# Rho Factor Exhibits a Predicted PrD Containing a Putative N-rich Amyloid Core

Because many of the prion-like polypeptides identified in eukaryotes are RNA binding proteins (King et al., 2012; Kim et al., 2013), we focused our attention in the transcription termination factor Rho (Rho). Rho is required for the factor-dependent transcription termination by an RNA polymerase in prokaryotes and is essential for the viability of the cell (Richardson, 1996; Cardinale et al., 2008; Washburn and Gottesman, 2011; Krishna Leela et al., 2013). Recent studies indicate that besides being a housekeeping gene, Rho can function as a gene regulator and participates in the control of prophage maintenance in bacterial genomes (Boudvillain et al., 2013; Menouni et al., 2013). Accordingly, it plays a critical role in determining what proteins are present in the cell, in what amounts and thus modulating the organism's phenotype.

PAPA predicts an 80 residues long PrD close to the Rho factor N-terminus, which resides in a longer intrinsically disordered region, as predicted with FoldIndex (Prilusky et al., 2005) (**Figure 1**). pWALTZ predicts the presence of three overlapping 21 residues long amyloid stretches comprising residues 90–110, 92–112, and 93–113 inside the identified Rho PrD (**Figure 1**). When we analyzed the location of structured, unstructured and PrD regions in Rho factor, we found that, overall, its topology resembles that observed in certain *bona fide* yeast prions, like Ure2p (**Figure 1**). Globular domains in prion-like proteins are responsible for their biological function. The Rho factor consists of six identical subunits, each containing three functional domains. The RNA binding site has been localized to the N-terminal portion of the protein, the ATP binding site is located in the central portion of the primary sequence, and subunit interaction sites have been proposed to reside in the C-terminal region (Geiselmann et al., 1993; Bogden et al., 1999). The interaction of Rho with RNA is critical to all the activities of the protein. Thus RNA binding is required to activate the RNAdependent ATPase activity of Rho. The predicted PrD and the RNA binding domain are contiguous in Rho, a topology that is also found in many eukaryotic prion-like proteins (King et al., 2012; Espinosa Angarica et al., 2013; Malinovska et al., 2013; Navarro et al., 2015).

The widely accepted "amyloid-stretch" hypothesis proposes that the amyloid potential of amyloidogenic proteins resides in short, highly amyloidogenic regions that act by nucleating the aggregation reaction (Ventura et al., 2004; Esteras-Chopo et al., 2005). We have recently proposed that this view also applies for prion-like proteins, explaining why all known prions adopt amyloid conformations in their propagative state (Sabate et al., 2015a). In order to assess if this is the case of Rho factor, we experimentally characterized the predicted central amyloid core of the prion domain (cPrD) using a synthetic peptide corresponding to sequence 92- NNNNSNFNNNSNNNSSFNNSN-112, with a 67% N content. Despite pWALTZ, which is specially intended to analyze PrDs, predicts that this N-rich sequence would endorse the surrounding PrD with significant amyloidogenic potential, wellcontrasted aggregation predictors like AGGRESCAN (Conchillo-Solé et al., 2007), TANGO (Fernandez-Escamilla et al., 2004) or FoldAmyloid (Garbuzynskiy et al., 2010) fail to predict any aggregation-prone region in this peptide and, indeed, they predict it to be soluble.

# Rho cPrD Forms **β**-sheet Enriched Aggregates

As a first step to experimentally characterize the selected cPrD we analyzed its *in vitro* aggregation properties. Rho cPrD was incubated at 25, 50, and 100 μM at 25◦C for 4, 48, and 120 h and aggregation from its initially soluble state was evaluated using synchronous light scattering (**Figure 2**). A concentration dependent scattering signal is observed after 4 h. However, the signal corresponding to the 25 and 50 μM solutions does not evolve significantly with time, whereas the scattering signal of the 100 μM peptide solution steadily increases to attain a maximum after 120 h (**Figure 2C**). Accordingly, unless otherwise indicated, all subsequent experiments were performed with the peptide at a concentration of 100 μM.

For most amyloids, the self-assembly reaction depends on the formation of intra-chain hydrophobic clusters (Hills and Brooks, 2007). However, Rho cPrD is a highly polar peptide, with less than 10% of its residues being hydrophobic. We explored the presence of exposed hydrophobic clusters in the aggregates formed by Rho cPrD at different times by measuring their binding to bis-ANS (**Figure 3**), a dye that increases its fluorescence emission upon interaction with these regions (Gohlke, 1972; de Groot et al., 2007; Zhou et al., 2012). The bis-ANS fluorescence emission maximum blue-shifts from 530 nm, in the absence of peptide, to 509 nm in the presence of the peptide after 4 h. This spectral change is even more pronounced after 48 h, even if the global intensity decreases. Bis-ANS fluorescence emission attains a maximum at 120 h, with its spectral maximum blue-shifted to 490 nm. These data clearly indicate that the two phenylalanine (F) residues in Rho cPrD play an important role on its aggregation reaction, leading to the formation of strong hydrophobic patches in the final aggregates.

FIGURE 2 | Aggregation of Rho cPrD as a function of the concentration and the incubation time. Aggregation changes were monitored by light scattering at different Rho cPrD concentrations 25 (doted line), 50 (dashed line), and 100 μM (solid line) incubated at 25◦C during (A) 4 h, (B) 120 h. The scattering at 4, 48, and 120 h for the 100 μM peptide solution is shown in (C).

The aggregation of proteins into amyloid fibrils results in the formation of intermolecular β–sheets (Nelson et al., 2005). To get insights into the secondary structure content of the assemblies formed by Rho cPrD, we analyzed the amide I region of the FTIR spectrum (1700–1600 cm−1) (**Figure 4**). This region corresponds to the absorption of the carbonyl peptide bond group of the protein main chain and is a sensitive marker of the protein secondary structure. Examination of the secondary structure of Rho cPrD peptide by deconvolved FTIR spectra allow us to assign the individual secondary structure elements and their relative contribution to the main absorbance signal at the beginning (4 h) and ending (120 h) of the aggregation reaction (**Figure 4**; **Table 1**). After 4 h of incubation the spectrum of Rho cPrD is dominated by a band at 1663 cm−1, corresponding to disordered structures, accounting for 74% of the total area. However, the presence of an inter-molecular β–sheet component at 1624 cm−1 is already observable at this time point. At the end of the reaction (120 h), the FTIR spectrum of Rho cPrD is dominated by a band at 1633 cm−1 attributable to β–sheet conformations. At this stage, the low frequency β–sheet components at 1607 and 1633 cm−1 together with the high frequency β–sheet component at 1676 cm−1 account for 77% of the total area, with disordered

conformations contributing only 23% of the signal. These spectral properties are compatible with the assembly of Rho cPrD into a highly β–sheet enriched amyloid-like structure.

# Rho cPrD Self-Assembles into Amyloid Fibrils

We used the amyloid-specific dyes CR, Th-T and Th-S to confirm that the detected β–sheet enriched aggregates were organized into amyloid-like suprastructures. The absorbance of CR increases and the spectrum maximum red shifts to 505–510 nm in the presence of peptide aggregates formed at 100 μM after 120 h of incubation at 25◦C (**Figure 5A**). This spectral change corresponds to that promoted by different amyloid proteins in the aggregated state (Klunk et al., 1989). Moreover, the difference spectrum between the dye in the presence and absence of aggregated peptide allows detecting the characteristic amyloid band at ∼541 nm (**Figure 5B**). The binding of Rho cPrD to CR at early time points is significantly lower.

Thioflavin-T fluorescence emission is enhanced in the presence of amyloid fibrils (LeVine, 1993; Sabate et al., 2013). The same behavior is observed upon incubation of Th-T with Rho cPrD (**Figure 5C**). In good agreement with light scattering signals, Th-T binding to peptide solutions increases with incubation time, the Th-T fluorescence at the 480 nm spectral maximum increasing 80-fold at 120 h. Furthermore, binding of Th-S to 120 h aggregates could be visualized by fluorescence microscopy (**Figure 5D**). Areas rich in fibrous material were stained with Th-S to yield green–yellow fluorescence against a dark background.

The dye binding results indicate that incubated Rho cPrD solutions contain detectable amounts of amyloid-like structure. To confirm this extent, the morphological features of the peptide assemblies in these samples were analyzed using TEM. As shown in **Figure 6**, we detect the presence of protein aggregates in all cases. Nevertheless, in good agreement with spectroscopic data, the size and morphology of the aggregates are significantly different. The peptide incubated for 4 h forms short, poorly ordered protofibrilar assemblies. These assemblies coexist with fibrilar structures at the 48 h, whereas only mature fibrils with a typical amyloid-like morphology are observed at the 120 h.

Seeded protein aggregation is a well-established mechanism for *in vivo* amyloid fibril formation and underlies prion propagation (Caughey, 2001; Wickner et al., 2001). The nucleation step of the amyloid assembly is shortened in the presence of preformed amyloid fibrils of the same protein that can act as nuclei for the subsequent polymerization reaction (Jarrett and Lansbury, 1992). Specific and short aggregationprone regions have been shown to play a crucial role in this process (Pastor et al., 2007; Sabate et al., 2012). To test whether preformed Rho cPrD fibrils can seed the aggregation of the correspondent soluble peptide, we followed the aggregation kinetics of the peptide at 100 μM in the presence and absence of 0.1% (w/w) preformed fibrils. As shown in **Figure 7**, the presence of fibrils strongly accelerated the formation of Th-T positive assemblies, raising the possibility that such specific amyloidpromoting interactions could also occur in the context of the complete Rho factor protein.

