Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Insights into intragenic and extragenic effectors of prion propagation using chimeric prion proteins

The study of fungal prion proteins affords remarkable opportunities to elucidate both intragenic and extragenic effectors of prion propagation. The yeast prion protein Sup35 and the self-perpetuating [PSI+] prion state is one of the best characterized fungal prions. While there is little sequence homology among known prion proteins, one region of striking similarity exists between Sup35p and the mammalian prion protein PrP. This region is comprised of roughly five octapeptide repeats of similar composition. The expansion of the repeat region in PrP is associated with inherited prion diseases. In order to learn more about the effects of PrP repeat expansions on the structural properties of a protein that undergoes a similar transition to a self-perpetuating aggregate, we generated chimeric Sup35-PrP proteins. Using both in vivo and in vitro systems we described the effect of repeat length on protein misfolding, aggregation, amyloid formation and amyloid stability. We found that repeat expansions in the chimeric prion proteins increase the propensity to initiate prion propagation and enhance the formation of amyloid fibers without significantly altering fiber stability.Key words: prion, yeast, sup35, PrP, nonsense suppression, translation termination, amyloid, repeatWe recently described a novel chimeric prion system that was designed to elucidate the consequences of one class of inherited prion disease mutations on protein folding.^1,2 We created a fusion between the mammalian prion protein PrP and the yeast prion protein Sup35p (Fig. 1). Sup35p is an essential translation termination factor in yeast. Interestingly, the majority of the protein can be sequestered into a self-propagating aggregate, the [PSI+] prion.³ Remarkably, when yeast are grown in normal laboratory conditions, the [PSI+] prion is not detrimental. In fact, the biological consequences of the switch from the [psi−] non-prion state to the [PSI+] prion state may be beneficial in terms of adaptation and evolution.⁴ Importantly, the prion state of Sup35p can be readily detected in vivo by monitoring the reduced function of the translation termination factor when the protein is propagating as a prion aggregate.³ In addition, several methods have been developed to not only follow the propagation of the prion, but also to control the propagation and promote prion induction and loss (curing).⁵ Therefore, in addition to simply being a fascinating biological problem in of itself, the [PSI+] prion in yeast affords the ability to further elucidate both intragenic and extragenic effectors of prion biology.Open in a separate windowFigure 1Schematic representation of the yeast protein Sup35p and the mammalian prion protein PrP highlighting the position of the oligopeptide repeat domain (ORD). The amino acid sequence represents the consensus for a single repeat. Numbers shown represent the amino acid position of the beginning and the end of each ORD. The numbers above the schematic represent the original PrP amino acid positioning and the numbers below represent the original Sup35p amino acid sequence positions.Several prions have now been identified and interestingly, there is little sequence homology between the proteins to suggest that only one type of sequence can form a self-propagating aggregate.^6–8 In vitro studies suggest that many proteins can form amyloids under the appropriate conditions.⁹ The fact that only a small percentage of proteins propagate as prions in vivo may be partly a consequence of physiological conditions being adequate to promote amyloid formation with those particular sequences. It is unclear what the precise distinction between prion and amyloid is at this time, but localization alone may preclude some amyloidogenic proteins from being “prion proteins” per se.¹⁰The sequence context that permits a protein to adopt a prion conformation in vivo is unclear. Several of the identified prion proteins have a domain that is enriched in glutamine and asparagine (Q/N) residues, but this is not true of all prion proteins.⁷ Our recent study demonstrates that the Q/N character of the Sup35p prion-forming domain can be significantly reduced, yet still propagate as a prion.¹ This was also found recently in another prion protein chimera created and expressed in yeast.⁶ These studies suggest that the lack of stable secondary structure may be one of the defining features of a prion-forming domain. One of the striking sequence similarities that does exist between two prion proteins occurs in an oligopeptide repeat region found in Sup35p and PrP.¹¹ Previous data clearly demonstrated that the Sup35p repeats are important for [PSI+] prion propagation.^12–15 The deletion of a single repeat from the wild type SUP35 sequence results in the loss of normal [PSI+] prion propagation.¹² Moreover, the addition of two extra repeats of Sup35p sequence served to enhance the formation of the [PSI+] prion.¹³ The expansion of the analogous repeat domain in the mammalian prion protein PrP is associated with an inherited form of prion disease.¹⁶ Since the repeat regions of Sup35p and PrP are similar in size and character, we wanted to determine if the Sup35p oligopeptide repeat region could be substituted with that of PrP. Indeed, the PrP repeats in the context of Sup35p supported the propagation of the [PSI+] prion in yeast.^1,17 Strikingly, we found phenotypic changes that occurred in a repeat length-dependent manner that suggested that the repeat expansions associated with disease result in an increase in the aggregation propensity but do not necessarily dictate only one type of aggregate structure.¹More recently, we verified some of these results in vitro.² These data are in agreement with other studies on the effect of repeat expansions.^18,19 Taking the analysis one step further, we demonstrated that the stability of the amyloid fibers formed with the repeat-expanded proteins did not differ significantly. A very interesting observation that we made was that the formation of amyloid fibers by the longest repeat-expanded chimera (SP14NM) followed drastically different kinetics compared to the chimera containing the wild type number of repeats (SP5NM).² In unseeded reactions, SP14NM did not show a lag phase during the course of fiber formation whereas SP5NM displayed a characteristic lag phase. Furthermore, the morphology of the amyloid fibers visualized by EM was different between SP14NM and SP5NM. SP14NM fibers were curvy and clumped but SP5NM fibers were long and straight. The correlation between the kinetics and the morphology of amyloid formation of SP14NM and SP5NM is reminiscent of fibers formed by β2-microglobulin (β2m) protein in different conditions.