Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria
© Macedo et al. 2015
Received: 6 July 2015
Accepted: 17 October 2015
Published: 4 November 2015
An increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrPC), into its misfolded pathogenic form (PrPSc) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation.
Here we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90–231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.
Overall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.
KeywordsMammalian prions Protein aggregation Protein conformation Inclusion bodies Amyloids E. coli
Protein aggregation is the hallmark of many neurodegenerative diseases, including Alzheimer’s (AD), Parkinson’s (PD), and the Transmissible Spongiform Encephalopathies (TSEs) , also termed prion diseases. The misfolding of a particular protein, i.e., the β-amyloid peptide (Aβ) for AD, α-synuclein (α-syn) for PD, and prion protein (PrP) for TSEs can lead to its abnormal accumulation in tissues, which usually comes along with severe cellular damages. Irrespectively of the misfolded protein sequence and structure, protein aggregation usually proceeds in a well-organized fashion to form amyloids in these diseases . Amyloid fibrils architecture is characterized by a β-sheet enriched core, which usually binds to Congo red (CR) and thioflavin-T (Th-T) dyes .
Inclusion bodies (IBs) formation in bacteria has long been regarded as an unspecific process resulting from the collapse of hydrophobic contacts between partially or totally unfolded species after protein synthesis at the ribosome . However, an increasing body of evidence indicates that the bacterial IBs formed by amyloidogenic proteins share a number of common structural features with the highly ordered and, in many cases, pathogenic amyloid fibrils [16–19]. Interestingly, it was shown that a specific domain of a bacterial DNA replication protein, the RepA-WH1, assembles into fibrils and, when expressed in E. coli, can lead to a peculiar amyloidosis through the inhibition of bacterial proliferation . These RepA-WH1 aggregated particles can be vertically transmitted across generations, thus this protein was characterized as a synthetic bacterial prionoid . Therefore, bacteria have become a simple model system to study intracellular protein aggregation under biologically relevant conditions that cannot be easily recapitulated in vitro, such as the presence of chaperones and proteases, molecular crowding, and the continuous synthesis of the protein in the ribosome [20–22].
Het-s, from the fungus Podospora anserina, was the first prion protein whose bacterial IBs were shown to display amyloid-like properties [23, 24]. The differential trait of these aggregates emerged when they were transfected into prion-free fungal strains, as they promoted prionic conversion . This result was later corroborated for the yeast prion Sup35 [25, 26]. The amyloid-like IBs of Sup35 induced the prion phenotype in prion-free yeast strains, the infectivity rate being modulated by the environmental conditions during the formation of IBs [25–27]. These observations provide perhaps the best evidence that the IBs molecular structure can recapitulate the architecture of amyloid fibrils, in such a way that even the infectious properties of amyloids, which depend on specific conformational features, seem to be conserved in the two type of aggregates.
It was previously shown that bacterially expressed recombinant murine PrP can be turned infectious in vitro causing prion pathology when inoculated in mice . Here, we address whether, like their fungal counterparts, mammalian PrP can form amyloid intracellular aggregates when expressed in bacteria. With this aim, we produced, purified and conformationally characterized the intracellular aggregates formed by the wild-type murine PrP encompassing residues 23–231 (PrPWT) and the C-terminal domain of murine PrP (PrP90–231) (Fig. 1). Our current study provides the first demonstration that recombinant murine PrPs can form amyloid structures inside bacterial IBs. Besides, although possessing similar secondary structure, PrPWT IBs and PrP90–231 IBs exhibit conformational diversity, as they bind CR and Th-T dyes to different extents, display distinct morphology, different stability and resistance against proteinase K proteolysis. These conformational differences result in different toxicity of the two PrP IBs resistant cores when added to neuroblastoma cells in culture.