# DISCUSSION

Prion-like proteins were initially thought to be restricted to mammals, resulting in transmissible pathologies (Aguzzi and Weissmann, 1998). Later on, the discovery of yeast prions (Wickner, 1994; Du et al., 2008; Patel et al., 2009; Rogoza et al., 2010) and more recently of prion-like proteins in multicellular eukaryotes, from snail to human (Maji et al., 2009; Heinrich and Lindquist, 2011; Majumdar et al., 2012; Tariq et al., 2013; Cai and Chen, 2014), suggest that prion-like mechanisms would sustain evolutionary conserved functions in eukaryotic kingdoms. Despite no bacterial prion-like protein has been characterized so far, computational predictions support the existence of a significant number of proteins with potential prionlike properties in bacterial proteomes (Espinosa Angarica et al., 2014; Iglesias et al., 2015). This is not surprising, since bacterial

#### TABLE 1 | Assignment of secondary structure components of Rho cPrD peptide in the amide I region of the FTIR spectra.

with Th-S and observed at 40X magnification by phase contrast and fluorescence microscopy displaying the green fluorescence characteristic of amyloid structures.

cells have been shown to support the formation of prion-like conformations of yeast prions (Sabaté et al., 2009; Garrity et al., 2010; Espargaró et al., 2012) and, more importantly, to propagate them for over a hundred generations, even when the cells can no longer make the protein that serves as the trigger for the initial conversion (Yuan et al., 2014), which suggests that functional prion-like mechanisms might be more ancient than previously

thought (Desantis et al., 2012). As a trend, prion-like sequences are predicted to be less abundant in bacteria than in eukaryotes (Espinosa Angarica et al., 2013), but, interestingly, pathogenic species seem to have a higher prion load than non-pathogenic ones. An exciting possibility is that these sequences represent a bet-hedging mechanism for pathogens, as suggested recently for yeast prions (Newby and Lindquist, 2013). These mechanisms are used to diversify microbial phenotypes. In fluctuating environments this allows a fraction of the population to survive in conditions when most would perish. This mechanism would permit certain cells to persist in strenuous environments like in the presence of antibiotics or to escape the immunogenic response, saving the population from extinction. Shuffling the states of multiple prion-like proteins would allow rapid phenotypic diversification.

Here, we addressed the presence of potential prion-like proteins in the proteome of the pathogen *C. botulinum* using a stringent approach in which both a long region displaying amino acid compositional similitude to *bona fide* prions (Toombs et al., 2010, 2012) and the presence of a specific nucleating sequence

inside it (Sabate et al., 2015b) should be present for a protein to be considered prion-like. This approach rendered a total of 54 candidates. Interestingly, the set of candidates is enriched in proteins that play a structural role and are linked to essential processes as cell wall metabolism or cellular shape maintenance. Although a more exhaustive analysis of these proteins is necessary, the data point to a possible relationship between the identified proteins and biofilm formation, which would confer a protecting strategy and facilitate the attachment of the bacteria to different surfaces. Indeed, the biofilms of a number of bacterial species have been shown to contain proteins in an amyloid conformation (Romero and Kolter, 2014) PrDs associated with proteins involved in survival and virulence were also found in *C. botulinum*. Sporulation and toxin production are powerful strategies that facilitate the invasion of new environments and bacterial survival in adverse conditions. In this context, proteins involved in spore formation and degradation, the degradationprotector NTNH in Botulism toxin and the cell-wall cross-linker L,D-transpeptidase develop non-essential functions, but facilitate the bacteria to remove toxic agents and evade the action of antibiotics or from harsh natural environmental conditions and toxic compounds (Biarrotte-Sorin et al., 2006; Magnet et al., 2007).

 This is also the function of the Rho factor in *C. botulinum,* for which we predict the existence of a highly scoring putative PrD at the N-terminus, adjacent to the RNA binding domain. It has been suggested that, in the prion-like state, transcriptional regulators may alter gene expression by creating diffusion barriers that restrict protein movement toward specific subcellular locations, by decreasing the effective concentration of the freely available pool of protein, or, on the contrary, by increasing the effective concentration in a certain location; this might result in enough functional diversity to create phenotypic divergence (Si, 2015). Interestingly enough, recent works have shown that Rho inhibition allows prophage maintenance, as a strategy to keep beneficial prophage genes, while silencing those likely to be deleterious (Cardinale et al., 2008; Menouni et al., 2013). Importantly, the pathogenic trait in *C. botulinum*, the botulinum neurotoxin, is mainly linked to a large plasmidome consisting of plasmids and circular prophages (Skarin and Segerman, 2014). Indeed, it has been recently shown that, in *E. coli*, mutations promoting adaptive properties, such us adaptation to thermal stress, converge to cluster either in the RNA polymerase complex or the termination factor Rho (Tenaillon et al., 2012; Rodriguez-Verdugo et al., 2014; Hug and Gaut, 2015). When we analyzed the mutations reported to occur specifically in Rho factor with our aggregation prediction algorithm AGGRESCAN (Conchillo-Solé et al., 2007), when found out that 72% of them endorse the terminator factor with increased aggregation-propensity, thus suggesting a link between the self-assembly of Rho and adaptation to changing environments.

We provide here strong evidence that detected PrD in Rho factor contains a short amyloid-like segment with the ability to potentially nucleate the Rho factor PrD assembly; however, it remains to be demonstrated if, in the case it occurs *in vivo,* the reaction would exhibit the reversibility required for considering this protein a *bona fide* prion.

In contrast to pWALTZ, conventional aggregation prediction algorithms do not capture the amyloidogenic potential of Rho cPrD. Because these latter algorithms usually display good accuracy when predicting the core of disease-linked amyloids (Sabate et al., 2015b), this suggests that the principles underlying their aggregation and that of Rho cPrD are somehow different. Indeed, the amyloid core of pathogenic proteins is usually very hydrophobic, whereas 90% of Rho cPrD sequence is made of N and S, and therefore polar, with only two hydrophobic residues. On the one hand, while a certain amyloid nucleation capacity favoring a sufficiently high aggregation rate is absolutely necessary, the final amyloid aggregate in a prion-like protein should at the same time display brittleness, a property that facilitates propagation. On the other hand, the protein should remain in a soluble state under physiological conditions, while keeping a cryptic amyloid capacity that allows it to self-assemble only in selected conditions. Both requirements imply that, in contrast to most amyloids, in PrDs, the aggregation reaction should not be nucleated by an extremely strong, and highly hydrophobic, amyloid core. We have proposed that the role of N residues in PrDs and their amyloid cores is to endorse these sequences with a basal aggregation propensity, while allowing them at the same time to remain soluble and disordered in normal cellular conditions (Sabate et al., 2015a; Zambrano et al., 2015). In contrast, the few hydrophobic residues found in these cores, especially aromatic ones, would play a key role in the initial amyloid oligomerization steps. This seems to be true for Rho cPrD since its assembly into amyloid-like fibrils is accompanied by an increase in the presence of hydrophobic clusters, as monitored by bis-ANS binding. It is very likely that, as described for amyloid peptides from the Sup35 prion (Balbirnie et al., 2001; Diaz-Avalos et al., 2003; van Der Wel et al., 2006; Zheng et al., 2006), complete hydrogen bonding of its N and S residues would also contribute to sustain the mature amyloid structure.

Aggregation constraints the evolution of proteins and accordingly nature have evolved different strategies to minimize protein aggregation in sequences and structures. Essentially, mutations that result in an increase in aggregation propensity tend to be purged out from the population, especially when they occur in a disordered context, since they are exposed

# REFERENCES

to solvent, being this the reason that intrinsically disordered protein segments are in general, very soluble (Santner et al., 2012; Uversky, 2013, 2015; Graña-Montes et al., 2014). In this context, the inherent amyloid potential of Rho cPrD strongly suggests that this protein segment, and the surrounding predicted PrD, are conserved because they serve functional purposes in *C. botulinum,* in agreement with the general view that PrDs are important for protein–protein interactions and provide the flexibility required to self-organizing macromolecular assemblies in living cells (Malinovska et al., 2013; Iglesias et al., 2015).

# CONCLUSION

Overall, despite the reversibility and the functionality of *C. botulinum* Rho factor self-assembly should still be validated, this study provides a first proof for the existence of amyloidogenic sequences embedded in the recurrent putative PrD identified in transcription regulators of pathogenic bacteria, a property that is compatible with them being biological capacitors that might respond to environmental conditions rewiring gene expression.

# AUTHOR CONTRIBUTIONS

Conception/design of the work: SV, IP; performed the experiments: IP, VI; generated and analyzed the data: IP, VI, SV; drafting the work: IP, VI, SV; final approval of the manuscript to be published: SV, IP.

# ACKNOWLEDGMENT

This work was funded by the Spanish Ministry of Economy and Competitiveness BFU2013-44763-P to SV.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal*.*frontiersin*.*org/article/10*.*3389/fmicb*.* 2015*.*01516

proteins: the Src homology 3 (SH3) case. *Proc. Natl. Acad. Sci. U.S.A.* 101, 7258–7263. doi: 10.1073/pnas.0308249101

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Pallarès, Iglesias and Ventura. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [*REP-PSI***+**]

#### *Fátima Gasset-Rosa† and Rafael Giraldo\**

*Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas – Consejo Superior de Investigaciones Científicas, Madrid, Spain*

#### *Edited by:*

*Salvador Ventura, Universitat Autonoma de Barcelona, Spain*

#### *Reviewed by:*

*Jesus R. Requena, University of Santiago de Compostela, Spain Galina Zhouravleva, Saint Petersburg State University, Russia*

#### *\*Correspondence:*

*Rafael Giraldo, Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas – Consejo Superior de Investigaciones Científicas, C/ Ramiro de Maeztu 9, E-28040 Madrid, Spain<EMAIL_ADDRESS>
#### *†Present address:*

*Fátima Gasset-Rosa, Department of Neurosciences, Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA*

#### *Specialty section:*

*This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology*

> *Received: 23 February 2015 Accepted: 29 March 2015 Published: 21 April 2015*

#### *Citation:*

*Gasset-Rosa F and Giraldo R (2015) Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [REP-PSI*+*]. Front. Microbiol. 6:311. doi: 10.3389/fmicb.2015.00311* The yeast translation termination factor Sup35p, by aggregating as the [*PSI*+] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the *Saccharomyces cerevisiae* proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs), which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [*REP-PSI*+], (i) was functional in a stop codon read-through assay in *S. cerevisiae*; (ii) generates weak phenotypic variants upon both its expression or transformation into [*psi*−] cells; (iii) these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and (iv) according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of *bona fide* yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [*PSI*+]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.

