Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

Amyloids are ordered protein aggregates typically associated with neurodegenerative diseases, such as Alzheimer disease and Parkinson disease, which usually result in cognitive impairment. However, the amyloid state of the neuronal RNA binding protein Orb2 shows a quite opposite behaviour: instead of impairment, it is associated with memory consolidation. Here, we have characterized the structural features of Orb2 from the monomer to the amyloid state in order to determine why it shows this unexplained puzzling behaviour. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids. However, Orb2 rapidly forms amyloids, and its toxic intermediates are transient. Furthermore, we find that an anti-amyloidogenic peptide that blocks the initial structural transitions in pathological amyloids also blocks Orb2 amyloid formation and memory consolidation. Our results provide structural insights into how Orb2 amyloids can mediate stable memory while being nontoxic and how amyloid-based diseases may affect memory processes. They also suggest a new pharmacological approach for blocking memory consolidation.

Competing interests: The authors declare competing financial interest: MCV and RH are co-inventors on an international patent application (reference EP15382176.4) covering the results contained in this article. Any potential income generated by exploitation of the patent rights and received by their employer, the CSIC, shall be shared with these authors according to Spanish law and the regulations of the CSIC.

Funding: This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid), Centro de Investigación Biomédica en Red sobre Enfermedades Respiratorias (CIBERES) to MM, CTQ2011-22514 to MB, Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency, Grants-in-Aid for Scientific Research on Innovative Areas (Synapse and Neurocircuit Pathology) from the Ministry of Education, Culture, Sports, Science, and Technology, Health Labour Sciences Research Grant for Research on Intractable Diseases from the Ministry of Health, Labour and Welfare, Japan to YN and a Ramon y Cajal contract from MICINN to SCT. The following fellowships were involved: Fundación Ferrer (Severo Ochoa fellowship) and Canon Foundation to RH, SIMR to LY and AM, MECD to AGP, MINECO to ES, and Japan Intractable Disease Research Foundation to MS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The nervous system is particularly susceptible to amyloid-driven diseases, some of which lead to severe cognitive deficits [ 22 ]. Currently, it remains unclear why some amyloids in general are detrimental for neurons, while a seemingly similar amyloid state of some neuronal CPEB proteins is critical for memory stabilization [ 9 , 11 ]. As with other biological conundrums, a structural/functional analysis of CPEB/Orb2 proteins may shed light on the features that distinguish functional from pathological amyloids, and it may also help us to understand the molecular basis of memory consolidation. Here, we have employed an array of in vitro (bulk and single-molecule biophysics as well as cell culture) and in vivo (Drosophila and yeast) techniques to characterize Orb2 amyloid both structurally and functionally. In addition to dissecting out the characteristics of the Q/N-rich PLD-containing region of Orb2, we have found that a known anti-amyloidogenic peptide inhibits some forms of memory consolidation in Drosophila. Furthermore, comparing with pathological amyloids, we found that although in solution Orb2 can form toxic oligomers, these toxic species rapidly progress to innocuous species. These transient Orb2 species are structurally similar to toxic Huntingtin aggregates and, when abundant, these two proteins form a heteromeric complex. These findings indicate that there are intrinsic structural constraints that prevent functional amyloids to dwell in the toxic conformation. These observations also provide clues as to how pathological amyloids may interfere with the function of those beneficial amyloids.

The Drosophila melanogaster orthologue of CPEB, Orb2, has two isoforms that are structurally similar to the ApCPEB isoform: Orb2A and Orb2B [ 17 , 18 ]. Both forms are expressed in the fly brain and they are required for long-term memory [ 11 , 17 , 19 ]. Orb2A, a synaptic protein, is scarce, and its availability is controlled by phosphorylation [ 19 , 20 ]. This isoform has eight amino acid residues (YNKFVNFI) preceding the PLD, which are critical for both the efficiency as well as the nature of the amyloid-like oligomers being formed [ 11 ]. The longer Orb2B isoform, with a region of 162 residues preceding the PLD, appears to act as a canonical CPEB [ 21 ], regulating translation via its RNA-binding domain [ 18 , 21 ]. The PLD of Orb2A has a low Q/N content compared to ApCPEB (23.5% versus 48.1%), it acts in long-term memory independently of its RNA-binding domain [ 21 ], and its mutations prevent memory consolidation [ 11 , 17 ].