Results and discussion
Aggregation of PrPWT and PrP90–231 into IBs in bacteria
The inherent aggregation propensity of amyloid proteins often results in their aggregation into insoluble IBs when they are produced in bacteria . In several cases, these intracellular aggregates have been shown to display amyloid-like properties. To verify if this is the case for mammalian prion proteins, we expressed the murine wild-type prion protein (PrPWT) encompassing residues 23-231 (PrPWT) and the C-terminal domain of murine PrP (PrP90–231) in bacteria and purified the resulting IBs. Both PrP forms [either the full-length, mature PrP (PrPWT) or the truncated fragment 90–231] can exist in vivo in healthy and diseased brain and have been extensively studied [30–32]. The N-terminal unstructured domain is proposed to participate in PrP physiological function because of its ability to bind to different classes of partners, including copper ions (Cu2+), glycosaminoglycans (GAGs), nucleic acids (NAs) and lipids [33–38]. It is proposed that PrPC acts as cell surface scaffold protein, gathering different partners in a macromolecular assembly to participate in cell signalling . PrPC undergoes endoproteolytic attack within its N-terminal domain, leading to the appearance of C-terminal fragments attached to the plasma membrane and soluble N-terminal peptides . Both in normal and pathological brains one of these cleavages occurs at position 90, thereby generating PrP90–231 C-terminal fragment. The truncated PrP encompassing residues 90–231 corresponds to the proteinase K-resistant core of the pathogenic PrPSc, referred also as PrPRes . Although the N-terminal domain appears to be unnecessary for prion propagation, since the fragment PrPRes is capable of transmitting prion disease in vivo ; this region may affect the pathways of prion misfolding and substantially impact PrPSc conformational diversity. Furthermore, in vivo studies have shown that mice expressing N-terminally truncated PrP develop disease more slowly and are less susceptible to infection than mice expressing PrPWT .
PrPWT and PrP90–231 form β-sheet enriched IBs
The aggregation of proteins into amyloid fibrils results in the formation of intermolecular β-sheets [16, 42]. Attenuated Total Reflectance–Fourier Transform Infrared spectroscopy (ATR-FTIR) permits addressing the structural characteristics of protein aggregates [43–46].
Assignment of secondary structure components of purified E. coli PrPWT and PrP90−231 IBs in the amide I region of ATR-FTIR spectra
Amide I ATR-FTIR spectra deconvolution and band assignment was done as described in the Methods section with OMNIC™ software. Band frequencies deviation: ±4 cm−1. The depicted wavenumbers refer to bands 1–5 (from the higher to the lower frequency) obtained from Fig. 3.
Stability of PrPs IBs towards chemical denaturation
PrPWT and PrP90–231 IBs bind to thioflavin-S in living cells
Amyloid properties of PrPWT and PrP90–231 IBs
We evaluated the binding of purified PrP IBs to the amyloid diagnostic dyes CR and Th-T to confirm that the prevalent β-sheet in these aggregates has an amyloid-like nature and to further explore if the IBs would present different amyloid properties. To evaluate the specific contribution of PrP IBs in these assays, relative to that of other proteins possibly present in this fraction, cells bearing the same plasmid without any insert were induced and the insoluble fraction purified in the same manner than those containing the PrP cDNAs and used as negative control.
ThT fluorescence emission increases significantly when the dye binds to amyloid fibrils . Both PrPs IBs promote a strong increase in Th-T fluorescence (Fig. 6b). In agreement with CR data, PrPWT IBs promote a larger increase in Th-T fluorescence (eightfold) than the promoted by PrP90–231 IBs (3.5-fold), whereas the negative control did not induce any significant increase in fluorescence emission relative to free Th-T (Fig. 6b). Although these two PrP IBs possesses similar secondary structure content, these data indicate that murine PrPWT and PrP90–231 adopt amyloid-like structures when they aggregate intracellularly in bacteria, displaying, however, different fibrillar structures. Indeed, it is known that mature fibrils of different PrP species, like hamster or mice, have similar secondary structures but show variation in fibrillar morphology .
Amyloid morphology of PrPs IBs
Amyloid seeding capacity of PrPWT IBs
We analysed the morphology of the aggregates present in the final reaction of seeded and unseeded kinetics by TEM. In agreement with spectroscopic data, the presence of PrPWT IBs and fibrils at the beginning of the reaction resulted in an increased number of amyloid fibrils in respect to non-seeded reactions or reactions seeded with PrP90–231 IBs. However, the acceleration of the fibrillation promoted by PrPWT fibrils and especially by PrPWT IBs results in the formation of apparently amorphous material tightly associated to the newly formed amyloid fibrils (Fig. 9). Interestingly, we could see after seeding PrP amyloid formation with PrPWT IBs the presence of twisted fibrils (Fig. 9). Several early prion studies reported that filamentous structures were found in scrapie-infected rodent brain [68, 69]. In 1981, one report has called attention to helical fibrils formed by the twisting of two or four filamentous structures; they were found in preparations from brains of scrapie-infected rodents . The ultrastructural morphology of these fibrils was reported to be different from that of many amyloids . Nowadays, there are many reports providing information about the PrP twisted fibrillar structures .
