Prion protein, PrPC, is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrPC in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline-regulated lentiviral vectors that upregulate or suppresses PrPC expression. Here, we show that expression of PrPC in pluripotent hESCs cultured under self-renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrPC in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, overexpression of PrPC in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrPC is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self-renewal and differentiation, and determining the fate of hESCs differentiation. The current study suggests that PrPC is at the cross-roads of several signaling pathways that regulate the switch between preservation of or departure from the self-renewal state, control cell proliferation activity and define stem cell fate.
human embryonic stem cells; prion protein; self-renewal; stem cell differentiation; stem cell fate
The transmissible agent of prion disease consists of prion protein in β-sheet rich state (PrPSc), which can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrPSc template. Here three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrPSc, whereas an alternative amyloid state, with a folding pattern different from that of PrPSc induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrPSc emerged. The dynamics between the two forms suggest that atypical PrPres gave rise to PrPSc. While no PrPSc was found in preparations of fibrils using Protein Misfolding Cyclic Amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb suggesting that atypical PrPres was the first product of PrPC misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrPSc-templated or spontaneous conversion of PrPC into PrPSc exists. This study provides compelling evidence that non-infectious amyloids with a structure different from that of PrPSc could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.
Recent studies demonstrated that the efficiency, rate and yield of prion amplification in vitro could be substantially improved by supplementing Protein Misfolding Cyclic Amplification (PMCA) with Teflon beads [Gonzalez-Montalban, et al. (2011) PLoS Pathog. 7, e1001277]. Here we employed the new PMCA format with beads (PMCAb) to gain insight into the mechanism of prion amplification. Using a panel of six hamster prion strains, the effect of beads on amplification was found to be strain-specific, with the largest improvements in efficiency observed for strains with the highest conformational stability. This result suggests a link between PrPSc conformational stability and its fragmentation rate and that beads improved amplification by assisting fragmentation. Furthermore, while exploring the PrPSc-independent bead effect mechanism, a synergy between the effects of RNA and beads on amplification was observed. Consistent with previous studies, amplification of all six hamster strains tested here was found to be RNA-dependent. Under sonication conditions used for PMCA, large RNA molecules were found to degrade into smaller fragments of a size that was previously shown to be the most effective in facilitating prion conversion. We speculate that sonication-induced changes in RNA size distribution could be one of the rate-limiting steps in prion amplification.
Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrPSc and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. Here we discuss a new replication mechanism designated as “deformed templating,” according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.
amyloid fibrils; infectivity; neurodegenerative diseases; prion diseases; prion protein; template-assisted mechanism
It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein (PrP). Several biophysical techniques including X-ray diffraction, CD, FTIR, hydrogen-deuterium exchange, proteinase K-digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary and quaternary structures. While both states displayed a 4.8 Å meridional X-ray diffraction typical for amyloid cross-β spines, they showed markedly different equatorial profiles suggesting different folding pattern of β-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-β structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 °C, only local unfolding was revealed, while individual state-specific cross-β features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-β structure. Both S- and R-fibrils, however, shared high conformational stability arguing that the energy landscape for protein folding and aggregation can contain several deep free energy minima.
amyloid fibrils; prion protein; X-ray diffraction; FTIR; hydrogen-deuterium exchange
Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23–231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31–231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31–231 (0.1–1%) in mixtures with full-length PrP 23–231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23–30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23–30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23–30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.
amyloid fibrils; fibril morphology; fibril polymorphism; prion protein; electron microscopy
Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian PrP under three different growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable was found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires cross-β structure. Using atomic force microscopy imaging we found that fibril fragmentation occurred in both directions, perpendicular to and along of fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins including α-B-crystalline, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.
amyloid fibrils; conformational stability; prion protein; fibril fragmentation; chaperones
The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into the disease-associated, transmissible form (PrPSc). PrPC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrPC and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrPC glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. The current study demonstrates that undersialylated PrPC is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrPC. As a result, PMCAb-derived PrPSc was less sialylated than brain-derived PrPSc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrPC using sialidase was found to increase the rate of PrPSc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrPC reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrPC was also found to control PrPSc glycoform ratio. A decrease in PrPC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrPSc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrPC differed from that of spleen-derived PrPC. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrPC, suggesting that Neu1 is not responsible for desialylation of PrPC. The current work highlights previously unappreciated role of PrPC sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.