One functional amyloid-like protein of particular interest is the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB family of proteins regulates the translation of dormant mRNAs [ 6 , 7 ], and some members of this family are involved in synaptic plasticity and long-lasting memory [ 8 , 9 ]. The CPEB isoforms share a common C-terminal catalytic region (RNA-binding domain), but they differ in the N-terminal region. Surprisingly, the N-terminus of some CPEB isoforms in Aplysia, Drosophila, and mouse have features characteristic of self-sustaining amyloidogenic (prion-like) proteins [ 9 – 13 ]. For example, the neuronal specific isoform of Aplysia CPEB (ApCPEB) has a glutamine-asparagine (Q/N)-rich N-terminal domain, which resembles a yeast prion-like domain (PLD) [ 14 ], and it is predicted to have conformational flexibility [ 10 ]. Indeed, ApCPEB undergoes a conformational transition to a β-sheet-rich state similar to that undertaken by other prion-like proteins [ 15 ]. In sensory neurons, the neurotransmitter serotonin controls the prion-like switch from the monomeric form to the self-sustaining oligomeric state, which is important for the serotonin-induced increase in synaptic strength [ 16 ]. Accordingly, it has been postulated that the switch to the oligomeric and self-perpetuating state contributes to the long-term maintenance of synapse-specific changes [ 16 ], providing a molecular mechanism for the persistence of memory [ 10 ].

Amyloids, whose cross-β fold has been postulated as the ancestral protein fold [ 1 , 2 ], were initially associated with fatal neurodegenerative disorders [ 3 , 4 ]. However, more than a century after the identification of these “pathological amyloids,” a growing list of so-called “functional amyloids” that fulfils a wide variety of physiological functions has recently emerged [ 5 ]. The discovery of functional amyloids raises the question of what causes such a strikingly distinct behaviour to that observed in pathological amyloids. Indeed, it remains unclear what features are shared by functional and pathological amyloids and what determines whether a particular amyloid is functional rather than toxic.

