Modulation of the Aggregation of the Prion-like Protein RepA-WH1 by

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Modulation of the aggregation of the prion-like protein RepA-WH1 by chaperones in a cell-free expression system and in cytomimetic lipid vesicles Cristina Fernandez, and Rafael Giraldo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00283 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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ACS Synthetic Biology

Modulation of the aggregation of the prion-like protein RepA-WH1 by chaperones in a cell-free expression system and in cytomimetic lipid vesicles

Cristina Fernández* and Rafael Giraldo Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas – CSIC, Madrid E28040, Spain *E-mail: [email protected]

Abstract The accumulation of aggregated forms of proteins as toxic species is associated with fatal diseases as amyloid proteinopathies. With the purpose of deconstructing the molecular mechanisms of these type of diseases through a Synthetic Biology approach, we are working with a model bacterial prion-like protein, RepA-WH1, expressed in a cell-free system. Our findings show that the Hsp70 chaperone from Escherichia coli, together with its Hsp40 and nucleotide exchange factor co-chaperones, modulate the aggregation of the prion-like protein in the cell-free system. Moreover, we observe the same effect by reconstructing the aggregation process inside lipid vesicles. Chaperones reduce the number of aggregates formed, matching previous findings in vivo. We expect that the in vitro approach reported here will help to achieve better understanding and control of amyloid proteinopathies.

Keywords Cell-free synthetic biology, prion-like protein, giant vesicles, DnaK-DnaJ-GrpE chaperones, RepA-WH1

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The formation of amyloid-like fibrils and aggregates by proteins is characteristic of several neurodegenerative diseases, such as Huntington´s, Parkinson´s, Alzheimer´s, prion-related diseases and amyotrophic lateral sclerosis (ALS).1-3 The proteins involved in these disorders vary considerably in their structure, function and size, but they form aggregates with a common, highly ordered cross-β sheet structure.4,5 Apart from mammals, proteins found in other organisms as insects, plants, yeast or bacteria can potentially adopt a similar amyloid architecture but with a functional role.6,7 RepA, the replication initiator protein of the Pseudomonas pPS10 plasmid,8 is a bacterial protein that forms an intracellular functional amyloid involved in the control of DNA replication.9 Moreover, the isolated N-terminal domain (WH1) of the protein, similarly to the mammalian proteins PrP or α-synuclein, assembles into fibrillar aggregates in vitro upon binding to DNA or acidic phospholipids.10-13 Not only RepAWH1 builds amyloid fibrils in vitro but also its expression in bacteria, as a fusion to the fluorescent protein tag mCherry, generates a reduction in cell proliferation as a consequence of the formation of amyloid aggregates in the cytosol.14-16 All the experimental evidence enables RepA-WH1 as a synthetic minimal model system to study protein amyloidosis. The central factor involved in protein folding, unfolding and remodeling aggregates in E. coli is the Hsp70 chaperone DnaK, jointly with its cochaperones Hsp40 (DnaJ) and nucleotide exchange factor (NEF, i.e., GrpE).17,18 In vitro experiments showed that DnaK disassembles RepA-WH1 amyloid fibers into oligomeric particles, whereas microfluidic experiments in vivo, pointed to a direct role of DnaK in remodeling proteotoxic aggregates of the RepA-WH1 prionoid into less cytotoxic species, which are readily transmissible to the progeny during bacterial cell division.16

One important tool in Synthetic Biology (SynBio) bottom-up approaches is the use of cell-free protein systems, i.e. the in vitro expression of recombinant proteins without the use of living cells. In recent years, there are coming up multiple applications of cell-free expression such us membrane proteins production,19 protein evolution20 or analysis of protein aggregation.21,22 In this work, using such SynBio approach, we have studied the aggregation propensity of two variants of the prion-like protein RepA-WH1 and the effect of the chaperone triad DnaK-DnaJ-GrpE on the aggregation. Such approach, including in vitro cell-free protein synthesis in the test tube or inside cytomimetic ACS Paragon Plus Environment

