteins involved in the recognition of nucleic acids, and the network formed was related to cell signaling pathways that are essential for cell survival, differentiation, proliferation and apoptosis (Fig. 3). This finding sheds light on an idea based on previous results that nucleic acids might be involved in the physiology or pathology of prions [7]. Another interactome analysis of PrPC included its mammalian paralogs, the doppel and shadoo proteins [82]. A similar physiological environment was described for the three proteins, as well as an interaction among them. Besides establishing an interactome network for these three paralogous proteins, this work has been able to confirm certain PrPC interactions that were previously suggested in other studies, including the interactions observed with the laminin receptor precursor, Na+/K+ ATPases and protein disulfide isomerases. These new approaches might be very helpful in the elucidation of the prion protein function, either in health or disease, by providing a wider perspective of its participation in vital cell processes, with many functions in different situations. 4. Prion traffic and topology – potential loci for interaction with partners PrPC mRNA is translated by ribosomes that are attached to the endoplasmic reticulum (ER), and the nascent protein is imported into the ER lumen, where it will be post-translationally processed (glycosylation, disulfide bond formation and C-terminal GPI attachment) and folded [83]. Precursor PrP has an N-terminal signal peptide that interacts with the translocation channel, directing

Fig. 3. Molecular network of PrPC and its interactors. The interactions between PrPC (shown in red) and different cell proteins, distributed according to their predicted subcellular localization, are shown. The PrPC interactors identified by microarray analysis are highlighted in blue and the ones circled in orange are involved with nucleic acid binding (Adapted from Ref. [59]).

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it to the ER lumen, and a C-terminal peptide sequence for the addition of the GPI anchor [41,84]. It was recently shown that, in addition to its signal for GPI addition, the GPI-sequence is important as an ER-targeting signal [85]. The processed protein is transferred throughout the Golgi, where it receives glycans and GPI modifications, and then reaches the extracellular leaflet of the cell membrane as a GPI-anchored protein [86]. In addition to N- and C-terminal peptide sequences, there is a third PrP region [111–134] that is rich in hydrophobic amino acids, which can also be found as a transmembrane domain (TMD). These three domains are important to determine PrP topology because they can influence protein translocation across the ER membrane. We can find four PrP topologies: PrPC or secPrP (found as a GPIanchored cell surface protein), CtmPrP (a transmembrane form with the C-terminus facing the exoplasmic milieu), NtmPrP (a transmembrane form with the N-terminus facing the exoplasmic milieu) and cyPrP (found in the cytosol) [87]. Ctm PrP and NtmPrP have the C- or N-terminal domains, respectively, and they are translocated into the ER lumen where they are integrated into the lipid bilayer through TDM [39]. Changes in the hydrophobicity of the TDM or near it and the interaction with the translocon channel significantly influence the generation of these forms [88]. The N-terminal signal sequence is important to direct the localization of the N-terminus to determine the proportions of both isoforms. When the N-terminus is translocated into the ER lumen before the TDM emerges from the ribosome, NtmPrP and sec PrP are favored. When these two regions are present in the translocon at the same time, the CtmPrP formation is favored [41]. Inefficiency in translocation into the ER lumen, due to the weakness of N-terminus signal peptide interaction with the translocon and of TDM biding, can dislocate PrP to the cytosol, generating cyPrP [41,89]. Other authors point to active retrotranslocation as being responsible for cyPrP production [90]. CyPrP is unglycosylated, contains the uncleaved signal peptide and increases in concentration during ER stress [91] when cells are in a reducing environment [92], or after proteosomal inhibition [93]. This form can also be localized to the nuclei of cells [94,95]. Ctm PrP and cyPrP have been associated with prion diseases. Increasing CtmPrP levels aggravate the neurodegeneration phenotype, and naturally occurring human PRNP mutations at the TDM cause disease when PrP is expressed in mice [88,96]. CyPrP was associated with neurodegeneration but was also shown to be non-toxic and even protective [97,98]. The majority of PrP molecules are synthesized as secPrP. PrPC reaches the extracellular membrane and, as a GPI-anchored protein, associates with specialized cholesterol and sphingolipid-rich microdomains called lipid rafts. This association is driven by its GPI anchor and by the first 28 amino acids at the N-terminal domain [99]. At the membrane, PrP can interact with the extracellular environment, and this interaction can be regulated by its endocytosis. It has therefore been suggested that endocytosis is crucial for PrPSc infection [100]. There is still a debate about the mechanism of PrPC endocytosis. Much evidence points to clathrin-mediated endocytosis (CME) as the pathway for PrPC endocytosis. PrPC is found in clathrin-coated pits [101,102], and it co-localizes with transferring receptor (transmembrane protein endocytosed by coated pits and excluded from lipid rafts) [103]. The raft-associated pathway caveolae are believed to be absent in neuronal cells, leading to the conclusion that CME is the main pathway in these cells [104]. Clathrin binds to a protein adapter that binds to cytoplasmic transmembrane proteins that present traffic motifs. This interaction generates coated pits and coated vesicles [105]. These proteins are subsequently sorted into endosomes (SEs) and delivered to the recycling endocytic compartment (REC) [106]. GPI-anchored proteins lack this cytoplasmic domain and thus have to interact with a cytoplasmic