Results

Orb2 Forms Canonical Amyloids Very Efficiently Both the endogenous and the recombinant Orb2 protein form sodium dodecyl sulphate (SDS) and urea-resistant oligomers that are characteristic of amyloids [11]. However, it remains unclear to what extent the Orb2 amyloid behaves as a typical pathological amyloid. Both recombinant Orb2A and Orb2B bind to thioflavin T (ThT) and Congo red (CR) dyes (Fig 1A–1C and S1A Fig), and this binding is inhibited by amyloid destabilizing reagents such as rottlerin [23] or the polyphenol (-)-epigallocatechin gallate (EGCG: S1B Fig) [24,25]. The soluble form of both full-length isoforms has a helix-rich secondary structure monitored by Circular Dichroism (CD) spectroscopy (Fig 1D). However, over time both isoforms become rich in amyloid-specific β-sheets, as evidenced by Fourier transform infrared (FTIR) spectroscopy (Fig 1E), which coincides with aggregation of the protein, as monitored by turbidimetry (Fig 1F). Furthermore, transmission electron microscopy (TEM) showed Orb2A to form spherical oligomers and typical unbranched amyloid fibers (Fig 1G). Interestingly, Orb2A adopted an amyloid structure more efficiently (without the typical lag phase) than other amyloids like the Aβ42 peptide or the yeast prion Sup35NM, with a t½ of ~15 min and at a protein concentration 8–10-fold lower (S1C Fig). Based on these observations, we conclude that, in isolation, Orb2A not only forms seemingly canonical amyloid structures but it also does so in an extremely efficient manner. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Orb2 forms self-propagating and canonical amyloids. (A) Pictograms showing the domain organization of Orb2A and Orb2B isoforms. PLD, prion-like domain, in red; RRM, RNA-binding domains, in yellow; ZnF, zinc-finger motif, in purple. The N-terminal amino acids preceding the PLD of Orb2A (eight amino acids) and Orb2B (162 amino acids) are represented in green and blue, respectively. (B) In the presence of both Orb2A and Orb2B, ThT (Proteostat) shows enhanced fluorescence emission at 485 nm over time, a typical feature of amyloids, although the kinetics is much faster for Orb2A and Orb2B than other amyloids (see S1 Fig). (C) Concentration (μM) of the azo-dye CR bound to the amyloid component formed by Orb2. The data are represented as the mean ± standard error of the mean (SEM): ***p < 0.001 (One-way ANOVA and Tukey post-test). The inset shows a 63x image of an Orb2A amyloid under a polarized light microscope. (D) The CD spectra of soluble, full-length isoforms of Orb2 showed a α-helix-rich secondary structure. Deconvolution of the CD spectrum by using algorithms in DICHROWEB showed that Orb2A has 6% more α-helix and 5% less random coil conformations than Orb2B. (E) Relative abundance of secondary structural elements determined using FTIR. These distributions are significantly different from one another and from a non-amyloidogenic control protein concanavalin A control with a p-value of < 0.001 (chi-square test). (F) Aggregation of both isoforms over the incubation period monitored by turbidimetry at 405 nm. (G) Representative electron micrograph of oligomers (asterisks) and amyloid fibers (arrow) formed by Orb2A. Scale bar: 0.2 μm. (H) Schematic representation of the constructs used in the yeast prion assay (left panel). Orb2A-Sup35C efficiently switches to the prion state. Randomly selected clones were replica plated either in complete yeast extract peptone dextrose (YPD) media or in media lacking adenine. Only Orb2A produced a high frequency of red Ade- and white Ade+ colonies (right panel). NM: N-terminal and medial regions; C: C-terminal region. The underlying data for panels in this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.1002361.g001

Prion-like Properties of Orb2A In order to determine whether Orb2A amyloid possesses the self-sustaining properties of prion-like proteins, we took advantage of the well-characterized yeast prion Sup35, a translation termination factor that can exist in two states: as an active monomeric state (nonprion) and as a less active amyloid state (prion) [26]. The conversion between those two states can be readily assessed by non-sense suppression of the mutant ade1-14 allele. In the nonprion state of Sup35, yeast colonies appear red in rich media, and they cannot grow in media containing a limiting amount of Adenine (-ade media), while in the prion-state, read-through translation turns the colonies white and cells can grow in -ade media. We substituted the NM prion domain of the Sup35 protein with the N-terminal domain of Orb2A, Orb2B or a paralogue of Orb2 in Drosophila, Orb1 (Fig 1H, left), and these chimeric constructs were expressed under the control of Sup35 promoter, representing the sole source of the Sup35 protein [27]. While all these chimeric proteins substituted the essential function of the Sup35 protein, only Sup35 carrying the N-terminal domain of Orb2A produced two readily distinguishable phenotypes: red Ade- [prion-] and white Ade+ [PRION+] colonies (Fig 1H, right). The Orb2A N-terminal domain produced the prion-like state with an unusually high frequency and perpetuated stably through hundreds of generations. Random selection of 100 colonies revealed that almost 50% of them spontaneously gave rise to white-pink Ade+ colonies for Orb2A, compared to ~1% for Orb2B and none for Orb1 (Fig 1H, right). Furthermore, the nonprion and prion-like states correlated with the monomeric and SDS-urea resistant amyloidogenic oligomeric states of Orb2, respectively (S1D Fig). These results indicate Orb2A is very efficient in adopting a self-sustaining amyloid-like state.