Keywords: amyloid cross-seeding, prion variants/strains, RepA-WH1 prionoid, [*REP-PSI***+**] prion, *Saccharomyces cerevisiae*, synthetic biology

# Introduction

Modularity is a basic principle of organization in proteins and their assemblies. In the case of the aggregation-prone amyloidogenic proteins, modularity comes from the existence of sequence stretches that become the β-strand building blocks in cross-β sheets (reviewed in Eisenberg and Jucker, 2012). Such stretches can either have hydrophobic or polar average residue compositions, but usually not both at the same time, thus posing constrains to homotypic interactions, which require chemically compatible side chains to cross-aggregate. Polar glutamine/asparagine (Q/N)-rich amyloidogenic sequences are a hallmark of prion domains in yeast (Alberti et al., 2009; reviewed in Liebman and Chernoff, 2012), in which cross-seeding has been well characterized, e.g., for the Rnq1p/[*PIN*+] prion nucleating the aggregation of Sup35p/[*PSI*+] (Vitrenko et al., 2007; Sharma and Liebman, 2013). In the translation releasing factor Sup35p, its prionforming domain (N) includes up to five and a half oligopeptide repeats (OPRs; **Figure 1A**; reviewed in Tessier and Lindquist, 2009). OPRs, while still assembled in the amyloid fibers, have been proposed to be the targets for the disaggregase chaperone Hsp104p (Chernoff et al., 1995) that, with the aid of Hsp70 chaperones Ssa1-4p (Shorter and Lindquist, 2008; Winkler et al., 2012), generate [*PSI*+] propagons, i.e., oligomeric primordia that are readily diffusible to the progeny (Cox et al., 2003; Derdowski et al., 2010). The modularity of Sup35p OPRs has allowed their partial or total replacement by heterologous sequences, such as the octapeptide repeats in the mammalian prion protein PrP (Parham et al., 2001; Dong et al., 2007; Tank et al., 2007), while keeping the resulting chimeras their original function as epigenetic determinants of reading-through stop codons in yeast. With the accumulated knowledge on Sup35p/[*PSI*+], this is probably the most suitable model system to address the molecular determinants of the prion condition in proteins (Sabate et al., 2015).

RepA, the DNA replication protein of the *Pseudomonas* plasmid pPS10, is one of the members best characterized of a protein family spread across a large group of plasmids from Gram-negative bacteria (reviewed in Giraldo and Fernández-Tresguerres, 2004). Soluble RepA dimers, which function as transcriptional self-repressors of the *repA* gene, undergo a large conformational change upon binding to specific DNA sequences from the plasmid replication origin, resulting in their dissociation into replication-competent RepA monomers (Díaz-López et al., 2003, 2006). Such structural change consists in an increase in β-sheet at the expense of the α-helical secondary structure component and affects WH1, the most Nterminal of the two 'winged-helix' domains in RepA, which is transformed from a dimerization domain into a secondary DNA binding module, ancillary to the main DNA recognition determinant, C-terminal WH2 (Giraldo et al., 1998, 2003). RepA monomers are aggregation-prone and, once a replication round is completed, the resulting plasmid copies are held together through interactions between RepA monomers bound to the replication origins of the two plasmid molecules, thus inhibiting further replication rounds (Gasset-Rosa et al., 2008a). Such complex interplay between transcriptional repression and DNA replication initiation/inhibition, focused further research on the molecular basis for the ligand (DNA) modulated balance between RepA solubility and functional aggregation. In RepA-WH1, a single hydrophobic sequence stretch partially folded as a α-helix changes its conformation, upon transient binding to specific DNA sequences, to assemble as a β-strand in amyloid fibers (Giraldo, 2007; Gasset-Rosa et al., 2008b). The RepA-WH1 domain, when expressed in *Escherichia coli* as a metastable fusion to the fluorescent protein mCherry, behaves as a synthetic prionoid, i.e., an amyloidogenic protein lacking infectivity (Aguzzi, 2009). Unlike prions in yeast, RepA-WH1 triggers a sort of intracellular amyloid 'proteinopathy' in its host (Fernández-Tresguerres et al., 2010), where the 'vertical' propagation (i.e., from mother cell to daughter cells) and the conformational selection of 'strains' of the prionoid are dependent on the Hsp70 chaperone DnaK, rather than on the Hsp104 ortholog ClpB (Gasset-Rosa et al., 2014). A recent electron microscopy reconstruction of the RepA-WH1 fibers, as templated *in vitro* on soluble molecules of the protein by amyloid seeds preformed *in vivo*, showed that the fibers are composed of intertwined amyloid tubules built by distorted protein monomers (Torreira et al., 2015).

Since the amyloidogenic sequence stretch in RepA-WH1 has clearly distinct features to those naturally found in yeast prions, – i.e., a single hydrophobic amyloidogenic stretch in a properly folded domain, rather than multiple repeats with polar residue composition in the frame of an intrinsically disordered tail – the bacterial prionoid is a suitable source of radically heterologous sequences to explore their ability to supplant the OPRs in Sup35p/[*PSI*+]. We report here that chimeras between Sup35p and at least three repeats of the hydrophobic amyloidogenic stretch in RepA-WH1 constitute novel functional prions in yeast that we have named [*REP-PSI*+]. Furthermore, [*REP-PSI*+] can cross-seed the propagation of [*PSI*+], but generating [*PSI*+] *WH*1, a new strain with very low mitotic stability (i.e., epigenetically unstable), as expected for a weak prion with severe limitations on the generation of propagons. These findings validate the possibility of tailoring yeast epigenetics by engineering aggregationprone hydrophobic amyloidogenic repeats within prion-forming domains.

# Materials and Methods

# Plasmid Constructs

Substitution of Sup35p-OPRs by Tandem Repeats of the Amyloidogenic Stretch in RepA-WH1 (WH1-R1-5)

The reporter plasmid pUKC1620 (Parham et al., 2001; Von der Haar et al., 2007), harboring a WT copy of the *SUP35* gene with its natural promoter, was used as template for PCR-amplification (*Pfu* DNApol) of N-*R6*-*sup35* using primers with BamHI (3- to the promoter) and EcoRV (annealing after the 5th repeat) ends. After digestion with those enzymes, the product replaced the BamHI-EcoRV *N-SUP35* fragment in the original plasmid, leading to pUKC\_*R5*. On this vector, *WH1-R1* was inserted through PCR, including the reverse primer (5- -CGAGgatatcC**G ATCAAAGACACAGCGCATAGCACTAG**ATTGAATTGCTGC TGAT), after the EcoRV site (lowercase), the complement of the sequence coding for the nine core amino-acid residues (LVLCAVSLI) in WH1(A31V) (bold; Giraldo, 2007). *WH1- R2* was built in a similar way, but the reverse primer had that sequence repeated twice with a TCC/Gly spacer. For pUKC\_*R0*, a construction lacking the five natural OPRs in Sup35p, the PstI site previous to the first OPR in the parental plasmid was mutated to EcoRV (QuikChange kit, Stratagene), followed by digestion with this enzyme and in-frame religation of the vector. *WH1-R1-3* were built

FIGURE 1 | Chimeras between the yeast prion protein Sup35p and the amyloidogenic stretch in the bacterial prionoid RepA-WH1. (A) Schematic representation of *Saccharomyces cerevisiae* eRF3 (Sup35p), with its domain composition and the N-terminal 51/2 OPRs highlighted.

(B) The R0 + WH1-R1-5 chimeras. Sup35p OPRs were totally replaced by 1–5 tandem repeats of the hydrophobic amyloidogenic stretch in the RepA-WH1 variant A31V (arrow; Giraldo, 2007). The heterologous sequences were engineered to fold as zigzag β-arcades (reviewed in Kajava et al., 2010), in which turns were made by inserting a Gly residue between the natural Asp (blue) C- and Arg (red) 'gatekeeper' N-ends from contiguous WH1 repeats. β-strand packing (faces of strands in black,

backs in gray) would be guided by interdigitation of chemically compatible side chains. β-arcades from distinct protein monomers would stack through main-chain hydrogen bonds into a parallel superpleated β-structure, as proposed for Sup35p/[*PSI*+] (reviewed in Kajava et al., 2010). In the resulting fiber, the engineered repeats should have to accommodate as a hydrophobic spine within an otherwise polar Q/N-rich axis. (C) The amyloidogenicity profiles (red plots) for the distinct chimeric N-domains, as estimated by the AGGRESCAN (left column; Conchillo-Solé et al., 2007) and WALTZ (right; Maurer-Stroh et al., 2010) algorithms, suggest that the synthetic peptide arrays assemble as amyloids. Horizontal lines: blue, aggregation hot spot threshold; green, sequence average.

#### by annealing the pre-phosphorylated (T4-PNK) oligos 5- - GGCCGCCTAGTGCTATGC**GCTGTGTCTTTGATCGAT**/5- - GCATAGCACTAGGCGGCC**ATCGATCAAAGACACAGC**

(complementary sequences in bold) followed by filling of the ends (Klenow DNApol) and self-ligation, a step that generated an extra terminal half repeat. Each repeat in the concatemers includes the natural C-terminal Asp and N-terminal Arg found in the RepA-WH1 amyloidogenic stretch (Giraldo, 2007) plus a linker Gly. Upon ligation of the concatemers mix at the EcoRV site in pUKC\_*R0* and transformation into *E. coli*, DNA sequencing identified clones with up to three RepA-WH1 repeats. Since *WH1-R4-5* were recalcitrant to be obtained through such approach, a fragment including three RepA-WH1 repeats was made through chemical synthesis (ATG:biosynthetics, Germany) and inserted into pUKC\_*R1-2*, in which EcoRV had been regenerated by site directed mutagenesis, to give pUKC\_*R4-5*.

## Plasmids for Protein Expression in Yeast under the pGAL1 Promoter

The full-length *WH1*(*Rn*)*-SUP35* chimeras were inserted into the pYeF2 vector (Cullin and Minvielle-Sebastia, 1994) after amplification by PCR, using as templates the pUKC series (see above) and primers having BamHI and NotI ends. These chimeras carried a C-terminal hemagglutinin (HA)-tag from the vector. For fluorescence microscopy observation, fusions were made between the NM-domains from the chimeras and the monomeric red fluorescent protein mCherry at their C-termini. With this purpose, each NM-domain was amplified by PCR on the pUKC plasmids, with oligonucleotides carrying BamHI and BspEI ends, and then ligated with a BspEI (5- )-NotI (3- ) mCherry fragment, amplified using as template pWH1(A31V)-mRFP (Fernández-Tresguerres et al., 2010). The BamHI-NotI fragments were then cloned into pYeF2 as above.