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transmembrane protein to be endocytosed. The same is believed to happen with PrPC [103]. The N-terminal region of PrPC was shown to be necessary and sufficient for prion CME and sorting from the plasma membrane [107,108]. Molecules with traffic motifs that are able to bind this region may assist in prion CME. LDL receptor-related protein 1 (LRP1) is known to bind GPI proteins and mediate CME [106]. LRP1 is able to bind surface heparan sulfate proteoglycans (HSPG), molecules that are thought to interact with the PrP N-terminus [65,109]. Thus, HSPG could act as co-receptors for LRP1 and PrPC [110]. There are also other candidates that assist prion endocytosis. The non-integrin 37 kDa laminin receptor precursor (LRP) and its mature 67 kDa laminin receptor (LR) are able to interact with PrPC along with the influence of sulfated glycans [75,111]. Endocytosis of PrPC is mediated by physiological concentrations of Cu2+ [112]. This effect seems to be modulated through the interaction of Cu2+ with octapeptide repeats, and this interaction was suggested to be necessary for this process because mutations of histidine residues abrogate PrPC endocytosis [113]. Zn2+ also has the ability to induce PrPC endocytosis [113]. To be internalized through CME, PrPC has to escape from the lipid raft region and interact with another partner; disruption of rafts lead to prion endocytosis, and Ctm PrP (PrP form not present in rafts) is constitutively internalized without Cu2+ [114]. Cu+2 induces PrPC to move laterally out of the rafts before internalization [103,114], allowing the interaction of PrP with a non-raft partner. While the interaction with the octapeptide repeat is important to dissociate PrPC from rafts [114], the polybasic N-terminal region is important to CME [107]. A conformational change induced by Cu2+ interaction could favor the interaction of the polybasic residues with other partners for CME [112]. LRP1 is required for Cu+2-mediated endocytosis [115]. Some authors have demonstrated the occurrence of PrPC internalization through clathrin-independent pathways. Although CME is the main mechanism of internalization, clathrin-independent mechanisms could account for part of this cellular process. Many GPI-anchored proteins are internalized via cholesterol-enriched raft caveolae domains [103]. PrPC was found trafficking through caveolae in CHO and microglia cells [116,117]. Recently, Kang et al. [118] showed in N2a cells that PrPC can be internalized via a clathrin-independent pathway that is associated with Arf6, and that this pathway can account for PrPC endocytosis in the absence of clathrin. Independent of the way the protein is internalized, it will converge to the early endosome and then to the late endosome to be degraded in the lysosome [119,120]. PrPC is also recycled back to the plasma membrane directly from early endosomes or transported first to the endosomal recycling compartment (ERC) [103,121]. In the case of PrPSc localization, it was found mainly to be intracellular, in late endosome/lysosome structures and recycling vesicles, with little localization at the cell surface [122– 124]. The cellular compartments involved in prion conversion have been investigated, and they are mainly compartments that allow the interaction between both isoforms. Recently, Marijanovic et al. [125] demonstrated that the ERC is the intracellular site of conversion. This result shows that molecules that are also found in this compartment could act as partners for prion conversion. As discussed before, the potential interaction of PrP with noncoding RNAs exerting a NA chaperone activity might explain the intracellular trafficking. Under some conditions, cytoplasmic PrP was shown to induce large ribonucleoprotein particles, which might perform post-transcriptional regulation [61].