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phospholipid vesicles, enables the identification of specific components of a complex process to unravel its basis and mechanism.23,24

Our former in vitro studies relied on using purified folded RepA-WH1 and the subsequent assay of ligand co-factors affecting its amyloidogenesis. Co-translational protein folding is a thermodynamic-driven process that is kinetically modulated at its local energy minima, resulting in aggregation as a side product.1,17 For addressing cotranslational RepA-WH1 folding and the role of (likely) its major modulator in vivo, the Hsp70 chaperone DnaK,16 in this study we have used a cell-free protein synthesis setup, the commercial PURE system, based on reconstitution of the transcriptional and translational machineries of E. coli.25,26 Western and dot-blot assays, with a specific antibody against oligomeric aggregates formed on-pathway to fibril formation, were employed in the analysis of the cell-free expression products. In addition, we have used, as cell-like compartments, giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) to reconstitute the aggregation process in a cytomimetic reaction environment. With this approach, we have successfully reproduced the aggregation of RepA-WH1 as observed in E. coli cells and have determined the effect on such process of the chaperone DnaK, together with its cochaperones DnaJ and GrpE. The results obtained may be of interest for the identification, using a simplified minimal approach, of factors affecting the aggregation of proteins involved in neurodegenerative diseases, e.g. chaperones, and for the screening of new compounds aiming to prevent it.

Results and Discussion Addressing co-translational RepA-WH1 aggregation in vitro We have evaluated in a cell-free system the aggregation propensity of two mutant variants of the prion-like protein RepA-WH1: A31V and ∆N37. In these proteins, the RepA-WH1 domain is fused through a flexible linker at their C-termini to the monomeric fluorescent protein mCherry. Previous work in bacteria has shown that WH1(A31V)-mCherry forms cytotoxic amyloid aggregates, while WH1(∆N37)mCherry, a deletion mutant lacking the amyloidogenic peptide stretch in RepA-WH1, forms non-cytotoxic inclusion bodies, established through non-amyloid hydrophobic interactions.14-16,27 The wild type form RepA-WH1-mCherry is weakly amyloidogenic



and, in bacteria, led to higher relative levels of soluble protein compared with the two mutant variants. 14,15

For our purpose, we built a set of plasmids to express the proteins under the control of the T7 promoter. This DNA was then used as a template for the PURE cell-free expression.25 Afterwards, the product(s) of the cell-free expression can be directly subjected to additional analysis, including quantitation by means of Western blot and dot-blot assays (Figure 1). To evaluate the solubility of proteins, we followed the method published by Niwa, et al. (2012),28 as described in Material and Methods. Moreover, we encapsulated the components inside GUVs to reconstitute the aggregation process in a confined space simulating a cell-like micro-environment. We used for this purpose GUVs made of POPC, a zwitterionic lipid that, contrary to acidic phospholipids,13 is not interacting with the protein in the conditions of the experiment (i.e., pH 7.5 – 8, the optimal pH for the cell-free expression). GUVs were prepared by means of a water-in-oil emulsion centrifugation method, as described.13 A schematic representation of the experimental procedure can be found in Figure 1. We finally evaluated the aggregation propensity of WH1(A31V/∆N37)-mCherry in the confined system by means of visual inspection in a confocal microscope. As a control we used both the soluble protein mCherry (Figure 2) and the wild type variant of RepA-WH1mCherry (Supporting Information).

[Figure 1]

Our experiments showed a clear difference between both variants: WH1(A31V)mCherry was partially expressed in a soluble state, although some aggregation was also evident, while WH1(∆N37)-mCherry was not soluble at the conditions tested (Figure 2). The wild-type form of RepA-WH1, when expressed with the cell-free system in test tubes, produces a higher level of soluble protein (Figure S1A). In addition, when the PURE system and the plasmid encoding for the prion like-protein were entrapped in giant liposomes (GUVs), we determined, using confocal microscopy, that the protein was indeed synthetized inside the lipid vesicles (Figure 2A). Nearly all WH1(∆N37)mCherry molecules formed large aggregates, while WH1(A31V)-mCherry aggregates were smaller and part of the protein remained soluble, resembling its behavior in vivo. 16 These results are in agreement with preliminary experimental work.29 ACS Paragon Plus Environment