### Plasmids for Protein Expression in *E. coli*

The fusions of *NM-WT*, *NM-R0*, *NM-R0* + *WH1-R2-4* to mCherry were cloned into the pRG vectors (*Ptac*, His10 N-tag; Fernández-Tresguerres et al., 2010) by PCR amplification, performed with primers with SacII (5- -NM) and XbaI (3- -mCherry) ends. All constructs were verified by DNA sequencing.

# *Saccharomyces cerevisiae* Strains

For screening prion-dependent translation through stop codons, it was used a derivative of the strain 74D-694: *MATa ade1-14*UGA *trp1-289 his3-*Δ*200 ura3-52 leu2-3*,*112 sup35::loxP* [pYK810] [*PSI*+] [*PIN*+]. For fluorescence microscopy, the YJT28 strain (-*ARS305::kanMX*, *ade2-1::ADE2*, W303-1a) was selected to suppress autogenous red fluorescence.

### Epigenetic Assay for Yeast Colony Color

The pUKCs derivatives (encoding the different chimeric alleles) were electroporated in a [*PSI*+] [*PIN*+] strain (see above) that initially carried pYK810, a plasmid bearing a copy of *SUP35* (Von der Haar et al., 2007). For displacing the resident pYK810, thus assuring that the incoming pUCKs were the only source of Sup35p (or its chimeras with the WH1 repeats), colonies growing in SD-His were replicated on the same medium containing 0.1% 5-FOA, thus counter-selecting for cells that carried pYK810 (*URA3*). Colonies were then plated on 1 /4 YPD and SD-adenine. For full development of color in colonies, agar plates were incubated for ≥72 h at 30◦C and then transferred to 4◦C for 24 h before photographic documentation.

# Protein Aggregate Transformation into [*psi***−**] Cells

Overnight cultures inoculated from pUKCs-carrying red [*psi*−] colonies, that have spontaneously lost the [*PSI*+] phenotype, were diluted 1/8 into 60 ml of YPD and grown to OD600 = 0.5. Cells were washed with water and 1 M sorbitol, resuspended in SCE buffer (1 M sorbitol, 100 mM Na-citrate pH 5.8, 10 mM EDTA, 10 mM DTT, 2 mg/ml lyticase), and then incubated at 30◦C for 1 h. The resulting spheroplasts were centrifuged and resuspended in SCT buffer (1 M sorbitol, 10 mM Tris. HCl pH 7.5, 10 mM CaCl2) and 100 μl were co-transformed with 2 μM of pYeF2 (Tanaka, 2010) as a marker (*URA3*), 10 μg of carrier ssDNA and 5 μl of whole cell extracts (i.e., the low-speed supernatants after cells lysis, see below) from pUKCs/[*REP-PSI*+] cells. Suspensions were incubated at room temperature (RT) with rotation mixing for 30 min. Then 44% PEG4000, 10 mM Tris. HCl pH 7.5, 10 mM CaCl2 buffer was added and further incubated for 45 min. Spheroplasts were then sedimented, resuspended in SOS medium (1 M sorbitol, 25% YPD, 7 mM CaCl2), added to 10 ml of top agar (SD-URA, 2% dextrose, 0.8% agar, 1 M sorbitol, 2% YPD), and platted on SD-URA agar. Incubation proceeded for ≥72 h at 30◦C. Large size colonies were selected and spotted on 1 /4 YPD agar. To address the stability of [*PSI*+], white colonies obtained after transformation of Sup35p-WT [*psi*−] cells with R0 + WH1-R3-5 protein extracts were grown in YPD at 30◦C overnight. They were then diluted to OD600 = 0.001, grown for 24 h, and 30 μl plated on 1 /4 YPD agar and incubated as above. Transformation and stability assays were performed independently twice.

# Aggregate Extraction and Sedimentation Assay

Two hundred ml cultures of yeast carrying the full-length protein chimeras cloned into pYeF2s (see above) were grown overnight in selective medium (SD-Ura) with glucose. Then cultures were diluted to OD600 = 0.07 in SD-Ura, but with 2% raffinose and 0.1% glucose, and grown to OD600 = 0.2, when protein expression was induced by adding 2% galactose and further incubated until OD600 = 2. Cells were then harvested and resuspended in 500 μl of 25 mM Tris. HCl pH 6.8, 250 mM NaCl, 5 mM EDTA, 10% glycerol (plus protease inhibitors; Roche). Lysis was then carried-out with glass beads (Lysing matrix C) in a MP FastPrep-24 homogenizer (five cycles, level 5, for 30 s at 4◦C). Cell debris was removed by a low-speed sedimentation step (600 × *g*, 3 min). Two hundred microliter of the resulting whole cell extracts were ultracentrifuged at 50,000 rpm (100,000 × *g*), for 15 min at 4◦C (Beckman Optima Max-XP, TLA100 rotor). Supernatants were collected and pellets were resuspended in 200 μl of the lysis solution. Proteins in equivalent volumes of supernatant and pellet fractions were analyzed by SDS-PAGE (10% polyacrylamide; 30 μg/lane) plus Western-blotting, using an anti-HA antibody (Roche, 1:1,000) and chemiluminescence detection (ECL2; Pierce).

# Semi-Denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)

Total cell lysates (45 μl, at 30 mg/ml) from yeast having the chimeras expressed from pYeF2 (see above) were mixed with 15 μl of loading buffer (TAE 2X, 20% glycerol, 8% sarkosyl, 0.5 g/l bromophenol blue, plus protease inhibitors). Samples were incubated at RT for 10 min, and electrophoresis performed in 1.5% agarose gels (TAE 1X, 0.1% SDS) at 100 V for 7.3 h, 10◦C (Molina-García and Gasset-Rosa, 2014). Proteins were then transferred to a PVDF membrane in a Trans-Blot device (Bio-Rad) in TAE 1X, 0.1% SDS, at 16 V for 15 h, 10◦C. Detection was performed with anti-HA (1:1,000).

# Visualization of Aggregates by Fluorescence Microscopy

### Overexpression of the NM-mCherry Chimeras

pYeF2s encoding the chimeras were transformed into the YJT28 strain and protein expression was carried out as described above. Culture aliquots were taken along 22 h for live cell observation.

#### Fluorescence Microscopy

It was performed with a Nikon Eclipse 90i microscope, equipped with CFI PLAN APO VC (NA 1.40) oil immersion objective and a Hamamatsu ORCA-R2 CCD camera. A red filter with excitation 543/22 and emission 593/40 was used. Differential interference contrast (DIC) images were also captured.

# Purification of His10-NM-mCherry Chimeras

Protein expression and purification were performed as described for His6-RepA-WH1 (Giraldo, 2007), but extending the Ni2+- IMAC gradient to 0.5 M imidazole. Protein stocks (30 μM) were kept at −70◦C in 0.1 M Na2SO4, 20 mM Na2HPO4 pH 6, 5 mM 2-mercaptoethanol, 10% glycerol.

### Amyloid Assembly of NM-mCherry Chimeras *In Vitro*

Protein chimeras (15 μM) were assembled *in vitro* by still incubation at 5◦C for a month, as described for RepA-WH1 (Giraldo, 2007), in 0.1 M Na2SO4, 60 mM Hepes pH 8, 8 mM MgSO4, 14% PEG4000, 6% MPD. Samples were examined in a JEOL JEM-1230 electron microscope.

### Circular Dicroism (CD) Spectroscopy

Spectra of the purified NM-mCherry chimeras (15 μM) were acquired in 0.1 M Na2SO4, 15 mM Na2HPO4 pH 6, at 5◦C, as described (Giraldo, 2007).

### Analytical Ultracentrifugation

NM-mCherry chimeras were dialyzed in 0.1 M Na2SO4, 20 mM Na2HPO4 pH 6, 5 mM 2-mercaptoethanol. Four hundred microliter of each sample were diluted to 0.8, 0.2, and 0.08 mg/ml and then centrifuged for 5 min at 13,000 rpm, 4◦C. The clarified supernatants were studied by sedimentation velocity. Centrifugation was carried out in a Beckman–Coulter XLI

analytical ultracentrifuge, at 48,000 rpm and 20◦C, measuring absorbance at 280 nm. Sedimentation coefficient distributions, c(s), were determined, with a confidence level of 0.68, using the SEDFIT 14.1 software (Schuck, 2000).

# Results

# Engineering Chimeras between the Amyloidogenic Stretch in RepA-WH1 and [*PSI***+**]

Structural modeling of the amyloid fibers assembled by the Ndomains of yeast prion proteins suggests that their basic building block might be a β-arch (reviewed in Kajava et al., 2010), in which adjacent Q/N-rich stretches would assemble as β-strands interdigitated through compatible side chains, while the intervening sequence would form a turn. If multiple stretches were present, as in Sup35p OPRs, β-arches would be further folded as β-arcades. The stacking of β-arcades from distinct protein monomers, stabilized through parallel main-chain hydrogen bonding, would result in a parallel superpleated β-structure (reviewed in Kajava et al., 2010). With a β-arcade based model in mind, we used a plasmid reporter system (Parham et al., 2001) in which all the OPRs in Sup35p were replaced by up to five tandem repeats of the hydrophobic amyloidogenic stretch found in the bacterial prionoid RepA-WH1 (Giraldo, 2007; WH1-R1-5; **Figure 1**).

