Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication.
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ABSTRACT:
SUBMITTER: Browning S
PROVIDER: S-EPMC3220511 | biostudies-literature | 2011 Nov
REPOSITORIES: biostudies-literature
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Project description:Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.
| S-EPMC3457318 | biostudies-literature
Project description:In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrP(Sc)) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrP(C)) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrP(Sc) molecules. Both RML- and 301C-derived prions containing unglycosylated PrP(Sc) molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrP(Sc) deposition and neuronal vacuolation. These results show that PrP(Sc) glycosylation is not necessary for strain-dependent prion neurotropism.
| S-EPMC2681931 | biostudies-literature
Project description:Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP(Sc), an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP(C)). Stable variations in PrP(Sc) conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP(Sc) quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP(Sc) quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP(Sc). To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP(Sc) tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP(Sc) aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP(Sc) quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
| S-EPMC3795044 | biostudies-literature
Project description:Interspecies prion transmission often leads to stable changes in physical and biological features of prion strains, a phenomenon referred to as a strain mutation. It remains unknown whether changes in the replication environment in the absence of changes in PrP primary structure can be a source of strain mutations. To approach this question, RNA content was altered in the course of amplification of hamster strains in serial protein misfolding cyclic amplification (sPMCAb). On adaptation to an RNA-depleted environment and then readaptation to an environment containing RNA, strain 263K gave rise to a novel PrP(Sc) conformation referred to as 263K(R+), which is characterized by very low conformational stability, high sensitivity to proteolytic digestion, and a replication rate of 10(6)-fold/PMCAb round, which exceeded that of 263K by almost 10(4)-fold. A series of PMCAb experiments revealed that 263K(R+) was lacking in brain-derived 263K material, but emerged de novo as a result of changes in RNA content. A similar transformation was also observed for strain Hyper, suggesting that this phenomenon was not limited to 263K. The current work demonstrates that dramatic PrP(Sc) transformations can be induced by changes in the prion replication environment and without changes in PrP primary structure.
| S-EPMC3752540 | biostudies-literature
Project description:The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.
| S-EPMC6013019 | biostudies-literature
Project description:The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.
| S-EPMC7383396 | biostudies-literature
Project description:Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.
| S-EPMC6134792 | biostudies-literature
Project description:Our discovery of dominant-negative inhibition of prion formation in cultured cells provided an explanation for the resistance of some sheep to scrapie and humans to Creutzfeldt-Jakob disease. To determine whether dominant-negative inhibition occurs in vivo, we produced transgenic (Tg) mice expressing prion protein (PrP) with either the Q167R or Q218K mutation alone or in combination with wild-type (wt) PrP. Tg(MoPrP,Q167R)Prnp(0/0) mice expressing mutant PrP at levels equal to non-Tg mice remained healthy for >550 days, indicating that inoculation with prions did not cause disease. Immunoblots of brain homogenates and histologic analysis did not reveal abnormalities. Tg(MoPrP,Q167R)Prnp(+/+) mice expressing both mutant and wt PrP did not exhibit neurologic dysfunction, but their brains revealed low levels of the PrP pathogenic isoform (PrP(Sc)), and sections showed numerous vacuoles and severe astrocytic gliosis at 300 days after inoculation. Both Tg(MoPrP,Q218K)Prnp(0/0) and Tg(MoPrP,Q218K)Prnp(+/+) mice expressing high levels of the transgene product remained healthy for >300 days after inoculation. Neither PrP(Sc) nor neuropathologic changes were found. Our studies demonstrate that although dominant-negative inhibition of wt PrP(Sc) formation occurs, expression of the dominant-negative PrP at the same level as wt PrP does not prevent prion formation completely. However, expression of dominant-negative PrP alone had no deleterious effects on the mice and did not support prion propagation.
| S-EPMC130589 | biostudies-literature
Project description:Mass per length (mpl) measurements on single amyloid fibrils that specifically propagate the [VH], [VK], and [VL] strains of the yeast prion [PSI] reveal unanticipated differences in their structures. Many fibrils have approximately 1.0 prion molecule per 4.7-A cross-beta repeat period, which is consistent with a self-replicating model built by parallel beta-sheet hydrogen-bonding of like prion peptide segments, but other fibrils are definitely heavier. The predominantly straight fibrils of the dominant [VH] strain have a bimodal mpl distribution, corresponding to components with approximately 1.0 and 1.2 prions per repeat. Fibrils of the weaker [VK] strain, which are almost all wavy, have a monodisperse mpl distribution with a mean of 1.15 prions per repeat. The recessive [VL] strain sample has approximately 1.05 prions per repeat in single fibrils and includes approximately 10% double fibrils, which are rare in the duplicate [VH] and [VK] samples. All of these samples were assembled from purified recombinant Sup35 prion protein by seeded growth on nuclei extracted from yeast bearing the three [PSI] strains. Infectious and noninfectious spontaneously assembled fibrils of the recombinant prion protein also display different heterogeneous morphologies. The strain-specific morphological differences we have observed directly confirm the structural prediction of the protein-only prion theory but do not have an obvious molecular explanation.
| S-EPMC1177419 | biostudies-literature
Project description:Amyloid polymorphism underlies the prion strain phenomenon where a single protein polypeptide adopts different chain-folding patterns to form self-propagating cross-beta structures. Three strains of the yeast prion [PSI], namely [VH], [VK], and [VL], have been previously characterized and are amyloid conformers of the yeast translation termination factor Sup35. Here we define specific sequences of the Sup35 protein that are necessary for in vivo propagation of each of these prion strains. By sequential substitution of residues 5-55 of Sup35 by proline and insertion of glycine at alternate sites in this segment, specific mutations have been identified that interfere selectively with the propagation of each of the three prion strains in yeast: the [VH] strain requires amino acid residues 7-21; [VK] requires residues 9-37; and [VL] requires residues 5 to at least 52. Minimal polypeptide segments capable of encoding prion conformations were defined by assembly of recombinant Sup35 fragments on purified prion nuclei to form amyloid fibers in vitro, whose infectivity was assayed in yeast. For the [VK] and [VL] strains, the minimal fragments approximately coincide with the strain-specific sequences defined by mutations of the N-terminal portion of the intact Sup35 (1-685); and for the [VH] strain, a longer Sup (1-53) fragment is required. Polymorphic structures of other amyloids might similarly involve different stretches of polypeptides to form cross-beta amyloid cores with distinct molecular recognition surfaces.
| S-EPMC2533192 | biostudies-literature