5. Prion strains and transmission barrier An interesting feature of the transmissible spongiform encephalopathies is the presence of prion strains. How a protein with the

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same primary sequence presents different pathogenic characteristics, such as incubation time [34], and physicochemical properties in humans and animals is still a mystery. Among the physicochemical properties, it has long been described that prion strains present different patterns of digestion by proteinase K, and the analysis of the banding profiles in western blotting is essential for their identification [126]. A possible explanation for the particular characteristic of each strain is the existence of different conformations for the prion protein [35,36], which could also happen due to post-translational modifications, such as the PrP glycosylation pattern [127]. The specific electrophoretic mobility of the three bands, relative to non-, mono- and di-glycosylated PrP forms, is also a characteristic of each TSE strain [126,128,129]. Moreover, these dissimilar conformations might lead to different recognition/binding of cellular factors that finally could generate a new infectious species. In vitro assays for prion conversion, mainly PMCA, have enlightened the studies of mammalian prion strains [130,131] more rapidly than time-consuming in vivo experiments. A new strain was fully created in vitro after replication of a mouse prion in the presence of hamster scrapie PrP [130]. This heterologous conversion showed that the final PrPSc displayed characteristics closer to the hamster PrP (the donor of infectious material) than to murine PrP (the receptor) [130]. Prion strains present selectivity to the cells that they infect and propagate in, showing a particular cellular tropism [132,133], and the presence of specific cellular molecular partners, such as nucleic acids [7,9], for PrP could also be responsible for the observed selectivity towards cellular infection. Long before the etiological agent of the TSEs had been identified, it was already known that inoculation of a foreign prion into a non-homologous species would not lead to disease progression, and this fact is called the species-barrier [134,135]. An exception to this is the occurrence of variant CJD in the middle of the 1990 decade, which is believed to be transmitted from cattle suffering from bovine spongiform encephalopathy to humans [136]. PMCA studies have also helped to investigate the transmission barrier [137], which can be generally overcome using transgenic animals expressing the same protein sequence as the one present in the infectious inoculum. The presence of polymorphisms along the prion protein primary sequence (i.e., specific amino acid residues that would account for the transmission barrier) is the main explanation for the occurrence of such events [138]. The fact that the same primary sequence is required for efficient conversion of the recipient PrP into the infectious PrP clarifies the fact that conformational differences between native and scrapie PrP would hinder contact between these molecules and further structural conversion. Besides that, the presence of a specific cellular component from the recipient animal that would interact with the PrPSc from the same species and help the conversion, as proposed by Telling et al. [139] after investigating prion transmission using transgenic animals, seems to be important to trigger disease propagation. Therefore, when a non-homologous PrP is present, the lack of the cellular-factor (formerly named factor-X) [140] would not lead to scrapie propagation. This factor, as discussed throughout this review, could be a protein [139,140], a glycosaminoglycan [141] or a nucleic acid molecule [7–9,11,12].