²⁰ At pH 3.6, β2m formed curvy, worm-like fibers with no apparent lag phase. In contrast, long, straight fibers were formed at pH 2.5 and had a distinct lag phase. Analysis of the β2m fibers formed at pH 3.6 using mass spectrometric techniques identified species ranging from monomer to 13-mer. This suggested that the fibers were formed by monomer addition. On the other hand, oligomers larger than tetramers were not formed during fiber formation at pH 2.5. Based on these data the authors propose that β2m forms fibers in a nucleation-independent manner at pH 3.6, but fiber formation at pH 2.5 follows a nucleation-dependent mechanism. We suggest that the mechanism underlying SP5NM and repeat-expanded SP14NM fiber formation is similar to β2m fibers formed at pH 2.5 and pH 3.6, respectively. It will be interesting to determine if disease-associated mutations in amyloidogenic proteins alter the pathway whereby amyloid formation occurs and how that process plays a role in pathogenesis.In our in vivo study,¹ we highlighted a unique feature of the longest Sup35-PrP chimera that related to the ability of the protein to adopt multiple self-perpetuating prion conformations more readily than wild type Sup35p. We suggest that this may be an important aspect of prion biology as it relates to inherited disease. If the repeat-expanded proteins can adopt multiple conformations that aggregate, then that may contribute to the large amount of variation observed in pathology and disease progression in this class of inherited prion diseases.^21,22We also found that the spontaneous conversion of the repeat-expanded Sup35-PrP chimera into a prion state was significantly increased. However, this conversion required another aggregated protein in vivo, the [RNQ+] prion. In vitro, the prion-forming domain of the chimera showed a similar trend with the longer repeat lengths enhancing the ability of the protein to form amyloid fibers. The chimera with repeat expansions (8, 11 or 14 repeats) formed fibers very quickly as compared to that with the wild type number of repeats (5). While this correlates with the in vivo data in that both systems demonstrate an increased level of conversion with the repeat expansion, the systems are very different with respect to their requirement for a different “seed” to initiate the prion conversion. So, how does the [RNQ+] prion influence [PSI+]? At the moment, that isn''t entirely clear. Susan Liebman and colleagues discovered another epigenetic factor in yeast, [PIN+], which was important for the de novo induction of [PSI+].^23–25 Several years later, the [RNQ+] prion²⁶ was found to be that factor in the commonly used [PSI+] laboratory strains, but they also found that the overexpression of other proteins could reproduce the effect.²⁵ Hence, [RNQ+] can be [PIN+], and may be the primary epigenetic element that influences [PSI+] induction in yeast, but need not be in every case. Two models were proposed to explain the ability of [RNQ+] to influence the induction of [PSI+].^25,27 One suggested that there is a direct templating effect where the aggregated state of the Rnq1 protein in the [RNQ+] prion serves as a seed for the direct physical association and aggregation of Sup35p and initiates [PSI+]. The second postulated that there is an inhibitor of aggregation in cells that is titrated out by the presence of another aggregated protein. Recent experimental evidence suggests that the templating model may explain at least part of the mechanism of action behind the [RNQ+] prion inducing the formation of [PSI+].^28,29Why is [RNQ+] required for the in vivo conversion of the repeatexpanded chimera that forms amyloid on its own very efficiently in vitro? Interestingly, we found that the [RNQ+] prion per se is not required. We overexpressed the Rnq1 protein from a constitutive high promoter (pGPD-RNQ1) and found that Rnq1p aggregated in the cells but did not induce the [RNQ+] prion. That is, the cells were still [rnq−] and did not genetically transmit the aggregated state of the protein. However, even these non-prion aggregates of Rnq1p served to enhance the induction of the chimeric prions. Therefore, either the [RNQ+] prion or an aggregate of Rnq1 protein is sufficient, which is in line with previous studies that demonstrated that some proteins that aggregate when overexpressed can also enhance the induction of [PSI+].²⁵ Also of note, recent data suggests that the requirement of [RNQ+] for the induction of Sup35p aggregation in vivo can be overcome by very long polyglutamine or glutamine/tyrosine stretches fused to the non-prion forming domain of Sup35p.³⁰ These fusions may alter protein-protein interactions or destabilize the non-prion structure of Sup35p in such a manner that the [RNQ+] prion seed is no longer required to form [PSI+] de novo. Indeed, the non-polymerizing state of some of the fusion proteins was shown to be very unstable.So, what is the important difference between our in vitro and in vivo systems in the prion conversion? Obviously there are many candidates. First, the full length Sup35 protein may alter the conversion properties since a large part of the molecule is the structured C terminal domain. The C terminal domain may influence the initiation of prion propagation in vivo and that is not a factor in the in vitro system. Second, the influences of co-translational folding and potentially some initial unfolding of the prion-forming domain are not present since the in vitro system starts with denatured protein. Third, the environmental influences are clearly different. The molecular crowding effects and chaperones that are required for prion propagation in vivo are not required for the formation of amyloid in vitro. Finally, it is unclear if amyloid structures similar to those formed with the prion-forming domain in vitro actually exist in yeast. Certainly there is some correlation between the structures since aggregated Sup35 protein from [PSI+] cell lysates can seed amyloid formation in vitro^31,32 and the fibers formed in vitro can be transformed into [psi−] cells and cause conversion to [PSI+].³³ Nevertheless, we find it interesting that the expansion of the repeat region can have a tremendous effect on amyloid formation in vitro yet still cannot overcome the requirement for [RNQ+] for conversion in vivo. The presence of co-aggregating or cross-seeding proteins may play a role in the sporadic appearance or progression of neurodegenerative diseases and the interconnected yeast prions [RNQ+] and [PSI+] may provide a model system for elucidating the mechanism underlying such effects.     