Boundaries and Conformational States of the Orb2A PLD A group of the prion-like proteins characterized to date contain a PLD that is disordered and frequently composed of a Q/N rich sequence with a low content of charged residues [28–30]. In Orb2A, the entire PLD is composed of an N-terminal Q/N-rich domain (88 residues with 35.2%Q+N, Fig 1A), which is followed by a 74-residue region containing few charged residues (6.8% compared to 21.6% in the rest of the Orb2A protein). PLDs often adopt distinct conformational states, such as coiled-coil rich [31] or stacked β-sheet rich structures [32–36]. The conformational switch can be gauged through their susceptibility to proteases [37]. To determine the region of Orb2A PLD that adopts distinct conformational states, we inserted the tobacco etch virus (TEV) protease recognition motif (ENLYFQG) at the N-terminus of EGFP-tagged Orb2 protein (Fig 2A), and we measured the accessibility of the TEV protease to these sites [38]. Insertion of the TEV protease sites at the positions indicated in Fig 2A did not alter the ability of Orb2 to oligomerize (S2A and S2B Fig) or bind to mRNA (S2C Fig). We found that the TEV protease site located outside the N-terminal of Orb2A or Orb2B (Orb2A162TEV, Orb2A216TEV and Orb2B370TEV) was cleaved efficiently and equivalently in both the oligomeric and the monomeric forms (Fig 2B). However, when the TEV site was located within the first 162 residues of Orb2A (Orb2A88TEV), or within the first 242 residues of Orb2B (Orb2B242TEV), most of the oligomers and a fraction of the monomers were not cleaved by TEV after 24h incubation with TEV protease (Fig 2B). This differential cleavage of monomers implies that: i) the 88 N-terminal residues are not inherently inaccessible; and ii) there is a conformational variability even among monomeric forms. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Organization of the Orb2 PLD. (A). Schematic representation of the location of the TEV protease recognition motif (ENLYFQG) inserted into Orb2. (B) EGFP-tagged Orb2A and Orb2B bearing TEV protease sites were expressed pan-neuronally, and immunopurified Orb2 was treated with TEV protease for 24 h. The left panel shows the schematic of the experiment. The cleaved C-terminus of different sizes derived from Orb2A are indicated with a bracket. The * indicates an EGFP reactive polypeptide most likely originated from degradation of the cleaved Orb2B370TEV C-terminus. (C) Schematic diagram of the epithelial fusion failure 1 (EFF-1) cell fusion “cytoduction” experiment in S2 cells. The expected outcomes in terms of the recruitment or nonrecruitment of oligomers are indicated. (D) Residues 88 to 162 are sufficient for recruitment into pre-existing oligomers. Full-length Orb2A-EGFP induced the Orb2A construct lacking the 88 N-terminal residues (Orb2AΔ1-88-Cherry) to oligomerize. Full-length Orb2A from D. willistoni failed to induce oligomerization of Orb2AΔ1-88-Cherry. (E) EGFP, Orb2B-EGFP, or Orb1-EGFP also failed to induce oligomerization of Orb2AΔ1-88-Cherry. Representative examples of fused cells are shown and the n for each experimental set is ≥10. Scale bar = 5 μm. https://doi.org/10.1371/journal.pbio.1002361.g002 To further explore the conformational variability in the monomer population and to rule out the possibility that resistant monomers were not simply oligomers that have dissociated, we obtained protein fractions containing primarily monomeric Orb2 by differential centrifugation. When the TEV protease site is within the first 162 residues of Orb2A, only a fraction of the monomeric protein is accessible to TEV (S3 Fig). These results suggest that the N-terminal Q/N rich region that ends at residue 162 is most likely the boundary of the PLD and that this domain can adopt at least two distinct conformational states: a protease-accessible monomeric state and a protease inaccessible state in both monomeric and oligomeric forms (Fig 2B and S3 Fig).