Using small amounts of the PrPWT IBs seeded solution for re-seeding PrPWT soluble protein results in formation of visible fibrillar material that, when analysed by TEM, displays typical amyloid morphology coexisting with more amorphous material, confirming thus the striking ability of PrPWT IBs to effectively propagate soluble protein conformational conversion into amyloid structures (Additional file 2).
PrPs IBs display a proteinase K resistant core
Prions are misfolded, self-propagating and infectious proteins. The bacterial inclusion bodies formed by fungal prions, such as HET-s PFD, Ure2p and Sup35-NM have been shown to display an amyloid fold and to be infective. This ability to embrace potentially harmful misfolded polypeptides into insoluble deposits seems to be a strategy mechanism perpetuated along the evolution from prokaryotic bacteria to highly complex eukaryotic organisms. We showed here for the first time that the IBs formed by mammalian prion proteins are also enriched in seeding competent amyloid-like structures, supporting the formation of prion-like conformations inside bacteria. Moreover, these aggregates display conformational diversity, thus becoming an interesting and simple model to study how this property can be modulated in vivo by the quality control machinery. Since PrP accumulates in IBs at high levels and these biological particles are easily purified, it is suggested that they might become a convenient source to obtain prion particles. It is clear that the bacterial cytosol where these aggregates are formed differs from that of eukaryotic cells; however, the potentiality of these inside-the-cell formed amyloid particles to adopt infective conformations is, in our opinion, much higher than the one of the aggregates formed by the purified recombinant protein in vitro after complete denaturation and refolding procedures. In addition, as already shown for other amyloids , PrP-producing bacterial cells can potentially be used for the easy and cheap screening of anti-aggregation compounds able to prevent intracellular PrP amyloid-like aggregation, being thus useful in the early stages of discovery of anti-prionic drugs.
Prion proteins expression and purification
E. coli C43 (DE3) cells were transformed with plasmids (pET-28 b) encoding the murine wild-type prion protein (PrPWT) encompassing residues 23–231 and the murine PrP C-terminal domain residues 90–231 (PrP90–231), both containing a histidine-tag. Cells were grown aerobically in liquid Luria–Bertani (LB) medium containing appropriate antibiotics in a rotary shaker at 37 °C and 250 RPM. Overnight cultures were diluted 100-fold in LB and allowed to grow to an OD600 = 0.7. At OD600 = 0.7, expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG). Cells were harvested after overnight induction (18–20 h), centrifuged, resuspended in 60 mL of buffer U (8 M urea, 10 mM Tris–HCl, 100 mM NaH2PO4, 10 mM reduced glutathione, pH 8.0). After sonication and centrifugation, the soluble protein fraction was added to 5 mL of HisTrap FF prepacked column (GE Healthcare) and washed with 30 mL of buffer U. On-column oxidative refolding was performed by applying for 2 h a 160 mL-gradient of buffer U to buffer B (10 mM Tris–HCl, 100 mM NaH2PO4, pH 8.0). Then, the column was washed with 50 mL buffer B. Unspecific bound proteins were removed from the column with 50 mL of 50 mM imidazole in buffer B. The recombinant proteins PrPWT and PrP90–231 were eluted with buffer E (10 mM Tris, 100 mM NaH2PO4, 750 mM imidazole, pH 5.8). Histidine tail-fused PrP was dialyzed against milliQ water at least two times. The histidine tail was removed from the prion protein using thrombin (1:1000). The cleavage reaction was carried out at room temperature for 2 h. After thrombin cleavage, the sample was repurified in the HisTrap column. Finally, the protein solution was dialyzed against milliQ water two times to remove any remaining salt. 15 % SDS-PAGE analysis revealed more than 95 % of purity.