The sensitive [SWI+] prion: New perspectives on yeast prion diversity

Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these “nouveau prions” has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI⁺], differs from the best studied, archetypal prion [PSI⁺] in several significant ways.¹ Notably, [SWI⁺] is highly sensitive to alterations in Hsp70 system chaperone activity and is lost upon growth at elevated temperatures. In that report we briefly noted a correlation amongst prions regarding amino acid composition, seed number and sensitivity to the activity of the Hsp70 chaperone system. Here we extend that analysis and put forth the idea that [SWI⁺] may be representative of a class of asparagine-rich yeast prions which also includes [URE3], [MOT3⁺] and [ISP⁺], distinct from the glutamine-rich prions such as [PSI⁺] and [RNQ⁺]. While much work remains, it is apparent that our understanding of the extent of the diversity of prion characteristics is in its infancy.Key words: Sis1, Hsp40, chromatin remodeling, Swi1, Ssa, heat-shock, protein misfolding, cell stress, Hsp 104, PINYeast prions are heritable elements, most of which are amyloid aggregates of single proteins. The three best studied yeast prions [PSI⁺], [RNQ⁺] (also called [PIN⁺]), and [URE3] are formed from amyloid aggregates of the cytosolic yeast proteins Sup35, Rnq1 and Ure2, respectively.² Yeast prions can spontaneously arise in an otherwise clonal cell population, a process referred to as prion formation or nucleation, but once formed their continued propagation is intimately related to molecular chaperone activity. Chaperone function is needed to fragment prion amyloids to create heritable seeds which can then be passed on to cell progeny, thus maintaining the prion in the cell line.³ Yeast prions vary in the steady-state number of heritable seeds per cell; having more seeds increases the chances of passing the prion to progeny and hence prions with higher seed numbers are more mitotically stable.^4–6The currently accepted model of prion fragmentation posits that components of the Hsp70 chaperone system work in congress with the disaggregase Hsp104.^1,7–9 Hsp70-type chaperones function by repeatedly binding and releasing client polypeptides in an ATP-dependent manner, a cycle that is tightly regulated by co-chaperone proteins (Fig. 1). J-proteins (Hsp40s) stimulate Hsp70 ATP hydrolysis and peptide binding via a conserved J-domain whereas nucleotide exchange factors (NEFs) stimulate ADP/ATP exchange, restoring the ATP-bound (peptide unbound) state. In prion fragmentation, the J-protein Sis1, the Hsp70 Ssa, and nucleotide exchange factors (NEFs) of the Sse family are co-chaperones required as partners for the Hsp70 Ssa. While chaperone proteins may have additional functions in prion biology, e.g., prion formation, these additional functions are still poorly understood.⁹Open in a separate windowFigure 1The Cyclic Hsp70 Chaperone System. Ssa (purple), the yeast cytosolic Hsp70, binds and releases client polypeptides (blue) in a regulated and ATP-dependent manner. J-proteins (aquamarine) including Sis1, Ydj1 and others, stimulate Ssa ATP hydrolysis by virtue of a conserved J-domain and thereby catalyze the “forward” direction of the cycle as indicated above. ADP•Ssa more stably associates with client polypeptides than the ATP-bound form and hence J-proteins favor the ADP•Ssa•Peptide complex. In some cases, J-proteins can also bind and deliver client polypeptides to Hsp70s via C-terminal domains (also shown above). Nucleotide exchange factors (NEFs), including the Sse proteins (dark blue) which share some structural homology with Ssa, catalyze the “reverse” direction of the cycle by facilitating ADP release and subsequent ATP binding, and thus favor an ATP•Ssa state with a dissociated peptide.In the past few years, the number of known yeast prions has rapidly grown such that, to date, a total of eight yeast prions have been identified and an additional 18 proteins have been annotated as putative prions.¹⁰ The biological and physical properties of these newly discovered prions are only beginning to be explored. We recently reported the results of an investigation into the biological properties of the prion [SWI⁺], which is formed from the chromatin-remodeling factor Swi1.¹ Swi1 is part of the SWI/SNF chromatin-remodeling complex that regulates the expression of approximately 6% of all yeast genes.¹¹ The presence of [SWI⁺] causes partial loss of SWI/SNF chromatin-remodeling function, resulting in the impaired ability to uptake certain sugars, among other phenotypes.¹¹ [SWI⁺] is a prion of particular interest because of its potential to alter global gene expression. Below we describe its intriguing interactions with molecular chaperone proteins and environmental stress, and the implications of these properties on yeast prion biology.     

Compositional Determinants of Prion Formation in Yeast

Numerous prions (infectious proteins) have been identified in yeast that result from the conversion of soluble proteins into β-sheet-rich amyloid-like protein aggregates. Yeast prion formation is driven primarily by amino acid composition. However, yeast prion domains are generally lacking in the bulky hydrophobic residues most strongly associated with amyloid formation and are instead enriched in glutamines and asparagines. Glutamine/asparagine-rich domains are thought to be involved in both disease-related and beneficial amyloid formation. These domains are overrepresented in eukaryotic genomes, but predictive methods have not yet been developed to efficiently distinguish between prion and nonprion glutamine/asparagine-rich domains. We have developed a novel in vivo assay to quantitatively assess how composition affects prion formation. Using our results, we have defined the compositional features that promote prion formation, allowing us to accurately distinguish between glutamine/asparagine-rich domains that can form prion-like aggregates and those that cannot. Additionally, our results explain why traditional amyloid prediction algorithms fail to accurately predict amyloid formation by the glutamine/asparagine-rich yeast prion domains.Amyloid fibers are associated with a large number of neurodegenerative diseases and systemic amyloidoses. Amyloid fibrils are rich in a cross-beta quaternary structure in which β-strands are perpendicular to the long axis of the fibril (8).