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[Figure 2]

DnaK (Hsp70) chaperone modulates the size of RepA-WH1 aggregates Based on the results described above, we decided to analyze the effect of the chaperone DnaK following the experimental procedure outlined in Figure 1. One of our goals was to determine possible differences in the action of DnaK on the expression of either cytotoxic amyloid aggregates, WH1(A31V)-mCherry, or a protein that forms non-toxic inclusion bodies, WH1(∆N37)-mCherry. Firstly, we performed the in vitro expression of RepA-WH1-mCherry variants in the absence of lipids and, by Western and dot-blots, we analyzed the aggregation propensity of the proteins. Conveniently, the PURE system does not include any chaperone in its formulation so we can evaluate the aggregation propensity of translated RepA-WH1 in the presence and absence of DnaK, a key modulator of the aggregation of RepA-WH1 in vivo,16 just by supplying the purified chaperone. As quantified in Western blots, the addition of DnaK increased to 70% the amount of soluble WH1(A31V)-mCherry (Figure 3A) and almost to 90% for the wild type protein (Figure S1, Figure S2). However, WH1(∆N37)-mCherry did not become soluble even at the highest concentration of DnaK tested (Figure 4).

[Figure 3] [Figure 4]

The cell-free expression samples were then analyzed in a dot-blot assay and incubated with B3h7, a monoclonal antibody specific for a non-native pro-amyloidogenic RepAWH1 conformation.30 DnaK (≥ 5 µM) reduced the presence of pro-amyloidogenic WH1(A31V)-mChery aggregates in the cell-free assay, as revealed by B3h7 (Figure 3B). This antibody does not recognize aggregates of WH1(∆N37)-mCherry,30 the deletion mutant31 lacking the amyloidogenic sequence in RepA-WH1.10,16 As a loading control we used anti-WH1,30 a polyclonal antibody that recognizes both the native (soluble) and aggregated conformations of RepA-WH1 in dot-blot assays, as well as its denatured state in Western blots.



When we expressed WH1(A31V)-mCherry inside GUVs, we noticed that the presence of DnaK reduced the number of aggregates, as observed in confocal microscopy sections (Figure 3C). This result points to a prevention of WH1(A31V)-mCherry aggregation by the Hsp70 chaperone. We also detected that there was a certain degree of heterogeneity between individual liposomes. This effect has been described previously in other studies, and attributed to the fact that some of the components necessary for protein expression are not enclosed at sufficient concentration to achieve the same levels of expression in all vesicles.32 As a control, we also expressed with the cell-free system the mCherry protein itself. We observed that it was uniformly distributed inside GUVs and by Western blotting mainly found in the soluble fraction (Figure 2).

The co-chaperones DnaJ (Hsp40) and GrpE (NEF) enhance remodeling of RepAWH1 aggregates Under normal physiological conditions, Hsp70 works in combination with two cochaperones, DnaJ (Hsp40) and GrpE (NEF), to activate Rep proteins for acting as DNA replication initiators.33 To investigate the role of DnaK on RepA-WH1-mCherry aggregation in a more cytomimetic context, we used a combination of the three chaperones with an optimized DnaK:DnaJ:GrpE ratio (5:1:1).34 The chaperone triad enhanced the recovery of WH1(A31V)-mCherry in the soluble fraction (Figure 5B) and decreased the formation of aggregates inside GUVs (Figure 5A). Cell-free expression of WH1(∆N37)-mCherry in the presence of DnaK+DnaJ+GrpE showed, both in solution and within GUVs, that together the three chaperones also worked more efficiently than DnaK alone to relieve the type of aggregates formed by WH1(∆N37)-mCherry (Figure 6).