## RepA-WH1 **+** Sup35p Chimeras are Functional in Yeast

The engineered chimeras were expressed in yeast from the *SUP35* promoter in a centromeric plasmid, and tested in an epigenetic red–white colony color assay (**Figure 2**): upon Sup35p aggregation as [*PSI*+], the reporter *ade1-14* allele, including a premature amber stop codon, is read-through by the ribosomes allowing for the synthesis of adenine, thus giving white color colonies on rich, unselective medium. Otherwise, soluble Sup35p efficiently terminates translation resulting in red [*psi*−] colonies by accumulation of the adenine precursor metabolite 5- -P-ribosyl-5-aminoimidazole (reviewed in Tessier and Lindquist, 2009). In the absence of endogenous Sup35p-WT, achieved in [*PSI*+] cells upon displacement of a resident *SUP35* plasmid by vectors encoding the chimeras, five of the native OPRs fused to two chimeric RepA-WH1 repeats (R5 + WH1-R2) were sufficient to yield a [*PSI*+]-like prion phenotype. However, with just one bacterial repeat (R5 + WH1-R1) the phenotype of this chimera was even weaker (more intense red color) than the R5 parental, suggesting that the insertion of a single hydrophobic stretch in the Q/N-repeats destabilized their assembly as amyloid. Interestingly, in the absence of any OPRs all the constructs including RepA-WH1 repeats (R0 + WH1-R1-5) gave a nearly WT phenotype, indicating that, in these chimeras, amyloids were successfully built with little interference between the RepA-WH1 and the Sup35p moieties (**Figure 2A**). In adeninedeficient media, all chimeras supported yeast growth, i.e., they read-through *ade1-14* thus restoring a functional pathway for adenine synthesis, albeit R5 + WH1-R1 led again to a poor phenotype (**Figure 2B**).

When, after several passages through liquid rich medium, yeast carrying the chimeras were plated on 1 /4 YPD agar (**Figure 2C**) red sectored colonies appeared if the number of WH1 peptide repeats was ≤2, indicating that these were unstable chimeric prions. The chimeric prions including ≥3 WH1 repeats, which we termed [*REP-PSI*+], exhibited two clearly different phenotypes, namely white and pink colonies (**Figure 2D**, top). To explore the mitotic stability of both phenotypes, single colonies of each kind were subcultured and plated again on 1 /4 YPD. White colonies showed a remarkable stability (frequency of conversion to red ≈10−6, matching that described for [*PSI*+]; as reviewed in Liebman and Chernoff, 2012; **Figure 2D**, left), whereas light pink colonies were very unstable, giving rise with high frequencies to dark pink (≈2 × 10−1) and red (≈10−2), or reverting to white (≈3 × 10−3), colonies (**Figure 2D**, right). Tentatively, the white phenotype was associated to a strong prion variant, *s*[*REP-PSI*+], the two pink tones to weak prion variants, *w1* and *w2*[*REP-PSI*+] (light or dark pink, respectively), and the red colonies to cured (i.e., having lost the prion phenotype) [*rep-psi*−].

#### [*REP-PSI***+**] Prion is Infectious and Templates on Sup35p a New Weak, Unstable Variant, [*PSI***+**] *WH1*

Yeast spheroplasts can be transformed with prion particles, either assembled *in vitro* or extracted from cultured cells (Tanaka, 2010). Spheroplasts prepared from red colonies, thus expressing either Sup35p-WT or the WH1-R3-5 chimeras in their non-prion form ([*psi*−] or [*rep-psi*−], respectively), were transformed with protein extracts from cells also carrying the chimeras but grown from white colonies, thus with a [*REP-PSI*+] phenotype. When the epigenetic phenotype of the transformants was assayed (**Figure 3A**), all colonies showed the white or the light pink stop codon read-through phenotype characteristic of the incoming prion aggregates (Sup35p-WT or the chimeras, respectively). The demonstration of the 'infectivity' of the R0 + WH1-R3-5 chimeras through the transformation of their aggregated forms qualifies [*REP-PSI*+] as a new synthetic yeast prion. However, after serial sub-culturing without selective pressure, in those clones expressing Sup35p-WT and cross-transformed with the WH1-R3-5 [*REP-PSI*+] chimeras, besides the pink phenotype of the incoming aggregates, red [*psi*−] colonies appeared frequently (**Figure 3B**). These results indicated that the WH1-R3-5 chimeras can template their conformation on Sup35p-WT to generate a new very weak prion strain (or ensemble of weak strains) that we have named [*PSI*+] *WH 1*.

# [*REP-PSI***+**] Prion Assembles Amyloids Larger than [*PSI***+**]

Biochemical analysis of the solubility of the engineered chimeric proteins, performed upon their overexpression from the *GAL1* promoter, showed that constructs R0 + WH1-R3-5 aggregated massively, whereas R0 + WH1-R1-2 were proteolytically more unstable (**Figure 4A**). Regarding the extra, higher mobility band observed in the SDS-PAGE of the whole cellular lysates for R0 + WH1-R1, including a single WH1 hydrophobic repeat in the context of the Sup35p Q/N-rich sequences might generate instability in the natural beta-arcades built by the prion,

which will thus become proteolysis-prone. Following this speculation, that higher mobility band would correspond to molecules of the chimera that have lost the bit N-terminal to the single engineered WH1 repeat. The R0 + WH1-R1-2 chimeras were further degraded during the manipulation of the cell lysates for ultracentrifugation analysis, in spite of performing the experiment at low temperature and supplying the samples with protease inhibitors. Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), a technique for the detection of amyloid aggregates in a broad range of sizes (Bagriantsev et al., 2006), was carried out by immediately running in the gel the WCL fractions under conditions that should denature proteases, thus reducing degradation of the R0 + WH1-R1-2 chimeras to a minimum. SDD-AGE revealed (**Figure 4B**) that the chimeras form two populations of aggregates according to their electrophoretic mobilities. The species with higher molecular weights were evident in R0 + WH1-R1-5, showing sizes in direct correlation with the increasing number of RepA-WH1 repeats, but were barely detectable for Sup35p-WT, R0 or the construct carrying R5 + WH1-R2. This result points to the construction of a distinct type of assembly by the chimeras in which the hydrophobic repeats have completely replaced the Q/N-OPRs. The presence of very high molecular weight species in SDD-AGE has been described in [*PSI*+] variants resistant to Hsp104 chaperonepromoted shearing, thus resulting in weak prion phenotypes with low mitotic stabilities (Derdowski et al., 2010; Alexandrov et al., 2012).

# RepA-WH1 Repeats in [*REP-PSI***+**] Alter the Structure of the Amyloids Assembled by the NM-Domains

The results above pointed to a singular structural arrangement in [*REP-PSI*+] for the hydrophobic RepA-WH1 OPRs within the flanking polar Q/N-rich sequences, which remained unaltered from the Sup35p N-domain. The N and M domains (**Figure 1A**) in the [*REP-PSI*+] chimeras were then fused to the monomeric red fluorescent protein mCherry and expressed in *E. coli*. Those expressed at sufficiently high levels were purified and their assembly morphologies (**Figure 5A**), average secondary structure compositions (**Figure 5B**), and association states (**Figure 5C**) were physically analyzed. NM-mCherry and NM-R0-mCherry controls were able to assemble into fibers under standard conditions (Giraldo, 2007; Fernández-Tresguerres et al., 2010; **Figure 5A**). However, NM-mCherry fusions carrying WH1-R2 or WH1-R4

detergent-resistant amyloid aggregates, with distinct average molecular weights (Low-MW and High-MW).

did not form fibers, but irregular oligomers whose average sizes (≤10 and ≈25 nm, respectively), directly correlated with the number of hydrophobic repeats. Such oligomers were compatible with the smaller aggregates detected by means of SDD-AGE for the [*REP-PSI*+] chimeras (**Figure 4B**).

Circular dichroism (CD) analysis of the purified proteins (**Figure 5B**) indicated a net increase in β-sheet structure (broad band at ≈220 nm) for NM-R2/4-mCherry compared with NMmCherry, whereas NM-R0-mCherry spectrum resembled the spectra of Q/N-rich peptides when forming coiled-coils (redshifted band at >225 nm; Fiumara et al., 2010). Since sedimentation velocity experiments (**Figure 5C**) showed that purified NM-R0-mCherry was a monomer (*s* = 2.3 S), such coiled-coil should be intramolecular. On the contrary, poly-dispersed aggregation was evident as multiple peaks with increasing sedimentation coefficients for NM-R2/4-mCherry and the WT control NM-mCherry. These *in vitro* experiments indicated that the chimeric [*REP-PSI*+] prions, carrying hydrophobic OPRs of bacterial origin, assemble as amyloid oligomers rather than fibers, as Sup35p/[*PSI*+] does.

# [*REP-PSI***+**] Prion Aggregates as Perivacuolar Foci *In Vivo*

The NM-mCherry fusion proteins were then expressed in yeast from a Gal-inducible plasmid (**Figure 5D**). When the N domain carried the Sup35p wild-type sequence, the characteristic ringlike aggregates appeared in the cytoplasm (Tyedmers et al., 2010), whereas if it lacked all the OPRs (NM-R0-mCherry) fluorescence labeling was found diffused. If the constructs included the N domain of the distinct [*REP-PSI*+] chimeras, aggregation appeared as multiple dots/foci whose sizes increased with the number of RepA-WH1 repeats. They were disposed around the vacuole (IPOD compartment), as previously described for mature NM-GFP amyloids (Tyedmers et al., 2010). Interestingly, the fraction of yeast cells expressing the NM-R2-mCherry chimera was significantly reduced, and the lysis of many cells became evident. It has been proposed that the cellular toxicity of hydrophobic amyloidogenic peptides is linked to their ability to assemble as oligomeric pores upon insertion into lipid bilayers (reviewed in Butterfield and Lashuel, 2010). It might be the case that NM-R2-mCherry, lacking the ability to further assemble into a stable parallel superpleated β-structure (**Figure 1B**), would have a preference for membrane targeting.

# Discussion

In this work, we have generated chimeras (**Figure 1**) by replacing the polar OPRs in Sup35p with tandem repeats of the hydrophobic amyloidogenic stretch found in the bacterial prionoid RepA-WH1 (Giraldo, 2007; Gasset-Rosa et al., 2008a, 2014). The resulting synthetic [*REP-PSI*+] prions are functional in yeast, generating both a strong [*PSI*+]–like phenotype and various weak phenotypes (**Figures 2** and **3**), which are compatible with a cloud of prions strains (Bateman and Wickner, 2013). These chimeric prions assemble themselves *in vitro* as discrete size particles (**Figure 5**), which probably would suffice to act as competent propagons *in vivo*.