[URE3] and [PSI⁺] are the prion (infectious protein) forms of the Saccharomyces cerevisiae proteins Ure2 and Sup35, respectively (61). Formation of both prions involves conversion of the native proteins into an infectious, amyloid form. Ure2 and Sup35 have served as powerful model systems for examining the basis for amyloid formation and propagation. Both proteins possess a well-ordered functional domain responsible for the normal function of the protein, while a functionally and structurally separate glutamine/asparagine (Q/N)-rich intrinsically disordered domain is necessary and sufficient for prion aggregation and propagation (4, 26, 27, 52, 53). Both proteins can form multiple prion variants, which are distinguished by the efficiency of prion propagation and by the precise structure of the amyloid core (14, 54).Five other prion proteins have also been identified in yeast: Rnq1 (13, 46), Swi1 (15), Cyc8 (33), Mca1 (30), and Mot3 (1). Numerous other proteins, including New1, contain domains that show prion activity when inserted in place of the Sup35 prion-forming domain (PFD) (1, 42). Each of these prion proteins contains a Q/N-rich PFD. Similar Q/N-rich domains are overrepresented in eukaryotic genomes (28), raising the intriguing possibility that prion-like structural conversions by Q/N-rich domains may be common in other eukaryotes. However, we currently have little ability to predict whether a given Q/N-rich domain can form prions.A variety of algorithms have been developed to predict a peptide''s propensity to form amyloid fibrils based on its amino acid sequence, including BETASCAN (6), TANGO (17), Zyggregator (51), SALSA (62), and PASTA (55). These algorithms have been successful at identifying regions prone to amyloid aggregation and predicting the effects of mutations on aggregation propensity for many amyloid-forming proteins. However, they have generally been quite ineffective for Q/N-rich amyloid domains such as the yeast PFDs. For example, using the statistical mechanics-based algorithm TANGO (17), which predicts aggregation propensity based on a peptide''s physicochemical properties, Linding et al. found that the Sup35 and Ure2 PFDs both completely lack predicted β-aggregation nuclei (24). Similarly, yeast PFDs are generally lacking in the hydrophobic residues predicted by algorithms such as Zyggregator to nucleate amyloid formation.Why are these algorithms so effective for many amyloid-forming proteins but not for yeast PFDs? For most amyloid proteins, amyloid formation is driven by short hydrophobic protein stretches, and increased hydrophobicity is correlated with an increased amyloid aggregation propensity (34). In contrast, the yeast PFDs are all highly polar domains, due largely to the high concentration of Q/N residues and the lack of hydrophobic residues. High Q/N content is clearly not a requirement for a domain to act as a prion in yeast, since neither the mammalian prion protein PrP nor the Podospora anserina prion protein HET-s is Q/N rich, yet fragments from both proteins can act as prions in yeast (49, 50). However, the significant compositional differences between the yeast PFDs and most other amyloid/prion proteins suggest that there may be two distinct classes of amyloid-forming proteins driven by different types of interactions. Specifically, Q/N residues, which are predicted to have a relatively low amyloid propensity in the context of hydrophobic amyloid domains (34), may promote amyloid formation when present at sufficiently high density. Stacking of Q/N residues to form polar zippers has been proposed to stabilize amyloid fibrils (35). Consistent with this hypothesis, mutational studies of Sup35 indicate that Q/N residues are critical for driving [PSI⁺] formation (12), and expanded poly-Q or poly-N tracts are sufficient to drive amyloid aggregation (36, 63). Therefore, this paper examines the sequence features that allow the polar, Q/N-rich yeast PFDs to form prions.Mutational studies of the PFDs of Ure2 and Sup35 have shown that amino acid composition is the predominant feature driving prion formation (40, 41). Due to the unique compositional biases observed in the yeast PFDs, algorithms have been developed to identify potential PFDs based solely on amino acid composition (19, 28, 42). These algorithms are designed to produce a list of potential prion proteins that meet a specific set of criteria (such as high Q/N content) but are not able to predict the prion propensity of each member of the list or to predict the effects of mutations on prion formation. A recent study by Alberti et al. was the first to systematically test whether compositional similarity to known PFDs is sufficient to distinguish between Q/N-rich proteins that form prions and those that do not. They developed a hidden Markov model to identify domains that are compositionally similar to known PFDs and then analyzed the 100 highest-scoring Q/N-rich domains in a series of in vivo and in vitro assays (1). Remarkably, they discovered 18 proteins with prion-like activity in all assays. However, an equal number, including some of the domains with greatest compositional similarity to known PFDs, showed no prion-like activity.This inability to distinguish between Q/N-rich proteins that form prions and those that do not might seem to suggest that amino acid composition is not an accurate predictor of prion propensity. However, an alternative explanation is that known yeast PFDs are not an ideal training set for a composition-based prediction algorithm, since yeast prions are likely not optimized for maximal prion propensity. It is unclear whether yeast prion formation is a beneficial phenomenon providing a mechanism to regulate protein activity or a detrimental phenomenon analogous to human amyloid disease. [PSI⁺] can increase resistance to certain stress conditions (56), but the failure to observe [PSI⁺] in wild yeast strains (29) argues that beneficial [PSI⁺] formation is at most a rare event. If yeast prions are diseases, the PFDs certainly would not be optimized for maximum prion potential. If prion formation is a beneficial event allowing for rapid conversion between active and inactive states, the prion potential of the PFD would be optimized such that the frequencies of prion formation and loss would yield the optimal balance of prion and nonprion cells (25). Thus, specific residues might be excluded from yeast PFDs either because they inhibit prion formation or because they too strongly promote prion formation; bioinformatic analysis can reveal which residues are excluded from yeast PFDs but not why they are excluded. Accurate prediction of prion propensity requires understanding which deviations from known prion-forming compositions will promote prion formation and which will inhibit.We have therefore developed the first in vivo method to quantitatively determine the prion propensity for each amino acid in the context of a Q/N-rich PFD. As expected, we found proline and charged residues to be strongly inhibitory to prion formation; but surprisingly, despite being largely underrepresented in yeast PFDs, hydrophobic residues strongly promoted prion formation. Furthermore, although Q/N residues dominate yeast PFDs, prion propensity appears relatively insensitive to the exact number of Q/N residues. Using these data, we were able to distinguish with approximately 90% accuracy between Q/N-rich domains that can form prion-like aggregates and those that cannot. These experiments provide the first detailed insight into the compositional requirements for yeast prion formation and illuminate the different methods by which Q/N- and non-Q/N-rich amyloidogenic proteins aggregate.     

Tracking protein aggregate interactions

Amyloid fibrils share a structural motif consisting of highly ordered β-sheets aligned perpendicular to the fibril axis.^{1, 2} At each fibril end, β-sheets provide a template for recruiting and converting monomers.³ Different amyloid fibrils often co-occur in the same individual, yet whether a protein aggregate aids or inhibits the assembly of a heterologous protein is unclear. In prion disease, diverse prion aggregate structures, known as strains, are thought to be the basis of disparate disease phenotypes in the same species expressing identical prion protein sequences.^4–7 Here we explore the interactions reported to occur when two distinct prion strains occur together in the central nervous system.Key words: prion, prions, strain, TSE, interaction, amyloid, LCP, neurodegeneration, aggregation     

Aerosols: An underestimated vehicle for transmission of prion diseases?