Dissection of the Orb2A PLD PLDs are often organized such that two distinct regions mediate the initiation of aggregation and recruitment to the aggregate for self-perpetuation. To determine whether the Orb2A PLD has this organization, we studied the recruitment of red-labelled proteins into EGFP-labelled preformed aggregates using a cell fusion-based assay [39]. Three distinct Orb2AΔPLD-Cherry fluorescent protein constructs that lacked specific regions of the PLD were generated: Orb2AΔ1-88-Cherry, Orb2AΔ88-162-Cherry, or Orb2AΔ1-162-Cherry. We fused S2 cells carrying Orb2A-EGFP to Orb2AΔPLD-Cherry expressing cells by inducing the expression of the Caenorabditis elegans epithelial fusion failure 1 (EFF-1) protein [39], which causes cell fusion via a homotypic interaction and the mixing of cytoplasmic but not nuclear components (Fig 2C). While Orb2AΔ1-88-Cherry expression appeared mostly diffuse by itself, in the fused cells this protein variant formed large puncta (Fig 2D). Orb2AΔ88-162-Cherry formed few puncta by itself but co-aggregated efficiently with full-length Orb2A-EGFP to form large fluorescence puncta. By contrast, Orb2AΔ1-162-Cherry, which lacks the entire PLD, failed to form any such aggregates (Fig 2D). Since only Orb2A-EGFP efficiently induced the formation of Orb2AΔ88-Cherry puncta but not EGFP, Orb2B-EGFP, or Orb1-EGFP, these results indicate that the intramolecular interaction is highly specific (Fig 2E). Surprisingly, the Orb2A protein from D. willistoni, which is ~81% identical to the D. melanogaster protein, forms puncta when it is expressed in S2 cells. However, Orb2A from D. willistoni failed to induce the aggregation of Orb2AΔ88-Cherry into puncta (Fig 2D), indicating that this is a species-specific process. These data suggest that the organization of Orb2A’s PLD resembles that of other prion-like proteins, whereby the first 88 residues are important for initiating aggregation, and residues 88–162 are important for the recruitment into preformed aggregates.