Our results suggest that cross-seeding of [*REP-PSI*+] through conformational templating, since the wild-type OPRs were absent from the chimeras, must be exerted by the flanking Q/Nrich sequences in the N-terminal domain that come from Sup35p-WT. This is compatible with findings showing that mutants including hydrophobic and/or aromatic residues at the N-terminus (residues 1–40) of Sup35p enhance [*PSI*+] nucleation, whereas aromatic side-chains at the OPRs promote chaperone-mediated propagation (Toombs et al., 2010, 2011; Alexandrov et al., 2012; Gonzalez-Nelson et al., 2014; MacLea et al., 2015). It is noteworthy that such studies were performed by

FIGURE 5 | Chimeric NM domains from [*REP-PSI***+**] fused to the mCherry reporter form oligomeric assemblies. (A) Purified His6-tagged NM-mCherry fusions (15 μM) were assembled *in vitro* and visualized by transmission electron microscopy. Insets are twofold magnifications of areas within dashed boxes. RepA-WH1 repeats drive the assembly of the NM-mCherry proteins into particles whose sizes increase with the number or repeats. (B) Circular dicroism (CD) spectra of the purified, preassembled NM-mCherry proteins (15 μM) showed an increase in β-sheet structure (broad minima at ≈220 nm) for the R0 + WH1-R2/4 chimeras. (C) Sedimentation velocity experiments were performed in an analytical ultracentrifuge with the same purified NM-mCherry proteins (10 μM) studied in (A,B). The analyses indicate poly-dispersed aggregation for the chimeras (i.e., multi-peaked profiles), with a direct correlation between the number of

RepA-WH1 repeats and the sedimentation coefficient values/number of peaks. However, the profiles for the R0 + WH1-R2/4 chimeras were simpler than that for the NM-Sup35 fusion, reflecting the ability of the latter to assemble fibers (A). (D) Epifluorescence microscopy imaging of yeast cells expressing the chimeric NM-mCherry fusions. Exposure times: 200 ms (Sup35-WT, R0, R0 + WH1-R1), 600 ms (R5 + WH1-R2, R0 + WH1-R3-5), and 2 s (R0 + WH1-R2). ND filter: 1/4. Left: superposition of the DIC and fluorescence images. Chimeras carrying R0 + WH1-R3-5 form foci (arrows) whose sizes increase with the number of WH1 repeats, whereas WH1-R1 is dispersed through the cytoplasm and R0 + WH1-R2 is cytotoxic (cell lysis). The fraction of cells expressing the chimeras (i.e., those fluorescence-labeled) is indicated (%); 200–400 cells of each type were counted.

mutating a single OPR out of five in Sup35p, yielding constructs somehow analogous to our unstable R5 + WH1-R1-2 chimeras, whereas in this work we have built a complete assembly made of up to five hydrophobic repeats (R0 + WH1-R1-5). According to the results discussed here, there seems to be in [*REP-PSI*+] a minimum threshold of three hydrophobic WH1 repeats in order to build a hydrophobic spine in the β-arcades sufficiently stable to surpass the control exerted by the surrounding natural polar sequences on prion nucleation and propagation. Thus, the engineered superpleated β-structure in [*REP-PSI*+] seems to behave as an orthogonal synthetic module functional in prion propagation, in the sense proposed by Toombs et al. (2012). It has also been described that point mutations replacing polar Q/Y residues by charged Lys in OPRs positions confronted in the β-arcades, thus leading to electrostatic repulsion, result in new prion variants, [*PSI*+] *M*1−*M*5, some of which are unstable, and template on Sup35p-WT a non-epigenetically heritable conformation (Bondarev et al., 2013, 2014). The 'prion no more' mutant G58D, which affects the second OPR (R2) in Sup35p, can be incorporated in WT aggregates, leading to increased frequency of fragmentation, thus compensating for deficiencies in propagation of weak [*PSI*+] variants but curing, as dominant-negative, strong prion variants (DiSalvo et al., 2011; Verges et al., 2011). We have shown here that even more extensive engineering of the OPRs can give way to a new prion, [*REP-PSI*+], that templates on Sup35p a new weak variant, [*PSI*+] *WH1*. Conversely, during the initial expression of the WH1-R1-5 chimeras from plasmids, the [*PSI*+] prion resident in the yeast cells, before the displacement of its encoding (*SUP35*) vector, could influence through templating the compatibility and selection of the distinct [*REP-PSI*+] variants described in this work. Cross-seeded aggregation between distinct RepA-WH1 variants has also been reported in bacteria (Molina-García and Giraldo, 2014). The possible contribution of the [*PSI*+]-ancillary prion Rnq1p/[*PIN*+] (Sharma and Liebman, 2013) to [*REP-PSI*+] nucleation will require further studies.

Interestingly, the R0 + WH1-R3-5 chimeras build oligomers rather than fibers, as Sup35p does: most probably, having a large hydrophobic beta-arcade inserted in the polar Q/N-rich prion domain will impose a hindrance to the assembly of long, structurally regular fibers. Similarly, the natural, non-Q/N-rich prion [*GAR*+] (Brown and Lindquist, 2009), which has recently been described to overcome glucose catabolite repression in *Saccharomyces cerevisiae* and other yeast (Jarosz et al., 2014), does not assemble as fibers but as oligomeric aggregates, as described here for the [*REP-PSI*+] chimeras. In yeast, under normal conditions, binding of Ssa1p (an Hsp70 chaperone) to Sup35p targets Hsp104p to [*PSI*+] aggregates for the generation of prion seeds (Winkler et al., 2012). Sup35p alleles defective in OPRs generate prions, such as [*PSI*+] -*22/69* (Borchsenius et al., 2001), that also build large aggregates behaving as unstable, weak prion variants (Tanaka et al., 2006; Derdowski et al., 2010). When the Q/N-rich

# References

Aguzzi, A. (2009). Beyond the prion principle. *Nature* 459, 924–925. doi: 10.1038/459924a

OPRs in *S. cerevisiae* Sup35p were replaced by heterologous non-Q/N sequences from other yeast species, the propagation of the resulting chimeric prion became independent on Hsp104p (Crist et al., 2003). The region between the OPRs and the initial residues in the medium (M) domain is the target recognized by Hsp104p in the Sup35p amyloids (Frederick et al., 2014). Besides this, the intermolecular contacts characteristic of weak [*PSI*+] variants exhibit an increased dependence on Hsp70s for Hsp104p-driven shearing, compared with those found in strong variants (DeSantis and Shorter, 2012). The propagation of [*GAR*+] is independent of the activity of the Hsp104 disaggregase, probably because the oligomeric nature of this prion allows for diffusion-driven propagation, but this becomes strictly dependent on Hsp70 (Brown and Lindquist, 2009; Jarosz et al., 2014). Interestingly, the bacterial Hsp104p orthologue, ClpB, does not contribute either to the propagation of the RepA-WH1 prionoid in *E. coli*, a function relying mainly on the Hsp70 chaperone DnaK (Gasset-Rosa et al., 2014). DnaK conformationally selects for an amyloid variant of RepA-WH1 with reduced toxicity and generates relatively small oligomeric particles, readily diffusible to the progeny (Gasset-Rosa et al., 2014). In the case of [*REP-PSI*+], the contribution of chaperones to its propagation in yeast remains to be explored.

Heterologous model systems have made fundamental contributions to the understanding of prion propagation. The expression of the yeast prion [*PSI*+] in bacteria has revealed that Sup35p aggregates as inclusion bodies which retain the ability to nucleate distinct strains (Garrity et al., 2010; Espargaró et al., 2012), and that, as in its original host, it still needs nucleation by [*PIN*+] (Garrity et al., 2010) and depends on Hsp104 for propagation (Yuan et al., 2014). In addition, [*PSI*+] has also been propagated in mammalian cells in culture, exhibiting the hallmarks of cytoplasmic inheritance (Krammer et al., 2009; Hofmann et al., 2013). The synthetic [*REP-PSI*+] prion, by expanding the repertoire of [*PSI*+] variants and rewiring amyloidogenic parts of Sup35p with alien, non-Q/N-rich sequences of bacterial origin, is a proof of concept for the feasibility of generating new phenotypic traits in prions. Engineering the consortium between [*REP-PSI*+] and its possible chaperone modulators will surely enable new trends in yeast epigenetics.

# Acknowledgments

We are grateful to M. Tuite for the gift of the pUKC1620 plasmid and the derivative of the strain 74D-694 used in the read-through assays, and J. A. Tercero for the strain YJT28. Thanks are also due to Y. Chernoff for advice on protein transformation into yeast and to J. R. Luque for performing the sedimentation velocity experiments at the CIB – CSIC analytical ultracentrifugation facility. This work has been supported by grants from Spanish MINECO (BIO2012-30852 and CSD2009-00088).

Alberti, S., Halfmann, R., King, O., Kapila, A., and Lindquist, S. (2009). A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. *Cell* 137, 146–158. doi: 10.1016/j.cell.2009. 02.044

structure using position-specific scoring matrices. *Nat. Methods* 7, 237–242. doi: 10.1038/nmeth.1432

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Gasset-Rosa and Giraldo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


2. Overview

Craniopharyngiomas (CPs) have had a prominent place in neurosurgery due to both the technical difficulty and controversy regarding the optimal treatment of these benign tumors. Harvey Cushing famously described craniopharyngiomas in 1936 as "the most forbidding of the intracranial tumors." Seventy years later, Rutka still wrote: "There is no other primary brain tumor that evokes more passion, emotion, and as a result, controversy than does the CP" [1]. Craniopharyngiomas comprise 1–2% of all brain tumors and occur in a bimodal distribution, with 40% of cases occurring between age 5–15 years and 60% occurring at ages >55 years.

The differential diagnosis for craniopharyngioma can include a variety of entities, including pituitary macroadenoma, metastasis, Rathke's cleft cyst, colloid cyst, glioma, meningioma, germinoma, abscess, sarcoid, or aneurysm. Imaging characteristics usually include a solid cystic lesion, speckled with calcifications in 50–80% of craniopharyngiomas (especially pediatric patients), as well a presentation with hypopituitarism and diabetes insipidus, which influence clinical thinking toward establishing this diagnosis.


**6.3.3 Data taking 2011**

Constantly upgrading and extending its hardware resources and updating the grid software, ALICE continued a successful LHC data handling campaign in 2011. By September, the total volume of the collected raw data was almost 1.7 PB with the first reconstruction pass completed. The year 2011 was marked by massive user analysis on the Grid. In May, the most important conference in the Heavy Ion Physics community, Quark Matter 2011 (QM2011) [66], took place and was preceded by an enormous end user analysis campaign. In average, 6 thousands end-user jobs were running all the time, which represents almost 30% of the CPU resources officially dedicated to ALICE (The number of running jobs is higher than that most of the time due to use of opportunistic resources). During the week before the QM2011, there was a peak with 20 thousands of concurrently running end-user jobs, see Figures 23,24. The number of active Grid users reached 411.