We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise—for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.Key words: prion, prion transmission, scrapie, chronic wasting diseases, CWD, Creutzfeldt-Jacob-disease, CJD, TSE, aerosol, pathogens, allergensPrions, the causative agents of transmissible spongiform encephalopathies, can be undoubtedly propagated from one individual organism to another. The specific routes of prion transmission have been subjected to intensive studies over the past two decades. Incidental and iatrogenic transmission has occurred through the intracerebral route in the case of Dura mater implants¹ and the parenteral route in the case of contaminated pituitary hormones.² In addition, the Bovine Spongiform Encephalopathy (BSE) disaster has provided grim evidence that prion can be transmitted enterally as well. Experimental transmission of prions has been routinely achieved via intraperitoneal and intravenous injection^3,4 but also through more exotic routes such as intralingual,⁵ intranerval⁶ and conjunctival inoculation⁷ and via the nasal cavity.⁸In all prion disease paradigms studied so far the propagation, accumulation and dissemination of the prion protein has been mostly shown to depend on a functional immune system.^9–12 This dependence of prion pathogenesis on the lymphoid compartment, however, is only true for peripheral routes of infection—whereas direct inoculation into the brain does not require any components of the adaptive or innate immune system.B cells in secondary lymphoid organs have been shown to be of importance for the neuroinvasion of the prion protein; in contrast, B lymphocytes in the blood do not appear to play a crucial role.^13–15A special role in prion pathogenesis can be assigned to follicular dendritic cells (FDC). The generation, maturation and function of FDC are dependent on cytokines and chemokines predominantly synthesized and secreted by B lymphocytes. Consistently with this role of B cells in prion pathogenesis, B cell deficient mice show a significantly impaired prion replication due to severely impaired maturation of FDCs.¹⁶ Other soluble and membrane-bound immune mediators such as lymphotoxin heterotrimers and TNFalpha^17,18 as well as components of the complement system^19,20 play an important role in prion pathogenesis.While prions mostly reside in tissues, prion infectivity has also been detected in a variety of body fluids including cerebrospinal fluid,²¹ blood,²² saliva,²³ milk²⁴ and urine.²⁵ Although shedding of prions may occur constitutively from these secretions and excretions, many of the latter phenomena are enhanced by chronic inflammatory processes such as granulomas²⁶ and follicular infiltrates,²⁷ which trigger the maturation of lymphotoxin-dependent, prion-replicating cells.²⁶ The presence of prions in fluids begs the question whether nebulization, and subsequent inhalation, of such fluids may trigger prion infections.Aerosols are finely dispersed particles originating from solid material or liquid using air or other gases as carriers. Natural examples of aerosols include dust (e.g., volcano ashes), smoke, haze and sprays (e.g., sneezing or sea water sprays from breaking waves). Aerosols might be formally categorized as primary or secondary, with primary aerosols being generated in mechanical or thermal processes e.g., by whirling up, impact on surfaces, or burning, whereas secondary aerosols are generated during chemical reactions or by using condensation nuclei.Primary aerosols play an important role in microbiology since they can act as efficacious vehicles for pollen, spores, algae, fungi, bacteria and viruses. Of medical importance are also dandruff, fragments of fur, hairs or skin and mites, which can all function as allergens and trigger e.g., allergic asthma.Moreover, aerosols are excellent vehicles for the transportation of drugs into the respiratory tract. The size of the individual droplets is crucial in specifying the target organs of aerosol. Particle sized 3–10 µm are generally deposited in the nasal cavity and in the throat, whereas smaller particles (e.g., 1 µm) tend to deposit within the lower airways. In rodents pulmonary deposition can reach 10%.^28,29 In humans, particles of 5 µm may reach the lung if inhaled orally, but deposition in the alveolar compartment after inhaling via the nose is highly unlikely.^28,29 For the reasons discussed above, we have become interested in exploring the transmission potential of aerosol-borne prions. Indeed, we found that mouse scrapie can be efficiently transmitted via aerosols.³⁰ In addition to results obtained by exposure to aerosols, we found that mice developed prion infections when inoculated intranasally.Interestingly, this route of transmission was entirely independent on immune cells as shown by challenging various transgenic mouse strains lacking defined functions of the immune system.Well-known examples of transmission of pathogens via aerosols are infections by respiratory viruses (e.g., influenza viruses, adenoviruses, rhinoviruses, coronaviruses) and bacterial diseases (e.g., legionellosis, pneumonic plague by Yersinia pestis, Q-fever by Coxiella burnettii, anthrax) and fungal diseases (particularly aspergillosis and candidosis). In stark contrast, aerosols have historically never been regarded as potential vectors for prion diseases—although very little data existed in favor or against this possibility. This attitude goes along with the implicit “conventional wisdom” that prions are not airborne diseases. However, the concept of “airborne disease” in all the bacterial, fungal and viral examples quoted above, encompasses three distinct phases: (1) release of the infectious agent into aerosols by an infected donor, (2) uptake by a healthy recipient and (3) establishment of disease. It is self-evident that little or no natural transmission between individuals will be observed if any one of these three steps is inefficient. The epidemiological evidence from human prion diseases seems to indicate, albeit indirectly, that step #1 does not occur in CJD patients—inter alia because there is a dearth of evidence of proximity clustering of sCJD.³¹ In the case of CWD the situation may be different since saliva and droppings, which might plausibly give rise to powerful aerosols under a variety of conditions, were found to harbor infectivity. Finally, milk from sheep affected by mastitis can carry scrapie infectivity and—again—could conceivably give rise to aerosols. Since both CWD and sheep scrapie can efficiently spread horizontally within animal collectives, it is extremely appealing to speculate whether aerosols may play a role in said transmission.In natural scrapie in sheep horizontal transmission of prion diseases has been long thought to arise from placental contamination. However, in mice suffering from nephritis prion infectivity is shed with the urine.²⁵ Furthermore, sheep having a mastitis can transmit infectious prions with milk.³²In Chronic Wasting disease (CWD) of deer several careful studies have been performed that, together with our present finding, depose in favor of airborne transmission in this naturally occurring disease. Indeed, CWD prions can be transmitted experimentally via aerosol and the nasal route to transgenic cervidized mice.³³ Although no anecdotal or epidemiological evidence has come forward that airborne transmission may be important for the spread of CWD, several lines of thought suggest that this possibility is not implausible. In deer, prions have been detected in urine, saliva, feces and blood of diseased animals. Moreover, it was claimed that pathological prion protein could be recovered from the environmental water in an endemic area.³⁴ Since all fluids can act as sources for the generation of aerosols, any of the body fluids mentioned above may represent the point of origin for airborne transmission of CWD prions.In this context, also the presence of infectious prions in blood of patients should be mentioned which was demonstrated by the transmission of vCJD by blood transfusions.