[Figure 5] [Figure 6]

Finally, as a control, we have also tested the chaperonin system GroEL+GroES (1:2), but we did not observe any effect on the solubility of the misfolded WH1(A31V)mCherry (Supporting Information, Figure S3) or WH1(∆N37)-mCherry (Supporting Information, Figure S4) proteins. This is not surprising, because these chaperonines


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work in conjunction as an ‘Anfinsen cage’ in the refolding of misfolded protein monomers, not on aggregates.17

Both the WH1(A31V)-mCherry hyper-amyloidogenic variant and WH1(∆N37)mCherry, in the absence of chaperones, were found aggregated, but with distinct appearance. For the WH1(∆N37)-mCherry mutant we observed aggregates of larger size, while WH1(A31V)-mCherry appeared partially soluble and forming smaller aggregates. The same aggregation phenotype is observed in living bacterial cells and attributed to prion-like particles and inclusion bodies, respectively.16 In vivo, mono/dipolar inclusion bodies formed by WH1(∆N37)-mCherry and, in general, by heterologous proteins expressed in E. coli, are large aggregates that asymmetrically distribute between daughter cells upon cell division, in such a way generating a subpopulation of cells freed of the burden imposed by aggregation.16,35 On the contrary, the smaller cytotoxic aggregates assembled by WH1(A31V)-mCherry efficiently propagate from mother to daughter cells, assuring a high penetrance in inheritance.16 In this respect, 'size (and number) matter' in the propagation of prion-like proteins,36,37 and our cytomimetic in vitro system successfully reproduces such relevant functional feature and correlates it with the expression/activity of an Hsp70 chaperone, which decreases the size of the aggregates.

In conclusion, the results presented in this study showed that it is possible to reproduce the RepA-WH1 aggregation observed in vivo with a cell-free system and in reconstituted, cytomimetic lipid vesicles. Moreover, in E. coli the Hsp70 chaperone DnaK, working together with its co-chaperones DnaJ and GrpE, has been shown to prevent protein aggregation of a hyper-amyloidogenic variant of this prion-like protein, and, to a lesser extent, of WH1(∆N37)-mCherry, which forms conventional inclusion bodies in vivo. The absence of endogenous chaperones qualifies the PURE system as an ideal tool for evaluating the role of chaperones in the modulation of the balance between protein folding and aggregation. 28 In previous publications by our group, we had shown that the prion-like protein RepAWH1 generates a unique amyloid proteinopathy in bacteria, having many features in common with the mitochondrial route described for many neurodegenerative diseases, Parkinson´s in particular. 27 Here we push the limits of such model of amyloid diseases



by reconstructing, through bottom-up SynBio approaches in vitro, an essential process in counteracting amyloid cytotoxicity.

Materials and Methods Plasmid construction Plasmids were constructed by consecutive ligation of PCR-amplified fragments carrying repA-WH1-mCherry, repA-WH1(A31V)-mCherry, repA-WH1(∆N37)-mCherry, or the mCherry red fluorescent monomeric protein alone, into the plasmid pET3d (Novagen) with a SacII site at the 5′end of the coding sequence and a BamHI site at the 3′end. The pET3d vector has T7 RNA polymerase promoter and terminator regions. The template DNA for the cell-free synthesis of the protein of interest must have downstream a ribosome-binding site, the open reading frame (ORF, in our case encoding a RepAWH1 variant) and a T7 terminator. Plasmids were purified using the High Pure plasmid isolation kit (Roche) and purity was analyzed using gel electrophoresis in a 0.8% agarose gel. Concentration of plasmids was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific).