A Single Point Mutation in Orb2A Affects Its Amyloidogenic and Prion-like Properties One surprising feature of PLDs is that although the domains can substitute each other, the amino acid sequence of the various PLDs are distinct. Therefore, it remains unclear to what extent individual amino acids at various positions contribute to the prion-like properties. Intriguingly, previously we found that a F to Y substitution at the 5th position of the Orb2A PLD—representing the addition of a single hydroxyl group—dramatically reduced Orb2 oligomerization and impaired memory consolidation [11]. This prompted us to determine the role of the 5th residue in amyloid formation and what aspects of amyloid formation (e.g., recruitment) and/or prion-like behaviour (i.e., self-sustaining properties) are affected by this mutation. To address these issues, the F5 residue was substituted for 18 different residues (Fig 3A), and Orb2A puncta stability was quantified using fluorescence recovery after photobleaching (FRAP). Remarkably, substitution of F5 with any residue except the highly hydrophobic ones (V, I, L, or W, only exception being E) strongly destabilized Orb2A oligomers (Fig 3B). ThT binding to Orb2A variants correlated with FRAP results, and the Orb2AF5Y mutant showed the weakest enhancement in ThT fluorescence (Fig 3C). To determine whether this 5th residue might also play an important role in oligomer packing, in addition to affecting the rate of Orb2A oligomerization, we performed fluorescence resonance energy transfer (FRET) assay. FRET efficiency depends on the distance between the fluorophores, which in turn depends on the orientation and packing of the protein subunits in the homo-oligomer. Surprisingly, only the F5L, F5W, F5S, F5Q and F5P (One-way ANOVA, p > 0.05) substitutions produced an average FRET efficiency similar to the efficiency observed in the wild-type protein. Moreover, the F5Y substitution produced aggregates with very variable FRET efficiency, whereas all other substitutions decreased the average FRET efficiency (*p < 0.05), suggesting that the nature of the 5th residue is important in oligomer packing (Fig 3D). Thus, the 5th residue of Orb2A seems to mediate a key intramolecular interaction important for the oligomerization process [40]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. The F5 residue in Orb2A is critical for Orb2 amyloid-like oligomer formation and self-sustaining prion-like properties. (A) Schematic representation of the experimental design and the positions analyzed by the indicated methods. (B) FRAP was used to measure the dynamic nature of the Orb2A-EGFP aggregates, showing that substitution of the 5th residue makes the aggregates more dynamic. (C) The F5Y mutation reduces the rate of amyloid formation, as gauged by ThT fluorescence. (D) FRET was used to measure the relative orientation and organization of the Orb2A proteins in the aggregate. For Fig 3B and 3D analysis, data are represented as mean ± SEM. We assumed statistical significance at *p < 0.05, **p < 0.01 and ***p < 0.001 (One-way ANOVA). (E) A single residue substitution in Orb2A, F5Y, dramatically reduced the ability to adopt the prion-like state. (F) Orb2A harbouring the F5Y mutation partitions less into the pellet fraction, and it forms less SDS-resistant oligomers than the wild type form. (G) Orb2AF5Y-EGFP did not induce oligomerization of Orb2A lacking the 88 N-terminal residues (Orb2AΔ1-88-Cherry). Scale bar: 5 μm. The underlying data for panels in this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.1002361.g003 We then asked whether this perturbation in oligomer packing and stability affected the prion-like behaviour of Orb2A (see Fig 1H). Remarkably, this single residue substitution dramatically interfered with the capacity of the Orb2A’s PLD to adopt a stable prion-like state, as concluded from a chimeric yeast prion assay (Fig 3E), coinciding with the reduction in the SDS-urea resistant amyloidogenic oligomeric states (Fig 3F). The Orb2AF5Y-EGFP mutant also impaired the formation of Orb2AΔ1-88-Cherry puncta (Fig 3G), and this PLD variant exhibited impaired capability to aggregate, as measured by turbidimetry and far-UV CD spectroscopy (S4A and S4B Fig). This mutant also has decreased ability to form amyloid components, according to a CR binding assay (S4C Fig) and oligomers/fibers, as assessed by TEM (S4D Fig). Taken together, these results corroborate the critical role of the F5 residue of Orb2A for its self-sustaining recruitment and amyloidogenic properties reported to be necessary for memory consolidation [11]. These results also suggest that there are inherent structural features determined by specific amino acid residues that control the Orb2A amyloid formation.

Orb2 Can Be Trapped by Pathological Amyloids Our observations suggest that in solution, Orb2 and polypeptides that form pathological amyloids sample similar conformational ensembles. We wondered whether in spite of being transient and rare, because of their structural similarity, these Orb2 transient toxic conformations could be hijacked by similar, yet longer-lived conformers from neurotoxic amyloids. To test this hypothesis, we coexpressed EGFP-tagged Orb2A protein with the Huntingtin protein-containing expanded polyQ repeats (HttQ128) in the larval motor neuron. HttQ128, unlike Orb2A, is neurotoxic, and its chronic expression produces lethality [50]. In contrast to when it is expressed alone, Orb2A formed larger fluorescence puncta in the presence of HttQ128, indicative of its aggregation, and in some cases these Orb2A puncta were surrounded by HttQ128 protein (Fig 5G and S11A Fig). The effect of HttQ128 on Orb2 appears to be specific, given that it had no obvious effect on other EGFP-tagged proteins, such as the synaptic protein synaptotagmin or the Golgi-associated protein GRASP-65, used as negative controls (Fig 5G and S11B Fig). Similarly, removal of the PLD from Orb2A abrogated its HttQ128-enhanced aggregation (Fig 5G). Since the structural properties of Orb2A are similar to those of the NM PLD of yeast prion Sup35, we also studied a chimeric protein in which the Orb2A PLD was substituted with the Sup35 PLD. Consistent with the structural studies, this chimeric construct was also recruited into the HttQ128 aggregates (Fig 5G and S11C Fig). These results suggest that pathological amyloids, when present in excess, can nucleate Orb2A aggregation, presumably by capturing the transient toxic conformers of Orb2 proteins.