^35,36 The growing body of evidence that prion transmission can be airborne—at least under certain conditions—dictates that the release of potentially contaminated aerosols should be avoided under all circumstances. In this context it is mandatory that reliable precautions be defined and followed in scientific and diagnostic laboratories. In particular, it is self-evident that safety cabinets should be used while processing brain and nerve tissue (or any other potentially contaminated tissue) of man and animals suspected with prion disease. Our experience shows that this necessity is generally very well-understood by prion scientists.A further stone of contention relates to the biosafety level of the laboratory environment. Because prions were hitherto considered not be airborne, so far no specific regulations have been implemented. As a consequence, prion laboratories have been mostly required to adhere to the category “BSL3**.” While it is understood that the airborne transmission of prions has thus far only been observed under extreme conditions, we feel that it is in order to critically reassess biosafety regulations in the light of the recent discoveries. In particular, one might consider implementing more stringent measures towards protecting workers within diagnostic and scientific laboratories from aerosols.The situation in slaughterhouses and plants handling potentially contaminated offal may be even more problematic. Although regulations in slaughterhouses dictate the use of protecting glasses and masks or, alternatively, visors the use of personal protecting equipment should be rigorously controlled. In addition, high-pressure cleaning devices produce massive aerosols and should be strictly avoided in areas of slaughterhouses where prion-containing material may be processed. Regulations concerning cleaning of heads from slaughtered animals do pay attention to aerosol avoidance, e.g., by allowing only water hoses without pressure.A case in point is the severe neurological syndrome arising in swine abattoir workers.³⁷ Here, an immune-mediated polyradiculoneuropathy was reported to be related to a process using high-pressure fluids to remove the brains of swine.³⁷ During this process, high amounts of swine brain tissue became aerosolized and were inhaled and/or gained access to the respiratory tract mucosa of abattoir workers, resulting in immunization with myelin constituents akin to experimental autoimmune encephalitis (EAE). Although significant physiological differences exist concerning breathing, where humans are regarded as mouth breathers and mice as nose breathers, many people indeed show nose breathing under no or only moderate body burden. Therefore, results obtained in mouse experiments might also be extrapolated to a considerable extent to the situation in man.In this context it is of importance to stress again that aerosols might be generated under various conditions and represent a normal entity of the environment in a variety of daily life situations.In our studies of airborne transmission of prion protein in mice³⁰ we took advantage of the fact that mice breathe exclusively through their nostrils^38,39 and therefore could be exposed in groups to aerosolized brain suspensions. Using this system, it was possible to vary both time of exposure as well as concentration of the prion load in the aerosol. We were surprised to discover that exposure times as short as 1 min were sufficient to achieve high attack rates. By extending the time of exposure it became obvious that incubation times were shortened. A possible alternative route of infection via the cornea or the conjunctiva was extremely unlikely, since newborn mice, whose eyelids were still closed, could also be infected. These findings show that the aerogenic transmission of prions is very efficient.But how do prions spread from the airways to the brain? Peripheral replication of prions in the lymphoid system—a characteristic of most other peripheral routes of transmission—appeared to be dispensable. Instead, the results argue for a direct pathway of brain invasion. One anatomical peculiarity of the nasal cavity is the “area cribriformis” of the olfactory epithelium. Here the olfactory bulb sprouts axons of olfactory receptor neurons passing through the cribriform plate of the ethmoidal bone to reach the olfactory mucosa where olfactory cilia extend representing non-myelinated nerve endings. Thus, open nerve endings are located in the nasal cavity through which aerosolized infectious prions might get access to the brain. In this context it is noteworthy that pathological prion protein was found in the olfactory cilia and basal cells of the olfactory mucosa of sCJD patients, as well as in the olfactory bulb and olfactory tract.^40,41 However, it was hitherto never clearly documented that olfactory receptor neurons represent an entry site for infectious prions; this might also be due to the sensitivity threshold of detection assays.In conclusion, aerosols can infect mice with a surprisingly high efficiency. Just how important a role is played by this newly recognized pathway of spread in natural transmission is, as of now, unclear and in need of further studies. Although it was not identified as a route of infection in epidemiological studies thus far, the worryingly high attack rate suggests that we would be well-advised to carefully avoid the inhalation of aerosols from prion-containing materials.     

RepA-WH1 prionoid: A synthetic amyloid proteinopathy in a minimalist host

The intricate complexity at the molecular and cellular levels of the processes leading to the development of amyloid proteinopathies is somehow counterbalanced by their common, universal structural basis. The later has fueled the quest for suitable model systems to study protein amyloidosis under quasi-physiological conditions in vitro and in simpler organisms in vivo. Yeast prions have provided several of such model systems, yielding invaluable insights on amyloid structure, dynamics and transmission. However, yeast prions, unlike mammalian PrP, do not elicit any proteinopathy. We have recently reported that engineering RepA-WH1, a bacterial DNA-toggled protein conformational switch (dWH1→mWH1) sharing some analogies with nucleic acid-promoted PrP^C→PrP^Sc replication, enables control on protein amyloidogenesis in vitro. Furthermore, RepA-WH1 gives way to a non-infectious, vertically-transmissible (from mother to daughter cells) amyloid proteinopathy in Escherichia coli. RepA-WH1 amyloid aggregates efficiently promote aging in bacteria, which exhibit a drastic lengthening in generation time, a limited number of division cycles and reduced fitness. The RepA-WH1 prionoid opens a direct means to untangle the general pathway(s) for protein amyloidosis in a host with reduced genome and proteome.Key words: RepA-WH1, bacterial prionoid, synthetic prionoid, amyloid proteinopathy, aging in bacteriaThe development of suitable model systems for the study of the complex neurodegenerative and systemic human diseases caused by the aggregation of proteins into amyloid cross-β assemblies has been successfully attempted in different ways.¹ From the point of view of the macromolecules involved, besides those proteins directly involved in amyloid diseases (Alzheimer''s β-amyloid and Tau, Creutzfeldt-Jakob''s PrP, Parkinson''s α-synuclein, Huntington''s huntingtin or β₂-microglobulin in dialysis-related amyloidosis), an ever increasing number of disease-unrelated proteins can be forced to unfold, and subsequently assemble, as amyloids under extreme, non-physiological physicochemical conditions. Both kinds of model proteins have been crucial to establish our current understanding of the common molecular basis for protein amyloidogenesis.¹ At the organisms side, although animal models, most notably mice, have returned invaluable information on protein amyloidosis, the complexity of the intricate regulatory and biochemical networks inherent to metazoans and their cultured cells, has hampered the outlining of a clear scenario on the mechanism(s) leading to cytotoxicity. Cytotoxicity can arise either from properties common to most amyloidogenic proteins, such as targeting of cell membranes by amyloid oligomers or co-aggregation of essential cell factors, or through pathways particular to each protein and its associated disease.