In vitro transcription and translation system For achieving coupled in vitro transcription and translation, the PURExpress protein synthesis kit (New England Biolabs, NEB # E6800S) was used. The kit contains two solutions: solution B, containing the T7 RNA polymerase, and solution A, containing the tRNAs and low-molecular weight cofactors. Reactions were carried out by mixing 1.5 µL of solution B with 2 µL of solution A along with the DNA template (0.6 µl at 100 ng/µl) and 0.25 µL of RNasin inhibitor (Promega), adjusted to a final volume of 5.5 µL with nuclease-free water. The reaction components were assembled on ice, and then, the reaction was initiated by incubation at 37°C. When indicated, purified DnaK was added at different concentrations (5, 10 or 15 µM). When required a DnaK+DnaJ+GrpE mix or GroEL+GroES chaperones (GeneFrontier) were added. ACS Paragon Plus Environment

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To evaluate the aggregation propensity, we use the method of Niwa et al., 2012.28 After cell-free expression, sample was diluted to 40 µl with outer buffer (0.1 M Na2SO4, 20 mM NaH2PO4, pH 7.5) and an aliquot (10 µl) was withdrawn from the Eppendorf tube and labelled as the total fraction (T). The remainder was centrifuged at 20,000g for 30 min (Optima MAX-XP Ultracentrifuge with a TL-100 rotor from Beckman) and the supernatant was recovered (sample S). The total and supernatant samples were analyzed by SDS-PAGE (12%) and Western blot. Band intensities were quantified by autoradiography. Solubility was defined as the ratio of the supernatant to the total protein.

Expression and purification of DnaK DnaK was purified from E. coli TG1 cells carrying the pMOB45-dnaK vector38 as follows. LB cultures were grown at 30oC up to OD600nm=0.45, at this optical density the temperature was shifted to 42oC for 1 h, afterwards incubation proceeded overnight at 30oC. Cells were harvested, resuspended in 50 mM Tris.HCl pH 8.0, 10% sucrose plus 1/8 volumes of 0.5 M KCl, 50 mM EDTA pH 8.0, 10 mM β-MeEtOH, 1% Brij-58, 0.1 mM spermidine, 1 mM p-NH2-benzamidine. Lysozyme (to 2 mg.mL-1) and RNase-A (to 0.1 mg.mL-1) were added and left on ice for 45 min, followed by 5 min at 37oC. Ultracentrifugation (Beckman Ti45, 30,000 rpm) was carried out for 45 min at 4oC. The supernatant was loaded in a SP-Sepharose (GE Healthcare) column equilibrated with buffer-A (0.025 M KCl, 20 mM HEPES pH 7.5, 1 mM β-MeEtOH, 0.1 mM EDTA, 10% glycerol). Flow-through was then loaded in a Q-Sepharose (GE Healthcare) column. A linear gradient was developed between buffer-A and this buffer supplemented with KCl to 0.75 M. Fractions containing DnaK were pooled and 1 mL aliquots were then loaded at 0.5 mL.min-1 in a Sephacryl S200-HR (GE Healthcare) 2.6x70 cm gel filtration column equilibrated in buffer-B (0.25 M KCl, 20 mM HEPES pH 7.5, 1 mM β-MeEtOH, 0.1 mM EDTA, 10% glycerol). DnaK peak fractions (95% pure) were ultra-filtrated (Amicon cell, 50 kDa cut-off membrane) and protein concentration

was

determined

(A280nm;

http://web.expasy.org/protparam).

Protein detection and quantitation by Western blot


ε280=14,650M-1.cm-1;


Protein expression levels were determined by means of Western blotting, incubating with primary antibodies against WH1 protein (mCherry fusions) or anti-His (mCherry alone) as follows: α-His, 1:5,000 (Sigma); α-WH1, 1:2,00030 for 2 h. A horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; Sigma) was subsequently incubated for 1 h. Chemiluminescence detection was performed with the ECL Prime kit (GE Healthcare), and band intensity analysis was carried out, with samples from three independent experiments, using ImageJ software.39 For the analysis with Image J several exposure times were taken with a ChemiDoc Imager (BioRad). We chose an exposure time that presented no signal saturation (overexposure), which could be detected by the software (Image Lab) when used in combination with the imager (ChemiDoc). The raw file was used for quantitative purposes. The analysis was performed using the rectangular selection tool in ImageJ. For background correction, an empty lane with no protein loaded was selected as background value, and then subtracted from each lane signal.