6. PrP structural features and conformational conversion catalyzed by polyanions The central event in prion pathogenesis is the conformational conversion of cellular prion protein (PrPC) into its pathological form (PrPSc), and there has been great interest in high-resolution structural studies to understand the molecular basis of this protein misfolding disease. Since the first structure determination of the recombinant C-terminal domain of mouse PrP in 1996 [142], the

three-dimensional structure of several PrPs from different species have been solved and compared [20,143] in order to dissect the effects of disease-causing mutations and the molecular mechanisms that underlie prion strains and the transmission barrier. The comparative analysis of PrP structures demonstrates that all prion proteins share a common architecture that is composed of two structurally and dynamically distinct portions of similar size. The N-terminal half forms a flexible, extended tail, while the C-terminal domain is structured and presents a globular fold of three alpha helices and a short, double-stranded, antiparallel beta-sheet. There is a single disulfide bridge, which connects two cysteine residues that are located in the second and third helices [143]. Structural superposition of the globular domains of different mammalian PrPs shows only minor conformational variations related to differences in primary structures, including distinct atom positions, surface charge potentials and local dynamics. It was hypothesized that such restricted conformational differences could account for the establishment of the prion transmission barrier and for the varied repertoire of PrP conformations (strains) that are generated from distinct polymorphic PrP isoforms [144]. The regions of higher flexibility in a protein are usually more prone to structural rearrangements. Therefore, pathological mutations or specific polymorphisms in PrPC sequences were initially expected to confer a local structural instability and to facilitate the conversion process. However, structural destabilization of PrP does not seem to be a general mechanism underlying the protein pathological conversion, as demonstrated by a biophysical analysis of the effect of hereditary prion disorder-associated mutations on the thermodynamic stability of the recombinant human PrP [145]. Moreover, it was shown that ovine PrP polymorphisms at positions 136, 154 and 171, which confer different susceptibility to scrapie, are associated with different H-bond patterns, but strikingly, the scrapie-resistant form presents a lower stability relative to the more susceptible isoforms [146]. Thus, structural factors other than the local conformational stability are likely to significantly influence the mechanism of PrP conversion. One such factor may be related to critical differences in PrP primary structures that generate distinct molecular interaction surfaces. This could affect not only the contact between the cellular and pathological forms of the prion protein, a fundamental step in the structural conversion, but also PrP interactions with specific cellular components (for example, other proteins, glycosaminoglycans or nucleic acids), which are speculated to play important roles in this process [7,9,139,140]. In fact, the surface region in the structure of the cellular form of PrP that includes the C-terminal part of helix 3 (residues 218–213) and the loop between strand 2 and helix 2 (residues 166–172) shows high species variability and has been related to the transmission barrier for prion diseases and to a possible interaction with ‘‘factor-X’’ [139,147]. Importantly, previous studies have shown that it is possible to successfully propagate prion infectivity using the in vitro PMCA reaction only in the presence of cofactors such as nucleic acids and lipids, which supports the idea that cellular partners may be important to drive PrP conversion in vivo [8,27,148]. Recently, Kim et al. [33] proposed a mechanism for prion propagation in vitro in the absence of any mammalian cofactor. The method consisted of a modified PMCA reaction, using a highly purified, recombinant hamster PrPC that was seeded with the proteinase K (PK)-resistant core of PrPSc isolated from brain homogenates of scrapie-infected hamsters. Based on these results, it could be speculated that cofactors might be more important for the initial formation of the PrPSc specimens than for the subsequent autocatalytic propagation of the misfolded form, and that, in this case, the infectious seed was sufficiently enriched with PrPSc particles to trigger the PrP conversion cascade. However, there is also a possibility that small RNA or DNA molecules that were present in the E. coli lysates and/or in the brain

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homogenates that were co-purified with one or both PrP forms affected the PMCA reaction. Further studies are necessary to address this issue. In addition to conformational diversity driving multiple prion protein interactions, variation in the electrostatic charge distribution on the molecular surface of different PrP structures (Fig. 4) is also likely to influence the intermolecular contacts of the prion protein and, as a result, the transmission barrier for TSEs [143]. Indeed, local variations in the surface charge seem to be crucial for the maintenance of the species barrier between humans and cattle because the prion proteins from both species do not significantly differ in any other structural feature [149]. Also, the single residue mutation of the familial Creutzfeldt–Jakob disease-related E200K variant of human PrP was shown to dramatically perturb the surface electrostatic potential of the protein, while the three-dimensional structure was not affected. Such change is thought to disturb the molecular contacts of the prion protein and to facilitate the conformational conversion by inducing abnormal interactions