The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into disease-associated, transmissible form (PrPSc). The amino acid sequence of PrPC and strain-specific structure of PrPSc are among the key parameters that control prion replication and transmission. The current study showed that PrPC posttranslational modification, specifically sialylation of N-linked glycans, plays a key role in regulating prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. A decrease in PrPC sialylation level increased the rate of prion replication in a strain-specific manner and reduced or eliminated a species barrier when prion replication was seeded by heterologous seeds. At the same time, a decrease in sialylation correlated with a drop in infectivity of PrPSc material produced in vitro. The current study also demonstrated that the PrPSc glycoform ratio, which is an important feature used for strain typing, is not only controlled by prion strain or host but also the sialylation status of PrPC. This study opens multiple new directions in prion research, including development of new therapeutic approaches.
Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogencity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides. However, AFM imaging on its own provides limited insight into conformation or composition of assemblies produced in the complex environment of a cell, or prepared from a mixture of proteins as a result of cross-seeding. In these cases, its combination with fluorescence microscopy (AFFM) increases its resolution.
Amyloids; Assembly; Atomic force microscopy; Atomic force fluorescence microscopy; Immunofluorescence; Oligomers
Proteins can be modified with eight homogenous ubiquitin chains linked by an isopeptide bond between the C-terminus of one ubiquitin and an amine from one of the seven lysines or the N-terminal methionine of the next ubiquitin. These topologically distinct ubiquitin chains signal for many essential cellular functions, such as protein degradation, cell cycle progression, DNA repair, and signal transduction. The lysine 48 (K48)-linked ubiquitin chain is one of the most abundant chains and a major proteasome-targeting signal in cells. Despite recent advancements in imaging linkage-specific polyubiquitin chains, no tool is available for imaging K48 chains in live cells. Here we report on a ubiquitination-induced fluorescence complementation (UiFC) assay for detecting K48 ubiquitin chains in vitro and in live cells. For this assay, two nonfluorescent fragments of a fluorescent protein were fused to the ubiquitin-interacting motifs (UIMs) of epsin1 protein. Upon simultaneous binding to a ubiquitin chain, the nonfluorescent fragments of the two fusion proteins are brought in close proximity to reconstitute fluorescence. When used in vitro, UiFC preferentially detected K48 ubiquitin chains with excellent signal-to-noise ratio. Time-lapse imaging revealed that UiFC is capable of monitoring increases in polyubiquitination induced by treatment with proteasome inhibitor, by agents that induce stress, and during mitophagy in live cells.
Misfolding and aggregation of prion protein (PrP) is related to several
neurodegenerative diseases in humans such as Creutzfeldt–Jacob disease,
fatal familial insomnia, and Gerstmann–Straussler–Sheinker
disease. Certain applications in prion area require recombinant PrP of high
purity and quality. Here, we report an experimental procedure for expression and
purification of full-length mammalian PrP. This protocol has been proved to
yield PrP of extremely high purity that lacks PrP adducts, which are normally
generated as a result of spontaneous oxidation or degradation. We also describe
methods for the preparation of amyloid fibrils from recombinant PrP in vitro.
Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of
Prpsc for development of prion diagnostics including the
generation of PrpSc-specific antibody.
Recombinant prion protein; Inclusion body; Protein purification; Amyloid fibrils; Conformational transiton; Prion diseases
The molecular mechanisms underlying structural diversity of amyloid fibrils or prion strains formed within the same primary structure is considered to be one of the most enigmatic questions in prion biology. We report here on the direct characterization of amyloid structures using novel spectroscopic technique, hydrogen-deuterium exchange ultraviolet Raman spectroscopy. This method enables us to assess the structural differences within highly ordered cross-β cores of two amyloid states produced within the same amino acid sequence of full-length mammalian prion protein. We found that while both amyloid states consisted of β-structures, their cross β-cores exhibited hydrogen bonding of different strengths. Moreover, Raman spectroscopy revealed that both amyloid states displayed equally narrow crystalline–like bands suggesting uniform structures of cross β-cores within each state. Taken together, these data suggest highly polymorphous fibrils can display highly uniform structure of their cross β-core and belong to the same prion strain.
Raman spectroscopy; atomic force microscopy; amyloid fibril; polymorphism; structure
Bioassay by end-point dilution has been employed for decades for routine determination of prion infectivity titer. Here we show that the new Protein Misfolding Cyclic Amplification with Teflon beads (PMCAb) can be used to estimate titers of the misfolded version of the prion protein (PrPSc) with a higher level of precision and in 3 to 6 days as opposed to two years, when compared with bioassay. For two hamster strains 263K and SSLOW, median infective doses (ID50) determined by PCMAb (PMCAb50) were found to be 1012.8 and 1012.2 per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding ID50 values measured by bioassay. These 102-103-fold differences could be attributed to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID50 and PMCAb50 could be due to higher rate of clearance of PrPSc seeds in animals versus PMCAb reactions. A well calibrated PMCAb reaction can be an efficient and cost effective method for the estimation of PrPSc titer.
Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrPSc) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrPSc particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrPSc was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrPSc comparing to those of brain-derived PrPSc. These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrPSc properties occurred during in vitro amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or ‘quasi-species’ and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.
The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrPSc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc template. Here we report that authentic PrPSc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrPSc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrPSc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrPSc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.
The transmissible agent of prion disease consists of a prion protein in its abnormal conformation (PrPSc), which replicates itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc. The current study reports that infectious prions and transmissible prion disease can be triggered in wild type animals by amyloid fibrils produced from recombinant prion prtotein, which are structurally different from PrPSc and lacks any detectable PrPSc particles. This work introduces a new hypothesis that transmissible prion diseases can be induced by prion protein structures different from that of authentic PrPSc and suggests that a new mechanism for triggering PrPSc formation different from the classical templating exists. The current work provides important new insight into the mechanisms underlying genesis and evolution of the transmissible states of the prion protein and has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.
We report the results of solid state nuclear magnetic (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23-231, Syrian hamster sequence). Measurements of intermolecular 13C-13C dipole-dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C-13C and 15N-13C solid state NMR spectra of a uniformly 15N,13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals can not be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173-224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90-231 of the human PrP sequence, formed under somewhat different conditions, suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.
amyloid; prion; cross-β; scrapie; dipolar recoupling; magic-angle spinning
According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.
For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrPC) at high levels confirming that cytotoxicity was in part PrPC-dependent. Silencing of PrPC expression by small hairpin RNAs designed to silence expression of human PrPC (shRNA-PrPC) deminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrPC-mediated and PrPC-independent mechanisms depends on the structure of the aggregates.
This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrPC expression can be exploited to reduce their deleterious effects.
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro.
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro. While PMCA has become an important tool in prion research, its application is limited because of low yield, poor efficiency and, sometimes, stochastic behavior. The current study introduces a new PMCA format that dramatically improves the efficiency, yield, and robustness of prion conversion in vitro and reduces the time of the reaction. These improvements have numerous implications. The method opens new opportunities for improving prion detection and for generating large amounts of PrPSc in vitro. Furthermore, the results demonstrate that in vitro conversion is not limited by lack of convertible PrPC nor by concentrations of cellular cofactors required for prion conversion.
Amyloid fibrils are highly ordered crystal-like structures. It is generally assumed that individual amyloid fibrils consist of conformationally uniform cross-β-sheet structures that enable the amyloids to replicate their individual conformations via a template-dependent mechanism. Recent studies revealed that amyloids are capable of accommodating a global conformational switch from one amyloid strain to another within individual fibrils. The current review highlights the high adaptation potential of amyloid structures and discusses the implication of these findings for several emerging issues including prion strain adaptation (i.e. gradual change in strain structure). It also proposes that the catalytic activity of an amyloid structure should be separated from its templating effect, and raises the question of strain classification according to their promiscuous or species-specific nature.
amyloid fibrils; conformational switch; prion protein; strain adaptation
Cyanobacteria account for 20–30% of Earth's primary photosynthetic productivity and convert solar energy into biomass-stored chemical energy at the rate of ∼450 TW . These single-cell microorganisms are resilient predecessors of all higher oxygenic phototrophs and can be found in self-sustaining, nitrogen-fixing communities the world over, from Antarctic glaciers to the Sahara desert .
Here we show that diverse genera of cyanobacteria including biofilm-forming and pelagic strains have a conserved light-dependent electrogenic activity, i.e. the ability to transfer electrons to their surroundings in response to illumination. Naturally-growing biofilm-forming photosynthetic consortia also displayed light-dependent electrogenic activity, demonstrating that this phenomenon is not limited to individual cultures. Treatment with site-specific inhibitors revealed the electrons originate at the photosynthetic electron transfer chain (P-ETC). Moreover, electrogenic activity was observed upon illumination only with blue or red but not green light confirming that P-ETC is the source of electrons. The yield of electrons harvested by extracellular electron acceptor to photons available for photosynthesis ranged from 0.05% to 0.3%, although the efficiency of electron harvesting likely varies depending on terminal electron acceptor.
The current study illustrates that cyanobacterial electrogenic activity is an important microbiological conduit of solar energy into the biosphere. The mechanism responsible for electrogenic activity in cyanobacteria appears to be fundamentally different from the one exploited in previously discovered electrogenic bacteria, such as Geobacter, where electrons are derived from oxidation of organic compounds and transported via a respiratory electron transfer chain (R-ETC) , . The electrogenic pathway of cyanobacteria might be exploited to develop light-sensitive devices or future technologies that convert solar energy into limited amounts of electricity in a self-sustainable, CO2-free manner.
Prions arise when the cellular prion protein (PrPC) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrPSc. Frequently, PrPSc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrPSc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrPSc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrPSc.
Prions are infectious proteins that cause heritable, sporadic, and transmissible diseases in humans and other mammals. These infectious proteins arise when the normal form of the prion protein (PrP) adopts a self-perpetuating conformation. This disease-causing PrP form is frequently distinguished from normal PrP by its resistance to digestion by proteases although considerable evidence shows that protease-sensitive prions occur naturally in humans and sheep. Here we describe the generation of novel protease-sensitive synthetic prions. After producing recombinant PrP of the wild-type mouse sequence in Escherichia coli, we polymerized the protein into an amyloid fiber conformation. Mice inoculated with these amyloid fibers developed extensive neurodegeneration characteristic of prion disease, but did not generate protease-resistant PrP. Prions from sick animals were transmitted to healthy animals, which likewise developed neurodegeneration but not protease-resistant prions. These novel synthetic prions demonstrate that truncated wild-type PrP can undergo a conformational change that becomes infectious yet the protein remains protease sensitive.
Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.
Prion disease; Generating prion infectivity; Prion strains; Prion neuropathology; Recombinant prion protein; Amyloid fibrils; Prion plaques
In contrast to most amyloidogenic proteins or peptides that do not contain any significant post-translational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant non-peptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with a N-myristoylamido-maleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher-order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher-order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure, and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core, but controls the mode of lateral assembly of filaments into higher-order fibrils.