Dot-blot assays Nitrocellulose membranes (0.45 µm ø pore; Bio-Rad) were set in a Bio-Dot microfiltration device (Bio-Rad). Wells were pre-equilibrated with 0.1 mM Na2SO4, 20 mM NaH2PO4 (pH 7.5). Protein samples (100 µl of 1/250 dilution of the total fraction of the PURExpress reactions, and subsequent 2-fold step dilutions) were diluted in 0.1 mM Na2SO4, 20 mM NaH2PO4 (pH 7.5) buffer and then spotted under gravity flow for 1 h. Blotted membranes were blocked in 5% powder milk overnight in Trisbuffered saline buffer (pH 7.0) containing 0.01% Tween-20 (TBS-T) and probed for two hours with the primary antibodies α-WH1 and B3h7 (1: 20,000 to 1: 6,000 in TBST) at 4 °C. The membranes were washed three times with TBS-T and then probed with the appropriate HRP-conjugated secondary antibodies (α-mouse/rabbit; 1:10,000) for 1 h. After three additional washes with TBS-T, chemiluminiscent detection was performed on X-ray films with the ECL Prime kit (GE Healthcare).

Giant vesicles preparation The giant unilamellar vesicles (GUVs) encapsulating the PURE system were prepared using the water-in-oil emulsion (w/o) transfer method, as described previously.13,40,41 In short, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, from Sigma) was dissolved in mineral oil (M5904, Sigma) at 0.5 mg/mL. In an Eppendorf tube, 200 µl of


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outer buffer (containing 400 mM of glucose, to achieve isotonic osmotic pressure) were added and on top of 200 µl of POPC in mineral oil. The tube with the solutions has to rest for one hour to allow the formation of a lipid monolayer. Aliquots of 5 µL of the PURE system solution, prepared as described above, were added to 200 µL of mineral oil. This mixture was repeatedly pipetted for several minutes to form w/o emulsions that were equilibrated on ice for 5 min. Then, 200 µL of the emulsion was placed gently on top of the monolayer previously formed and the tube was centrifuged at 2,000 rpm and 20°C for 10 min. The mineral oil on top was removed and the GUVs are washed with the outer solution two times centrifuging at 2,000 rpm. GUVs were collected from the bottom of the Eppendorf tube and incubated for 3 h at 37°C.

Visualizing GUVs with fluorescence microscopy A laser scanning confocal microscope (LEICA TCS SP2, Leica) equipped with a 63× oil immersion objective was used to image the GUVs. The laser line used for mCherry was 514 nm. All measurements were performed at room temperature (20°C).



Figures

Figure 1. Schematic representation of the experimental procedure followed for the analysis of the aggregation of RepA-WH1 and the study of the effect of chaperones. The first step consists in the mixing of the components in a test tube (template DNA, PURExpress components and, when required, the addition of chaperones). Afterwards, two directions are followed: 1. The incubation in the test tube for the expression of the protein, for 3 h at 37°C. After incubation sample was centrifuged at 20,000g for 30 min. The total fraction (T) was analyzed in a dot-blot and the same sample and the supernatant (S) in a Western blot. 2. The encapsulation in giant vesicles by the doubleemulsion method and subsequently the incubation of giant vesicles for 3h at 37°C. Protein expression inside liposomes was observed using confocal microscopy.


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Figure 2. Aggregation propensity of variants of RepA-WH1. A) Expression of RepAWH1 inside GUVs. mCherry protein was expressed as a control. pET-mCherry, pETA31V-mCherry and pET-∆N37-mCherry are the plasmids used as templates for the cell-free expression to produce the corresponding proteins: mCherry, WH1(A31V)mCherry and WH1(∆N37)-mCherry. B) Example of Western blots after cell-free expression.29 Total fraction (T) and soluble sample (S) after centrifugation at 20,000g for 30 min. mCherry protein was detected using α-His antibody, whereas the variants WH1(A31V)-mCherry and WH1(∆N37)-mCherry were detected with a specific antibody against RepA-WH1 protein (α-WH1). C) Western blot quantification of the soluble fraction obtained after cell-free expression of the control and different variants of the prion-like protein RepA-WH1. Experiments were performed in triplicate.