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of E200K PrPC with molecular partners that are implicated in the pathogenesis of prion disorders [150]. In a recent study, Wen and collaborators [151] determined the solution NMR structures of the recombinant Rabbit prion protein, RaPrPC-(91–228), and its S173N variant. This work attempted to determine why rabbits are resistant to transmissible spongiform encephalopathies. As can be visualized in Fig. 4, the structure of RaPrPC is quite similar to that of other species, and the only remarkable change is in the charge distribution of RaPrPC. Electrostatic interactions are crucial for DNA or RNA recognition by proteins. Our structural studies [47] confirmed, by computational modeling [58], that the DNA-PrPC interaction includes an important segment of the globular domain and the unstructured N-terminal domain. The differences in potential DNA-binding sites and charge distributions among PrPs from different species are shown in Fig. 4. Although significant progress has been made over the last few years regarding the structural analyses of the cellular forms of PrPs,

Fig. 4. Molecular surface graphs evidencing the differences in the predicted DNA-binding sites (left panel, colored blue) and the electrostatic potential distribution (right panel, colored blue for positive charge and red for negative charge) among PrP molecules from distinct mammalian species (PDB codes: 1QM1, 1DWZ, 1XYX, 1B10 and 2FJ3 for human, cattle, mouse, Syrian hamster and rabbit PrPs, respectively). Interestingly, PrP from rabbit, which constitutes, to date, the only known TSE agent-resistant species, presents a relatively small putative DNA-contacting surface. DNA-binding predictions were performed using DISPLAR (Ref. [58]). All surface graphs were generated using MolMol (Koradi et al., Journal of Molecular Graphics, 1996).

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very little is known about the structural features of the corresponding pathological forms as a result of the heterogeneity and insolubility of the samples purified from the brains of infected animals, which are not appropriate for structural studies by highresolution methods (NMR or X-ray diffraction) [144,152]. Lower resolution techniques for structural analyses (e.g., FT-IR, CD) have been alternatively used, and it was demonstrated that PrPSc presents higher amounts of beta-structure (up to 50%) relative to PrPC, though a considerable part of the alpha-helical content seems to be preserved [21,153]. Importantly, FT-IR spectroscopy has also shown that the PrPSc beta-sheet conformation varies among different prion strains [35,154,155], supporting the idea that each strain corresponds to a distinct conformation that a specific PrP molecule can assume. Although the high-resolution structure of PrPSc has not been reported yet, two different structural models for PrPSc have been proposed. The spiral protofibril model [156] is based upon molecular dynamics simulations of the segment 109–219 in the hamster PrP variant D147N under amyloidogenic conditions, while the beta-helix model [157] was built by threading a portion of the mouse prion sequence (residues 89–175) through a lefthanded beta-helical structure that is supposed to assemble into a trimeric structure. Each model is supported by different experimental data, and further studies are necessary for a better structural characterization of prion aggregates [144,158]. Solid-state NMR techniques have significantly contributed to the characterizations of amyloidogenic proteins [159] and can potentially be used to provide further valuable information on PrPSc structure. Future studies along this direction will be critical for understanding the unique features of prion diseases, like PrP conformational conversion, strain diversity, the species barrier and prion infectivity.

7. Diagnostics and therapeutics for prion diseases Methodologies that allow for early disease diagnosis are very important to provide effective treatment. For now, there is no effective diagnostic strategy to identify neurodegenerative disorders in the early stages of development. Commonly, they are noticed only after the first clinical symptoms appear. At this point, treatment – when and if there is one available – can only go as far as palliative measures. A complete diagnosis depends on the biochemical assays that are possible only through post-mortem biopsy of the brain [160]. Those circumstances are not different in the specific case of prion disorders. Currently, available tests still rely upon the detection of PK-resistant PrPSc [161]. Over the years, a series of PrPSc detection methods were developed toward the emergence of an effective diagnostic approach. Detection protocols have evolved greatly, such as prion antibodies [162], PMCA assays, and development of biomarkers [163]. Despite all of the efforts, those methodologies are not yet available as diagnostic tools. Most of them are still only used in research labs due to high financial costs and the need of further technology development. We expect great developments in this field in the years to come. Concerning the therapeutics for prion diseases, a series of compounds have been tested to reverse or prevent prion aggregation and deposition by directly or indirectly affecting PrPC conversion into PrPSc [164]. Unfortunately, these promising compounds were only effective in cultured cells and did not evolve much after that. They were proven to be ineffective for treating sick animals. Quinacrine in particular, aside from being highly hapatotoxic, was ineffective in CJD patients [165–167]. Recent studies provided new perspectives in the field of antiprion drug therapy. Quinacrine-derived compounds are still being considered as potential drugs for treating Alzheimer and prion dis-

eases [168]. However, it was recently observed that quinacrine, when administered at a 300 mg/day dosage, was reasonably tolerated, but the clinical course of the disease was not really affected [169]. The anti-scrapie activity of antimalarial compounds, such as quinolines, has stimulated studies with aminoquinoline derivatives [170]. It was found that 4-amino-7-chloroquinoline and N-(7chloro-4-quinolinyl)-1,2-ethanediamine significantly inhibited the aggregation of prion peptides. Yeast-based screening assays revealed the anti-scrapie efficacy of the antihypertensive drug guanabenz (GA), and further investigations demonstrated its activity against prions in transgenic mouse-based in vivo assays [171]. Polyamines are also being revisited, and permanently-charged, branched polyamines were reported to decrease prion levels in neuroblastoma cells with reduced intrinsic toxicity [172]. Besides drug therapies, other approaches have been developed in the attempt to prevent and treat prion diseases. These other strategies are aimed at clearance of PrPSc and depletion of PrPC molecules so that they are no longer available for conversion [173]. An interesting example of these efforts is presented in a manuscript published in 2007 by Deryl S. Spinner and coworkers. They reported that the immunization of Prnp+/+ and Prnp-/ (knockout) mice with CpG oligodeoxynucleotides enhanced the humoral immune response and the production of antibodies against prion protein PrPSc [174]. These results represent an important step toward the production of vaccines against prion diseases [174]. Nucleic acids have also been reported to prevent prion misfolding and propagation in infected cells [18]. Therefore, modified oligonucleotides are excellent candidates for diagnostic and therapeutic procedures [18]. Degenerate oligonucleotides have been reported to reduce PrPSc formation in vivo [54]. PrP interaction with DNA led to the identification of anti-DNA antibodies that specifically recognize PrPSc from human brain tissues [175]. Selected DNA thioaptamers bind with high affinities to different species of PrP [55], and phosphorothioate DNA can decrease PrPSc content in infected neuroblastoma cells [56]. Another approach that was utilized in our laboratory was to use naphthalene derivatives, such as the compound 4,40 -dianilino-1,10 -binaphthyl-5,50 sulfonate (bis-ANS), which inhibits the aggregation of the Syrian hamster PrP peptide ShaPrP(109–149) [176]. We found that bisANS competed for the same site of DNA-binding. Another interesting approach is the utilization of RNA interference (RNAi). The results include inhibition of PrPSc propagation in cultured cells [177]. In vivo, RNAi has been applied to decrease PrPC levels in mice, cattle and goats [178,179]. Recently, White and coworkers reported neuronal rescue from early dysfunction and prolonged mouse survival after RNAi treatment [180]. Despite all of these efforts, there is no available therapy for prion diseases. This might be due to the fact that the molecular mechanisms that lead to PrP conversion into the scrapie form are still unknown. Moreover, there is also a lack of knowledge about the species that account for the pathogenesis of prion diseases. Once we have a full picture of the disease generation and progression and understand if the presence of PrPSc or the lack of functional PrPC leads to neurodegeneration, new compounds could be designed and evaluated as effective therapies.

8. Concluding remarks Prion biology has undergone excellent progress in recent years. However, the mechanisms by which PrPC and adjuvant factors, such as nucleic acids and glycosaminoglycans, lead to misfolding and conversion into infectious prions have not been completely elucidated. Numerous studies using biochemical, biophysical and

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cell biology methods have produced a consensus that prion protein binds to nucleic acids with affinities in the nanomolar range and that they act as adjuvants to the pathogenic conversion. The catalytic effects of RNAs, DNAs or GAGs on the PrPC ? PrPSc conversion would depend on the sequence and structure of the target molecules. The potential therapeutic use of modified nucleic acids and GAGs has been demonstrated by different groups. The nucleic acid-binding properties of the prion protein (both RNA and DNA) might have broader implications for its native function than for disease. The great abundance of RNA in the cytosol that acts in a variety of cellular processes may be a clue to the physiological target of the prion protein. Altogether, the recent biological and structural findings that prion protein binds to RNAs, DNAs and GAGs provide evidence that nucleic acids can stimulate the conversion of the cellular prion form into the infectious one. Particularly, the structural studies on the interaction between prion protein and PrP domains with nucleic acids [11,17,47], sulfated glycans [181] and protein partners, such as the co-chaperone hop/STI1 [182], promises to open new avenues for the design of compounds targeting prion diseases. The stimulation at the level of in vitro protease-resistant prion protein (PrPres) amplification by nucleic acids [29,30,50] is consistent with the hypothesis of nucleic acid catalytic action. However, many pieces of the prion puzzle are not yet in place. All this evidence leaves no doubt that prions have other additional accomplices that chaperone the activity of converting the normal, cellular form of protein into the disease-causing isoform. A recent intriguing result was described by Collinge’s group [183] showing an apparent ’’spontaneous generation’’ of prions from normal brain tissue coating a metal surface. The researchers hypothesize that the novo formation of prions are helped by bound cofactors. Here, we reviewed the experimental methodology and results that seek to understand how nucleic acids and GAGs bind to prions as well as the resulting implications for cellular toxicity and prion conversion. There are, however, many questions that remain to be explored. The formation of a complex between non-infectious PrP and RNA may be just part of the story. The connection between PrPC, NA and PrPSc could be a side effect of the prion protein’s physiology. The implications of these interactions are causing a paradigm shift in the area of prion research, and we can anticipate new findings in the near future. Acknowledgments This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Ministerio da Saude (MS-DECIT), the National Institute of Science and Technology for Structural Biology and Bioimaging, (CNPq INCT Program), and by ABC-UNESCO-L’Oréal. References [1] A. Aguzzi, A.M. Calella, Physiol. Rev. 89 (2009) 1105–1152. [2] R.S. Knight, R.G. Will, J. Neurol. Neurosurg. Psychiatry 75 (Suppl. 1) (2004) i36–i42. [3] I. Vorberg, M.H. Groschup, E. Pfaff, S.A. Priola, J. Virol. 77 (2003) 2003–2009. [4] S.B. Prusiner, D.C. Bolton, D.F. Groth, K.A. Bowman, S.P. Cochran, M.P. McKinley, Biochemistry 21 (1982) 6942–6950. [5] J.S. Griffith, Nature 215 (1967) 1043–1044. [6] S.B. Prusiner, Proc. Natl. Acad. Sci. USA 95 (1998) 13363–13383. [7] J.L. Silva, L.M. Lima, D. Foguel, Y. Cordeiro, Trends Biochem. Sci. 33 (2008) 132–140. [8] N.R. Deleault, B.T. Harris, J.R. Rees, S. Supattapone, Proc. Natl. Acad. Sci. USA 104 (2007) 9741–9746. [9] J.L. Silva, T.C. Vieira, M.P. Gomes, A.P. Bom, L.M. Lima, M.S. Freitas, D. Ishimaru, Y. Cordeiro, D. Foguel, Acc. Chem. Res. 43 (2010) 271–279. [10] C. Wong, L.W. Xiong, M. Horiuchi, L. Raymond, K. Wehrly, B. Chesebro, B. Caughey, EMBO J. 20 (2001) 377–386. [11] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. Foguel, J.L. Silva, J. Biol. Chem. 276 (2001) 49400–49409.

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