Fig. 23. Total network traffic at the ALICE Storage Elements - 2010/2011

middleware, see section 3, is mature and only a few changes are necessary.

jobs. As a result, the ALICE Grid in a sense has been working as a cloud.

In total, the ALICE sites were running in average 21 thousands of jobs with peaks up to 35 thousands (Figure 21). The resources ratio remained 50% delivered by Tier-0 and Tier-1s to 50% delivered by Tier-2s. Altogether, 69 sites were active in the operations. The sites' availability and operability kept very stable throughout the year. The gLite (now EMI)

Grid Computing in High Energy Physics Experiments 211

In the beginning of the 2011 campaign, there was a concern that the storage would be saturated. In fact the storage infrastructure was performing basically without problems supporting the enormous load from the end-user analysis and was getting ready for the Pb-Pb operations. The network situation, as was already mentioned for the WLCG in general, has been excellent and allowed for the operation scenario where the hierarchical tiered structure got blurred, the sites of all levels were well interconnected and running a similar mixture of

Fig. 24. End-user jobs profile - 2011

Fig. 21. ALICE running jobs profile 2010/2011.

Fig. 22. Network traffic OUT by analysis jobs - 2010

30 Will-be-set-by-IN-TECH

Fig. 21. ALICE running jobs profile 2010/2011.

Fig. 22. Network traffic OUT by analysis jobs - 2010

Fig. 23. Total network traffic at the ALICE Storage Elements - 2010/2011

Fig. 24. End-user jobs profile - 2011

In total, the ALICE sites were running in average 21 thousands of jobs with peaks up to 35 thousands (Figure 21). The resources ratio remained 50% delivered by Tier-0 and Tier-1s to 50% delivered by Tier-2s. Altogether, 69 sites were active in the operations. The sites' availability and operability kept very stable throughout the year. The gLite (now EMI) middleware, see section 3, is mature and only a few changes are necessary.

In the beginning of the 2011 campaign, there was a concern that the storage would be saturated. In fact the storage infrastructure was performing basically without problems supporting the enormous load from the end-user analysis and was getting ready for the Pb-Pb operations. The network situation, as was already mentioned for the WLCG in general, has been excellent and allowed for the operation scenario where the hierarchical tiered structure got blurred, the sites of all levels were well interconnected and running a similar mixture of jobs. As a result, the ALICE Grid in a sense has been working as a cloud.

continuously developing into the future absorbing and giving rise to new technologies, like the advances in networking, storage systems and inter-operability between Grids and Clouds

Grid Computing in High Energy Physics Experiments 213

Managing the real data taking and processing in 2009-2011 provided basic experience and a starting point for new developments. The excellent performance of the network which was by far not anticipated in the time of writing the WLCG (C)TDR shifted the original concept of computing models based on hierarchical architecture to a more symmetrical mesh-like scenario. In the original design, the jobs are sent to sites holding the required data sets and there are multiple copies of data spread over the system due to anticipation that network will be unreliable or insufficient. It turned out that some data sets were placed on sites and never

Based on the existing excellent network reliability and growing throughput, the data models start to change along a dynamical scenario. This includes sending data to a site just before a job requires it, or reading files remotely over the network, use remote (WAN) I/O to the running processes. Certainly, fetching over the network one needed data file from a given data set which can contain hundreds of files is more effective than a massive data sets deployment

The evolution of the data management strategies is ongoing. It goes towards caching of data rather than strict planned placement. As mentioned, the preferences go to fetching a file over the network when a job needs it and to a kind of intelligent data pre-placement. The remote access to data (either by caching on demand and/or by remote file access) should be

To improve the performance of the WLCG-operated network infrastructure, the topology of LHC Open Network Environment (LHCONE [24]) is being developed and built. This should be complementary to the existing OPN infrastructure providing the inter-connectivity between Tier-2s and Tier-1s and between Tier-2s themselves without putting an additional load on the existing NREN infrastructures. As we learned during the last years, the network

During the 2010 data taking the available resources were sufficient to cover the needs of experiments, but during 2011 the computing slots as well as the storage capacities at sites started to be full. Since the experience clearly shows that delivery of the Physics results is limited by resources, the experiments are facing a necessity of more efficient usage of existing resources. There are task forces studying the possibility of using the next generations computing and storage technologies. There is for instance a question of using multicore processors which might go into the high performance computing market while WLCG prefers

and will spare storage resources and bring less network load.

is extremely important and better connected countries do better.

[70,71].

touched.

implemented.

**7.2 Network**

**7.3 Resources**

usage of commodity hardware.

**7.1 Data management**


*3.4. Banning Diesel Vehicles*

Perhaps, surprisingly, analysis of the survey results revealed that the vast majority of respondents would support the ban. About 65% of the sample population agreed with it, as opposed to 20% who would not be in favour. Nearly 15% of the sample population said they "neither agree nor disagree" with the ban, which suggests that they might be open to new policies and would not be considered problematic or non-compliant should such a policy come to be enforced.

A selection of the respondents are cited below expressing their views about the ban:

"I have only recently found out how bad diesel cars can be. It is a great initiative to ban them, but the government should give diesel car owners a huge incentive to compensate for the purchase of a new car that meets their requirements." (male, 42)

"Ban diesel cars, educate people to shift from cars to public transport, and increase diesel prices" (female, 36)

"Getting rid of diesel is just a start, we should work towards banning all private cars as soon as possible, including electrics which waste as much space." (female, 28)

"I would support more punitive measures including the expansion of the Congestion Charge Zone/London LEZ and even higher costs for diesel engines in Parking Control Zones. Also, a London based scrappage scheme which should incentivise diesel car users with active travel-based solutions, e.g., trading your diesel car for a new bike/public transport season ticket etc." (male, 37).

Of those surveyed, almost 13% advocated the ban when asked about recommendations for improving air quality in London in the open survey question. Therefore, support for a policy enforcing a ban is a conclusion that can legitimately be drawn from the data analysed in this research.

It is believed that banning diesel vehicles would create a positive impact on the environment by reducing the amount of air pollutants in the atmosphere. In Delhi, for example, slightly less than half of the diesel vehicles were replaced by compressed natural gas (CNG) alternatives [59]. Interestingly, in just one year, ambient air pollutants were reduced significantly. The case study reported reductions in sulphur dioxide (SO2), carbon monoxide (CO), and NOx by 22%, 10%, and 6%, respectively [59]. We can speculate about what the impact would have been if those diesel vehicles were replaced by zero emission EVs. Likewise, in the context of London, if a total ban on diesel vehicles is implemented, a positive impact would be seen in terms of reductions in pollutant concentrations, similar to, or possibly better than, those reported in the Delhi case study, since they would be replaced by EVs rather than CNG, which represent a more attractive alternative in terms of emissions produced.

Experts argue [7] that banning diesel vehicles would cause a substantial reduction in the NOx and PM2.5 emissions of 47% and 52%, respectively on the 2015 levels by the year 2030. Reductions in the NOx and PM2.5 levels would contribute to savings totalling 300 Disability Adjusted Life Years (DALYs). One DALY can be defined as the loss of the equivalent of one year of full health [64]. By the year 2024, diesel vehicles would be responsible for about 6300 DALYs. In the light of these findings, we can claim with confidence, that mitigation measures, i.e., banning diesel vehicles, should be considered as soon as possible. It is further argued that the ban would not only improve air quality and people's health but would also increase the uptake of EVs, especially if the appropriate infrastructure is in place [7].

Although some scholars [16] claim that there is little point in banning individual vehicles from certain roads because doing so would only improve air quality on those particular roads, but could consequently displace the pollution onto other roads, it can be argued that the ban first needs to be tested on specific roads for a certain period of time before implementing it across the entire city. People on low incomes who use their vehicles for their daily commute to work would be the group affected most by the ban since they may struggle to purchase newer models. If the ban is proven to achieve the desired outcomes in respect to improved air quality, then those people could be o ffered incentives to switch to more environmentally-friendly vehicles or, for instance, benefit from reduced public transport fares.

If policymakers also advocate implementing the ban but have concerns regarding the public acceptability, then the policy could be introduced gradually or on a trial basis. For instance, policymakers could initially enforce a ban on the most polluted roads by restricting the entry of diesel vehicles only. They could also enforce a ban in di fferent areas for a limited amount of time, and then if this was found to result in significant reductions in pollutants levels by monitoring progress, the ban could be extended to more areas of London, thereby increasing the public confidence in respect to any proposed ban.

Overall, it is highly desirable to phase-out all old diesel vehicles since they cause higher levels of pollution than newer models [2,37,56]. In addition, new buses and taxis operating in London are more environmentally benign and thus should also be considered as an alternative to current diesel-fuelled vehicles [17,37].

### *3.5. Type of Vehicle Owned by Participants*

The results of this survey question provide an insight into the proportion of car users in addition to the engine type of their vehicles. As can be seen from the analysis in Figure 2, at least 61% of the respondents were car users, while 39% were non-car users. It can be assumed that some respondents were also car users but did not own a car. The following questions were aimed at eliciting whether car drivers would be willing to switch from ICE vehicles to the use of EVs as the 2040 ban would apply to both petrol and diesel vehicles. If the number of car drivers in this survey had been very low, then the survey analysis would be inadequate because car drivers are the targeted group in respect to their attitudes towards the forthcoming ban and their willingness to shift to more environmentally-friendly alternatives. Therefore, as more than 61% of the respondents were car drivers, the analysis can be considered valid, although it may be desirable to increase the sample size in further research.

In 2016, there were 39% diesel vehicles, 60% petrol vehicles and 1.3% other vehicle types (EVs and hybrids) in Great Britain [3]. These statistics are consistent with, and to some extent similar to, the survey results in terms of the proportion of each vehicle type among the population, which suggests that the survey succeeded in reaching the targeted groups and being representative of the overall population.