PrPWT and PrP90–231 IBs extraction
IBs were purified from IPTG-induced cells harbouring the pET-28(b)/PrPWT plasmid, the pET-28(b)/PrP90–231 plasmid and vector alone by detergent-based procedures. IBs were purified from induced cell extracts by detergent-based procedures as previously described . Briefly, cells in a 10 mL culture were harvested by centrifugation at 12,000g (at 4 °C) for 15 min and resuspended in 200 µL of lysis buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 100 mM NaCl), plus 30 µL of 100 mM protease inhibitor PMSF and 6 µL of a 10 mg/mL lysozyme solution. After 30 min of incubation at 37 °C under gentle agitation, Nonidet-P40 was added at 1 % (v/v) and the mixture was incubated at 4 °C for 30 min. Then, DNase I and RNase were added to a final concentration of 25 μg/mL and 3 µL of 1 M MgSO4 was added. The resulting mixture was further incubated at 37 °C for 30 min. Protein aggregates were separated by centrifugation at 12,000g at 4 °C for 15 min. Finally, IBs were washed once with the same buffer containing 0.5 % Triton X-100 and once with phosphate buffered saline (PBS). After a final centrifugation at 12,000g for 15 min, pellets were stored at −20 °C until analysis. The frozen pellets were reconstituted in PBS. SDS-PAGE analysis revealed that in all cases the murine prion proteins were the major polypeptidic components of the aggregates. Prion proteins concentration in IBs was estimated using image densitometry software ImageJ in the SDS-PAGE gel analysis in comparison with the respective dosed purified protein. We performed the same procedures with cells extracts of bacteria containing an empty plasmid as control IBs.
Secondary structure determination
Attenuated total reflectance (ATR)-Fourier Transformed Infrared spectroscopy analyses of PrPWT and PrP90–231 IBs were performed using a Nicolet 6700 IR spectrometer (Thermo Scientific, USA) equipped with an ATR accessory. Dried samples were applied directly to the ATR crystal to be analysed. Each spectrum consisted of 128 accumulated scans, measured at a spectral resolution of 4 cm−1 within the mid-IR range (4000–675 cm−1). Fourier deconvolution of the FTIR spectra was performed with a resolution enhancement factor of 1.6 and a bandwidth of 21 cm−1. Peak position and curve fitting were determined with OMNIC™ software v. 8.0 (Thermo Scientific WI, USA) with a mixed Gaussian-Lorenztian function, allowing assignment of different secondary structure components in the amide I range (1700–1600 cm−1) [47, 48, 67].
Congo red binding
To get insights into the amyloid nature of the PrPWT and PrP90–231 IBs, CR binding assays were performed. The interaction of 20 μM CR with the purified IBs (final OD600: 0.1) was tested using a Cary100 UV/Vis spectrophotometer (Varian, Palo Alto, CA, USA). CR binding was quantified by the equation: CR bound = Abs540nm/25,295 − ABS477nm/46,306 . The extent of amyloid structure was measured by the increase of CR bound to PrP IBs in relation to control IBs [IBs purified from IPTG-induced cells harbouring only the pET-28(b) plasmid].
Thioflavin T (Th-T) binding assay
Th-T binding was used to probe amyloid presence in the samples. Incubation of 30 μM Th-T with PrPWT IBs and PrP90–231 IBs (final OD600: 0.1) or the correspondent amyloid fibrils was recorded using a Jasco FP-8200 spectrofluorometer (Jasco Inc, MD, USA) with an excitation wavelength of 445 nm and emission range from 480 to 580 nm at 37 °C in PBS. Five individual scans were averaged for each measurement. The intensity of the spectra at the 482 nm maximum was recorded as an indication of the extent of amyloid conformation in the aggregates.
Thioflavin-S binding in living cells
Detection of thioflavin-S (Th-S) binding was performed in living cells expressing PrPWT, or PrP90–231 and control non-induced cells. Bacterial cells were washed with PBS buffer and diluted at an OD600 of 0.1. Th-S was added at 125 µM final concentration; cells were then incubated for 1 h and washed twice with PBS. Cells were placed on top of a microscope slide and covered with a cover slip. Photographs were acquired at 40-fold magnification under UV light in a Leica fluorescence microscope (Leica DMRB, Heidelberg, Germany).
For stability assays, purified PrPWT and PrP90–231 IBs were prepared at final OD350 = 1 in PBS containing selected concentrations of urea ranging from 0 to 8 M. The reactions were allowed to reach equilibrium by incubating them for 12 h at room temperature. The fraction of soluble protein (fS) was calculated from the fitted values using equation: fS = 1 − ((yS − y)/(yS − yA)), where yS and yA are the absorbance at 350 nm of the soluble and aggregated protein, respectively, and y is the absorbance of the protein solution as a function of the denaturant concentration. The value m1/2 was calculated as the denaturant concentration at which fS = 1/2. OD350 changes were monitored with a Cary400 Varian spectrophotometer.
Transmission electron microscopy (TEM)
Each sample (20 μL) was applied to a carbon coated copper grid, and after 5 min the grid was washed with MilliQ water. Samples were stained with 2 % (w/v) uranyl acetate for 1 min and then washed again. Images were collected on a Jeol 1200 microscope (Boston, MA, USA) operating at 80 kV.
In vitro conversion of PrP into amyloid fibrils
To target amyloid fibril formation, PrP solutions were prepared immediately before use by resuspending lyophilized purified PrPWT powder in 4 M GdnHCl, 0.02 M thiourea, and 0.1 M MES, pH 6.0, in a protocol adapted from previous studies . Samples were centrifuged at 12,000g for 5 min and the protein concentration was determined by its extinction coefficient at 280 nm (63,495 M−1 cm−1), calculated from the PrPWT primary sequence in http://web.expasy.org/protparam/. The fibrillation reactions of 0.5 mg/mL PrP were carried out in 1.5-ml conical low-binding plastic tubes up to a total reaction volume of 0.6 ml at 37 °C with continuous shaking at 600 rpm for at least 3 days using an Eppendorf Thermomixer Comfort (Eppendorf, USA). Aliquots from each sample were taken over time.
PrP aggregation departing from monomeric recombinant PrP was monitored by measuring the transition from non-aggregated to aggregated state by following light scattering at 350 nm in a Jasco FP-8200 spectrofluorometer (Jasco Inc, MD, USA). The polymerization reactions showed typical nucleation-elongation kinetics of amyloid formation. The reactions were carried out with 0.5 mg/mL of soluble purified PrPWT in 4 M GdnHCl, 0.02 M thiourea, and 0.1 M MES pH 6.0 using 1 cm-path length quartz cuvette in a total reaction volume of 1 mL at 37 °C with continuous shaking at 600 rpm using micro-stir bars. In the seeding assays, a solution of PrPWT IBs resuspended in PBS with OD350 of 10.0 were sonicated for 10 min, and then diluted 100-fold (final OD350 = 0.1) at the beginning of the reaction. The seeding ability of 2 % preformed fibrils (after 10 min of sonication) was also evaluated. Cross-seeding assays were performed in the same manner by adding a sonicated solution of PrP90–231 IBs (final OD350 = 0.1) to initially soluble PrPWT.
Confocal microscopy and image processing
Confocal images of human neuroblastoma (SH-SY5Y) cell cultures were captured in complete medium at 37 °C, using a laser scanning confocal microscope (Leica TCS SP2 AOBS equipped with a HCX PL APO 63 × 1.4 oil, immersion objective, Germany). Briefly, SH-SY5Y cells were seeded in 35 cm2 plates (Mat Tek) with approximately 30 % of confluence in complete medium and incubated for 72 h in the presence of sterile PBS buffer + PK (positive control) and the PK-resistant core of the PrPs IBs. Proteinase K was inactivated by boiling all solutions before applying them to cultured cells. Cells were incubated with 0.5 μg/mL SYTO green and 10 μg/mL propidium iodide (PI) (Molecular Probes) for 15 min at 37 °C and washed twice with PBS buffer. Cell morphology was analysed by confocal fluorescence microscopy using an orange diode (588–715 nm emission collected) and a UV laser (excited at 350 nm and collected at 405 nm). Two independent experiments, both in duplicate were done and the entire field of each plate was observed at the microscope.
Proteinase K (PK) resistance assay
The PK concentration in this assay was optimized in preliminary experiments (not shown). PrPWT IBs and PrP90–231 IBs at final OD350 of 0.5 were incubated with PK (Sigma-Aldrich, USA) at final concentration of 2.5 μg/mL in PBS for 1 h at 37 °C. Aliquots of PK digestion were taken at every 10 min and the reaction quenched by the addition of the same amount of 4 times concentrated denaturing sample buffer. Samples were heated at 95 °C for 5 min and analysed by Tris–Glycine SDS-PAGE. The assay with soluble purified recombinant PrP was performed in the same manner .
BM carried out most of the experiments and drafted the manuscript. RS and SN participated in the experimental work. SV and YC supervised the project and revised the manuscript. All authors read and approved the final manuscript.
We thank Pablo Castro for help with microscopy analysis and we also thank Dr. Byron Caughey (RML, NIH) for providing us the rabbit polyclonal anti-PrP antibody R20. This work was supported by Ministry of Education of Brazil [CAPES process number: 99999.002869/2014-04 to the fellow student B.M]; and by Ministerio de Economia y Competividad, Spain [BFU2013-44763P to S.V.]; by ICREA [ICREA Academia 2009 to S.V.] and by FAPERJ, CNPq and INBEB from Brazil to Y.C.
The authors declare that they have no competing interests.
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