A Small Anti-amyloidogenic Peptide Blocks Orb2 Amyloidogenesis Polyglutamine-binding peptide 1 (QBP1) is a known inhibitor of the amyloidogenesis of HttQ expansions. This hydrophobic peptide binds monomeric expanded polyQ proteins and blocks the initial critical β-conformational transition of these species, suppressing their subsequent oligomerization and fibrillogenesis, and consequently, the associated cytotoxicity and neurodegeneration [41,49,51–53]. QBP1 acts as a polyvalent anti-amyloidogenic agent on several amyloids [41], and thus, we examined the potential inhibitory effect of the minimal active core of QBP1 (Ac-WKWWPGIF-NH 2 ) [54] on Orb2A amyloid formation. The conformational similarity at the monomer level between Orb2 and HttQ prompted us to further examine whether QBP1 could inhibit Orb2A amyloid formation. Isothermal titration calorimetry (ITC) revealed that QBP1 physically interacts with Orb2A, and that complex formation, a slow event, was exothermic in nature (Fig 6A). Under similar conditions, a scrambled version of QBP1 (SCR, Ac-WPIWKGWF-NH 2 ) [54] interacted poorly, serving as a negative control in the subsequent experiments (Fig 6A). Unlike the SCR, incubation with QBP1 inhibited Orb2A PLD aggregation and amyloidogenesis, as monitored by turbidimetry and CR binding (Fig 6B and 6C). Far-UV CD spectroscopy revealed that QBP1, but not SCR, suppressed Orb2A PLD signal loss due to protein precipitation (Fig 6D). Furthermore, TEM showed a significant reduction in the formation of both oligomers and fibers in the presence of QBP1 but not the SCR (Fig 6E) compared to Orb2A PLD alone (see Fig 4C). Consistent with these results, the SMFS analysis revealed that Orb2A PLD treated with QBP1 formed fewer M conformers (from 37.7% to 16.1%), shifting the NM/M proportion towards an increased population of NM conformers, while SCR had no effect on this initial conformational transition (Fig 6F and S12 Fig). Together, these results indicate that QBP1 interferes with Orb2 amyloid formation in vitro and suggest that early in amyloidogenesis, Orb2 and some pathological amyloid-forming polypeptides adopt similar conformations that are recognized and blocked by QBP1. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. QBP1 interferes with Orb2 amyloid formation in vitro. (A) Representative calorimetric traces for the interaction of full-length Orb2A with the QBP1 and SCR peptides. Traces in orange and pink correspond to the heat released upon injection of Orb2A into the ITC cell loaded with an excess of QBP1 or SCR, respectively, while black, blue, and red traces correspond to the Orb2A, QBP1, and SCR dilutions, respectively. (B) QBP1 but not SCR drastically reduced the turbidity at 405 nm of aged Orb2A PLD. (C) QBP1 reduces the quantity of amyloid formed as shown by measuring the CR bound to Orb2A PLD. The data are represented as the mean ± SEM: *p < 0.05 and ***p < 0.001 (One-way ANOVA and Tukey post-test). (D) Far-UV CD spectroscopy in the absence or presence of QBP1 or SCR indicates that QBP1 blocks protein precipitation over time of Orb2A PLD, but SCR does not. (E) Representative electron micrographs show that oligomers (asterisks) and amyloid fibers (arrows) of Orb2A PLD were drastically reduced when incubated with QBP1 (left) but not with the SCR (right). Scale bars: 1 μm. (F) The conformational polymorphism of Orb2A PLD is strongly diminished in the presence of a 1:10 molar excess of QBP1, which decreases the M frequency relative to the NM conformers (QBP1, n = 112 and SCR, n = 101). The underlying data for panels in this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.1002361.g006