² The key to such a riddle relies on comprehensive systems biology analyses, but also on resorting to experimental models with their number of potential variables (proteins and their interactions) drastically reduced, but yet showing the same (cytotoxic) response.Since the discovery of the Ure2p/[URE3⁺] and Sup35p/[PSI⁺] prions in yeast, these (relatively) simple eukaryotic microorganisms have been instrumental in addressing the molecular basis for amyloid conformational templating, structural polymorphism and cell-to-cell transmissibility.^3–5 However, two limitations to the applicability of yeast prions as universal models for amyloidosis are noteworthy: (1) the amyloidogenic sequence stretches in yeast prions are consistently Gln/Asn-rich, unlike most proteins involved in amyloid proteinopathies (which bear hydrophobic stretches) with the exception of the proteins involved in Huntington disease and in related ataxias; (2) even more importantly, while yeast prions are the epigenetic determinants of distinct, mildly advantageous phenotypes that improve adaptability to environmental challenges,^6,7 they are not the causative agents of a proteinopathy in yeast albeit, when overexpressed, Sup35p/[PSI⁺] becomes detrimental for cell growth. Although this prion has recently been successfully propagated in Escherichia coli,⁸ it still does not behave as a proper pathogenic agent in this microorganism. Natural amyloids have also been described and characterized in bacteria such as E. coli (curli/CsgA)⁹ and Pseudomonas (FapC),¹⁰ but, invariantly, they are extracellularly secreted and functional in scaffolding cellular consortia such as biofilms. A case apart is posed by inclusion bodies, intracellular protein aggregates accumulated in bacterial cytoplasm upon heterologous expression of recombinant proteins, which exhibit some amyloid features¹¹ but with a discrete detrimental effect on cell fitness.^12,13     

Alimentary prion infections: Touchdown in the intestine

Neurodegenerative diseases are caused by proteinaceous aggregates, usually consisting of misfolded proteins which are often typified by a high proportion of β-sheets that accumulate in the central nervous system. These diseases, including Morbus Alzheimer, Parkinson disease and Transmissible Spongiform Encephalopathies (TSEs)—also termed prion disorders—afflict a substantial proportion of the human population and, as such, the etiology and pathogenesis of these diseases has been the focus of mounting research. Although many of these diseases arise from genetic mutations or are sporadic in nature, the possible horizontal transmissibility of neurodegenerative diseases poses a great threat to population health. In this article we discuss recent studies that suggest that the “non-transmissible” status bestowed upon Alzheimer and Parkinson diseases may need to be revised as these diseases have been successfully induced through tissue transplants. Furthermore, we highlight the importance of investigating the “natural” mechanism of prion transmission including peroral and perenteral transmission, proposed routes of gastrointestinal uptake and neuroinvasion of ingested infectious prion proteins. We examine the multitude of factors which may influence oral transmissibility and discuss the zoonotic threats that Chronic Wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE) and Scrapie may pose resulting in vCJD or related disorders. In addition, we suggest that the 37 kDa/67 kDa laminin receptor on the cell surface of enterocytes, a major cell population in the intestine, may play an important role in the intestinal pathophysiology of alimentary prion infections.Key words: prion, 37 kDa/67 kDa laminin receptor, CJD, BSE, CWD, scrapie, Alzheimer disease, Parkinson disease, intestine, enterocytesMany different mechanisms exist which underlie the etiology of the numerous neurodegenerative diseases affecting the human population. Amongst the most prominent are Morbus Alzheimer, prion disorders, Parkinson disease, Chorea Huntington, frontotemporal dementia and amylotrophic lateral sclerosis. The molecular mechanisms underlying these diseases vary; however, all neurodegenerative diseases share a common feature: they are caused by protein aggregation. The only neurodegenerative diseases proven to be transmissible are prion disorders. In contrast to frontotemporal dementia, recent evidence suggests that Alzheimer and Parkinson diseases may also be transmissible. Pre-symptomatic Alzheimer disease (APP23) mice exhibited an increase in the Alzheimer phenotype when brain homogenate of autopsied human Alzheimer disease patients and older, amyloid beta- (Aβ-) laden APP23 mice was injected into their hippocampi.¹ These findings suggest that the Aβ-abundant brain homogenate of Alzheimer disease patients may possess the ability to induce or supplement the overproduction of Aβ, possibly leading to the onset of Alzheimer disease.The pathological feature associated with Parkinson disease is the formation of Lewy bodies in cell bodies and neuronal processes in the brain.² The main component of these protein aggregates is α-synuclein (reviewed in ref. ²). Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy embryonic neurons that had been grafted onto the brain tissue of the patients several years before (prior to said examination).^3–5 It may thus be proposed that α-synuclein transmission is possible from diseased to healthy neurons, suggesting that Parkinson disease may be transmissible from a Parkinson disease patient to a healthy individual. These findings imply that Alzheimer and Parkinson diseases may be transmissible through tissue transplants and the use of contaminated surgical tools.⁶Prion disorders, also termed Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative diseases that affect the central nervous system (CNS) of multiple animal species. In lieu of the social, economic and political ramifications of such infections, as well as the possible intra- and interspecies transmissibility of such disorders, various routes of experimental transmission have been investigated including intracerebral, intraperitoneal, intraventricular, intraocular, intraspinal and subcutaneous injections (reviewed in ref. ^7–9). However, such routes of transmission are not representative of the “natural” mechanism as the majority of prion disorders are contracted through ingestion of infectious prion (PrP^Sc) containing material. Thus, the peroral and perenteral prion transmission is of greatest consequence with respect to TSE disease establishment. Moreover, the presence of PrP^Sc in the buccal cavity of scrapie-infected sheep¹⁰ (reviewed in ref. ¹¹) and the possible horizontal transfer as a result hereof, as may be similarly proposed for animals suffering from other TSEs, may further contribute to the oral transmissibility of TSEs.A number of model systems have been employed to study TSE transmissibility. Owing to ethical constraints, TSE transmissibility to humans via the oral route may not be directly investigated and as a result hereof, alternative model systems are needed. These may include the use of transgenic mice, cell lines which are permissive to infection¹² and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. ⁹).Intestinal entry of PrP^Sc has been proposed to occur via two pathways, the membranous (M) cell-dependent and M cell-independent pathways (Fig. 1).^13,14 The former involves endocytic M (microfold)-cells, which cover the intestinal lymphoid follicles (Peyer''s patches)¹⁴ and may take up prions and thereby facilitate the translocation of these proteins across the intestinal epithelium into the lymphoid tissues (reviewed in ref. ⁹) as has been demonstrated in a cellular model.