**Figure 2.** Type of vehicle owned.

### *3.6. Shifting to Electric Vehicles*

Remarkably, over 68% of car drivers said they would agree to replace their current vehicles with EVs, as opposed to 20% who would refuse. Just under 12% of the respondents claimed to "neither agree nor disagree" with replacing their vehicles with EVs. It may be possible to persuade this group to switch to EVs by providing scrappage schemes or incentives to purchase them [37,56]. Therefore, one implication of this is that the government should provide a scrappage scheme for old vehicles to encourage both those who "disagree" and those who "neither agree nor disagree" to consider this option.

When participants were asked about their recommendations for tackling air pollution, nearly 12% responded that the adoption of EVs would provide a solution. Furthermore, about 8% of the participants said that incentives are necessary in order to persuade people to switch to EVs, while others complained about the high prices of EVs. It is therefore proposed that the government and Mayor of London should incentivise the purchase of EVs to increase their uptake rate. Below is a selection of the participants' opinions:

"London's air quality could be improved by the government giving incentives and benefits when people purchase an electric car." (male, 50).

"Diesel cars should be a relic of the past. We need to improve the performance of electric vehicles and lower their cost so that they are more widely adopted." (female, 39).

The KWT was employed to test the relationship between the willingness to switch to EVs and the likelihood of supporting the ban. The results revealed that an H value of 13.07 was obtained, as highlighted in Table 2, suggesting that a statistically significant relationship exists. As such, those who are willing to replace their diesel/petrol vehicles with electric ones are more likely to support the ban. Therefore, if the Mayor wishes to implement this policy soon, it is advised that the incentives for choosing EVs be put in place [37]. This programme could also be stimulated by the market. For instance, car manufacturers should be encouraged to be ambitious to produce more EVs and fewer ICE ones. After all, as discussed in the analysis, over 68% of car users agree on the need to shift to EVs.

As stated in the MTS [17], the goal is for all newly registered vehicles in London to be zero emissions so as to achieve the zero-emission road transport by 2050. Therefore, it is vital to discuss which factors would influence the acceptance of EVs in the capital to rapidly increase their adoption as a way to address air pollution.

Fundamentally, moving towards a more sustainable transport system through the uptake of EVs is critically linked to the level of public awareness and concern about the environmental impacts of ICE vehicles as well as EVs. For instance, Khaola et al. [65] investigated a possible correlation between people's concern for the environment and their attitudes towards green products (e.g., EVs) in general and found that there is a strong correlation between them. Additionally, Dogan and Ozmen [66] surveyed 752 respondents to investigate how the environmental concern would affect participants' attitudes towards buying EVs, and they discovered that participants with higher levels of environmental concern were more interested in EVs and more likely to purchase them.

Furthermore, it is argued that those who seek additional knowledge about EVs may be strongly influenced in terms of positive attitudes towards EVs. Many authors argue [20,67] that having a prior knowledge about EVs can make a huge di fference regarding the decision-making process of buying EVs compared to those who have little or no knowledge.

Another factor that is believed to influence the adoption of EVs is incentives [68]. Studies show that providing incentives exerts a positive influence on purchasing EVs [69,70] given that the higher the incentive, the more likely people will purchase an EV. Incentives can be provided in di fferent forms such as tax rebates and subsidies on purchase [71]. A tax rebate of £1000, for example, means that individuals will have a £1000 reduction in their tax when they purchase an EV. A study that was conducted in the United States showed that \$1000 in tax rebate resulted in a 2.6% increase in EVs sales [71]. It is suggested that a similar method be used in London to encourage more people to purchase zero emission vehicles as an alternative to more polluting ones.

Although EVs are being introduced as an alternative to break fuel dependence and tackle other environmental problems [67], it is imperative to acknowledge that non-exhaust emissions should be also be taken into account when considering how best to address air pollution. There is a substantial volume of literature that has reached the conclusion that the volume of emissions from non-exhaust sources is much greater than emissions from vehicles exhausts [12,72,73]. Therefore, the goal of zero-emission road transport cited in the MTS is most likely to be achieved when there is almost no car use. Otherwise, it would be very challenging to realise this ambitious vision set out by the Mayor in the event that private cars, taxis, and buses continue to operate on London's roads. However, the phasing-out of ICE vehicles would significantly improve the NOx reductions [7] since EVs do not emit those kinds of pollutants.

### *3.7. Influence on which Kind of Vehicle to Purchase*

The findings from Figure 3 highlight the importance of providing information (i.e., raising awareness) about the harm caused by diesel vehicles, as the participants showed positive attitudes towards low emission vehicles such as hybrids and EVs. Interestingly, about 60% of those surveyed said that they would purchase a low-emission vehicle including hybrids and EVs. Furthermore, about 4% of the respondents stated that they would not purchase any vehicle because they prefer to use other types of public transport such as trains, buses, and cycling. In addition, the most remarkable observation to emerge from comparing Figures 2 and 3 is that the percentages of diesel and petrol vehicles dropped by more than half, from 18% to 8% and from 37% to 17%, respectively.

**Figure 3.** Which kind of vehicle to purchase in the future.

The KWT was also employed here to ascertain the e ffect of providing information on people's decision-making when buying a new vehicle. The test results presented in Table 1 reveal that providing people with relevant information about the potential harm caused by emissions from diesel vehicles will influence their decision-making process and, ultimately, lead them to make more environmentally friendly choices. It might be argued that those who are considering purchasing a more environmentally friendly vehicle are more likely to agree with the ban on diesel vehicles. Therefore, it is essential that the motor industries get involved [37] with this movement towards a sustainable zero-carbon road transport system by providing more environmentally friendly vehicles to help more people to make the shift away from ICE vehicles. This view was also expressed by one survey respondent:

"I think the car industry needs to be the leader. Technology can be used to assist in sustainable & cost-effective vehicles and transport modes." (female, 29).

Below is a selection of the participants' views about, and recommendations for, improving the local air quality in London:

"Air quality could be improved by creating more green spaces and trees as well as encouraging people to use public transport. Electric vehicles are the future, but at the moment they do not seem to be viable on a large scale due to their high cost." (male, 25).

"Encourage people to use environmentally friendly vehicles by reducing their price." (male, 19).

Although the findings in Figure 3 indicate positive attitudes towards different types of EVs, many researchers [74–76] believe that the high cost of purchase represents a barrier to the adoption of EVs, which concurs with the views cited above. Additionally, several authors [67,68] have identified other potential barriers to the adoption of EVs, such as their limited range, as well as style, size, performance and safety.

Nonetheless, some of these barriers could potentially be overcome by driver training, for example to address the problem of EV's limited mileage range [67]. Moreover, it is important to understand the specific needs and requirements of Londoners in relation to a vehicle. This could help to develop effective policies and stepping-stone guidelines for car manufacturers so as to overcome some of the aforementioned barriers. Two key factors could foster the acceptance of EVs, namely providing incentives in different forms, and raising consciousness about and concern for the environment through awareness campaigns. Acceptance can also be increased by advertising and marketing, which might include word-of-mouth recommendation [77]. Additionally, Globisch et al. [78] stressed the importance of providing EV users with the necessary information, support and assistance to overcome technical failures, as a way to foster the EV deployment. They also found that focusing on the vehicle design (perceived enjoyment) of EVs can significantly influence the choice of the car purchased.

Intervention by policymakers could also make a difference [79]. For instance, a new policy could be introduced stipulating that, for every sports car sold by manufacturers, five EVs should be sold. This would even-out the total amount of emissions produced by a particular car manufacturer since sports cars emit more pollutants than EVs [80]. It would also potentially encourage car manufacturers to set ambitious goals in terms of increasing the EV sales using different methods such as awareness-raising campaigns, and more efficient marketing tools, as well as providing customised assistance to their users.

### **4. Conclusions and Policy Implications**

Using London as a case study, this paper explored the public opinion and respondents' views regarding the forthcoming ban on diesel vehicles, examined whether the ban could be implemented earlier than 2040, and set out to discover Londoners' attitudes towards switching to EVs.

The analysis from the KWT revealed that being concerned about the environment, together with an appropriate level of awareness surrounding its vulnerability to certain hazards, could play a huge role in supporting the ban. In order to facilitate the implementation of the ban, it is highly recommended that Londoners' level of awareness about the harm caused by diesel vehicles needs to be increased, which can be achieved by awareness-raising campaigns. Findings from the survey also revealed that the majority of participants "agree" with the ban, suggesting that the policy can be implemented well before 2040. These results have been very encouraging, and the discussion proposed several courses of action for the policy of banning diesel vehicles.

This work has shown substantial evidence that the majority of car drivers—slightly less than 70%—would agree to switch from driving ICE vehicles to EVs. However, some participants raised concerns about the high price of EVs whereas others proposed that the government should provide

incentives for people to switch to EVs, which suggests that Londoners may not be willing to pay more for a private transport. A further important implication is that Londoners must be provided with incentives tailored to their needs, as well as ways of increasing their awareness of and concern for the environment as a solution to facilitate the acceptance of EVs. Another factor likely to enhance the EV policy and uptake rate is the banning of diesel vehicles, if the appropriate infrastructure is provided. This may include creating su fficient charging stations and extending the installation of charging points to cover more residential areas as well as carparks [20,67,81].

Our findings will be useful for the Mayor of London and policymakers to push the Agenda 2030 forward so as to achieve the goals of sustainable cities and communities as well as climate action [82]. This paper has led us to conclude that the ban should be enforced as soon as possible since the majority of Londoners appear to support the initiative, according to the sample in this research. We would argue that even if CO2 emissions were to be controlled and reduced, the overall climate change e ffect would still exist because of the time scales involved, meaning that it takes a long time to reverse the severe impacts of poor air quality [18]. In order to achieve the desired outcomes for air quality in the long-term, critical decisions must be taken and "aggressive" policies implemented now to determine the quality of London's air in the future.

**Author Contributions:** M.S. primarily ran the modelling, produced the figures and tables, designed the questionnaire, collected the surveys, and co-wrote the manuscript. M.C. designed the questionnaire, further interpreted the modelling results, commented on the interview results, and edited the manuscript. Y.Z., C.P., X.G., and Y.L. commented on and edited the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank the two special issue editors and the three anonymous reviewers for their suggestions regarding the improvement of the initial draft. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Conflicts of Interest:** The authors declare no conflict of interest.