Figure 3. DnaK chaperone modulates the aggregation of WH1(A31V)-mCherry. A) Western blot analysis of the aggregation of WH1(A31V)-mCherry in the presence or absence of DnaK (5 µM). Peak area of bands was determined by densitometry (ImageJ software). Experiments were performed in triplicate; a representative blot image is shown. B) Dot-blot in the presence of DnaK probed with a specific anti-amyloid oligomer conformational antibody (B3h7). As a loading control, α-WH1 was used. C) Confocal fluorescence microscopy of WH1(A31V)-mCherry expression inside GUVs, adding increasing concentrations of DnaK (5, 10, and 15 µM). White arrows point to some large aggregates. Three individual vesicles are presented of each experimental condition.


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Figure 4. DnaK by itself is not able to rescue efficiently the aggregation of WH1(∆N37)-mCherry. A) WH1(∆N37)-mCherry expressed inside GUVs in the absence and presence of 15 µM DnaK. Three examples at each condition are shown. B) Western blot analysis of the total (T) and soluble (S) samples. A representative blot image is shown in the presence or absence of DnaK. No DnaK-promoted WH1(∆N37)mCherry solubilization was observed after centrifugation at 20,000g, but a decreased in the size of the aggregated particles according to (A). Some traces of soluble protein were detected with the addition of DnaK when centrifugation was performed at a lower speed (8,000g). C) Quantification of Western blot analysis of the aggregation of WH1(∆N37)-mCherry in the presence or absence of DnaK, centrifugation was performed at 8,000g. Peak area of bands was determined by densitometry. Experiments were performed in triplicate.



Figure 5. The co-chaperones DnaJ and GrpE help DnaK in remodeling WH1(A31V)mCherry aggregates efficiently. A) Confocal fluorescence microscopy of WH1(A31V)mCherry expression inside GUVs without chaperones and in the presence of a mixture of DnaK+DnaJ+GrpE at 5:1:1 and 10:2:2 µM, respectively. Three individual vesicles are presented of each experimental condition. B) Western blot analysis of the aggregation of WH1(A31V)-mCherry in the presence of DnaK+DnaJ+GrpE mix at 5:1:1 µM, respectively. Experiments were performed in triplicate.


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Figure 6. DnaK, in combination with DnaJ and GrpE, counteracts aggregation of WH1(∆N37)-mCherry. A) WH1(∆N37)-mCherry expressed inside GUVs in the presence of a mixture of DnaK+DnaJ+GrpE at two concentrations: 5:1:1 or 10:2:2 µM of each chaperone respectively. Three examples at each condition are shown. B) Western blot quantification of the soluble fraction obtained after centrifugation at 20,000g in the absence or presence of a mixture of DnaK+DnaJ+GrpE (5:1:1 µM).

Abbreviations POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) GUVs (Giant Unilamellar Vesicles) Hsp70 (heat-shock protein 70) Hsp40 (heat-shock protein 40) NEF (nucleotide exchange factor)

Acknowledgements The authors would like to thank all members of their group for support and M. Jiménez for helpful discussions. We also thank G. Rivas for critical reading of the manuscript and the Reviewers for helpful suggestions. We are indebted to A. Serrano for help in plasmid constructions and to M.T. Seisdedos and G. Elvira from the Confocal Laser Microscopy Facility at CIB-CSIC for assistance in imaging. This work has been financed by grants BIO2015-68730-R and BFU2015-72271-EXP to R.G..



Supporting Information Figures S1- S4 (PDF)

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“For Table of Contents Use Only”

Modulation of the aggregation of the prion-like protein RepA-WH1 by chaperones in a cell-free expression system and in cytomimetic lipid vesicles

Cristina Fernández* and Rafael Giraldo


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Modulation of the Aggregation of the Prion-like Protein RepA-WH1 by

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