¹³ Following such uptake by the M cells, the prions may subsequently pass to the dendritic cells and follicular dendritic cells (FDCs) (Fig. 1), which allow for prion transport to the mesenteric lymph nodes and replication, respectively.¹⁵ The prion proteins may subsequently gain access to the enteric nervous system (ENS) and ultimately the central nervous system (CNS).¹⁵Open in a separate windowFigure 1Proposed routes of gastrointestinal entry of ingested infectious prions (PrP^Sc) as well as possible pathways of amplification and transport to the central nervous system.However, prion intestinal translocation has been observed in the absence of M cells and has been demonstrated to be as a result of the action of polar, 37 kDa/67 kDa LRP/LR (non-integrin laminin receptor; reviewed in ref. ^16–18) expressing enterocytes. Enterocytes are the major cell population of the intestinal epithelium and due to their ability to endocytose pathogens, nutrients and macromolecules,¹⁹ it has been proposed that these cells may represent a major entry site for alimentary prions (Fig. 1).Since enterocyte prion uptake has been demonstrated to be dependent on the presence of LRP/LR on the apical brush border of the cells,^14,20 the interaction between varying prion protein strains and the receptor^21–23 may be employed as a model system to study possible oral transmissibility of prion disorders across species as well as the intestinal pathophysiology of alimentary prion infections.²⁴ Moreover, the blockage of such interactions through the use of anti-LRP/LR specific antibodies has been reported to reduce PrP^Sc endocytosis¹⁹ and thus these antibodies may serve as potential therapeutics to prevent infectious prion internalization and thereby prevent prion infections. It must be emphasized that the adhesion of prion proteins to cells is not solely dependent on the LRP/LR-PrP^Sc interactions;²⁴ however, this interaction is of importance with regards to internalization and subsequent pathogenesis.We applied the aforementioned cell model to study the possible oral transmission of PrP^BSE, PrP^CWD and ovine PrP^Sc to cervids, cattle, swine and humans.²⁴ The direct transmission of the aforementioned animal prion disorders to humans as a result of dietary exposure and the possible establishment of zoonotic diseases is of great public concern. It must however be emphasized that the study investigated the co-localization of LRP/LR and various prion strains and not the actual internalization process.PrP^BSE was shown to co-localize with LRP/LR on human enterocytes²⁴, thereby suggesting that PrP^BSE is transmissible to humans via the oral route which is widely accepted as the manner by which variant CJD originated. This suspicion was previously investigated using a macaque model, which was successfully perorally infected by BSE-contaminated material and subsequently lead to the development of a prion disorder that resembles vCJD.²⁵ These results, due to the evolutionary relatedness between macaques and humans, allowed researchers to confirm the oral transmissibility of PrP^BSE to humans. PrP^BSE may also potentially lead to prion disorder establishment in swine,²⁴ livestock of great economic and social importance.The prion disorder affecting elk, mule deer and white-tailed deer is termed CWD. Cases of the disease are most prevalent in the US but are also evident in Canada and South Korea.^26,27 As the infectious prion isoform is reported to be present in the blood²⁸ and skeletal muscle,²⁹ hunting, consumption of wild venison and contact with other animal products derived from CWD-infected elk and deer may thereby pose a public health risk. Our studies demonstrate that PrP^CWD co-localizes with LRP/LR on human enterocytes²⁴ thereby suggesting a possible oral transmissibilty of this TSE to humans. This is, however, inconsistent with results obtained during intra-cerebral inoculation of the brains and spinal cords of transgenic mice overexpressing the human cellular prion protein (PrP^c),^26,27 which is essential for TSE disease establishment and progression. Further, discrepancies have also been reported with respect to non-human primates, as squirrel monkeys have been successfully intracerebrally inoculated with mule-deer prion homogenates,³⁰ while cynolmolgus macaques were resistant to infection.³¹ CWD has been transmitted to ferrets, minks and goats³² and as these animals may serve as domestic animals or livestock, secondary transmission from such animals to humans, through direct contact or ingestion of infected material, may be an additional risk factor that merits further scientific investigation.Ovine PrP^Sc co-localization with LRP/LR on human and bovine enterocytes may be indicative of the infectious agents'' ability to effect cross-species infections. The oral transmissibility of Scrapie has been confirmed in hamsters fed with sheep-scrapie-infected material.³³The discrepancies with regards to the transmissibility of certain infectious prion proteins when assessed by different model systems may be due to the experimental transmission route employed. Oral exposure often results in significantly prolonged incubation times when compared to intracerebral inoculation techniques and thus failure of transgenic mice and normal experimental animals to develop disease phenotypes after being fed TSE-contaminated material may not necessarily indicate that the infection process failed.¹⁴ Apart from the route of infection, numerous other factors may influence transmission between species, including dose, PrP polymorphisms and genetic factors, the prion strain employed as well as the efficacy of prion transport to the CNS.³⁴ The degree of homology between the PrP^c protein in the animals serving as the infectious prion source and recipient has also been described as a feature limiting cross-species transmission.³⁴ The negative results, as referred to above, obtained upon prion-protein inoculation of animal models may have resulted due to the slow rate at which the infectious prion induces conformational conversion of the endogenous PrP^c in the animal cells and this in turn results in low levels of infectious prion replication and symptom development.²⁷Furthermore, even in the event that certain prion disorders are not directly transmissible to humans, most are transmissible to at least a single species of domestic animal or livestock. The infectious agents properties may be altered in the secondary host such that it becomes transmissible to humans (reviewed in ref. ³⁵). Thus, interspecies transmission between animals may indirectly influence human health.It is noteworthy to add that although the oral route of PrP^Sc transmission may result in prolonged incubation times, it may broaden the range of susceptible hosts. A common constituent of food is ferritin, a protein that is resistant to digestive enzyme hydrolysis and, due to its homology across species, it may serve as co-transporter of PrP^Sc and facilitate enterocyte internalization of the infectious prion.³⁶ It may thus be proposed that prion internalization may occur via a ferritin-PrP^Sc complex even in the absence of co-localization between the infectious agent and LRP/LR such that many more cross-species infections (provided that the other infection factors are favorable) may be probable.³⁷ In addition, digestive enzymes in the gastrointestinal tract facilitate PrP^Sc binding to the intestinal epithelium and subsequent intestinal uptake³⁶ and thus depending on the individuals'' digestive processes, the susceptibility to infection and the rate of disease development may vary accordingly. As a result hereof, though laboratory experiments in cell-culture and animal models may render a particular prion disorder non-infectious to humans, this may not be true for all individuals.In lieu of the above statements, with particular reference to inconsistencies in reported results and the multiple factors influencing oral transmissibility of TSEs, further transmission studies are required to evaluate the zoonotic threat which CWD, BSE and Scrapie may pose through ingestion.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival