Dataset Information

Modeling PrPSc Generation Through Deformed Templating.

ABSTRACT: Deformed templating is the process by which self-replicating protein conformations with a given cross-? folding pattern can seed formation of an alternative self-replicating state with different cross-? folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a bona fide infectious conformer, PrPSc through deformed templating. The process can take many rounds of replication (if taking place in vitro) or even several passages of the evolving PrP conformers through successive brains if in vivo, through experimental transmission. In all cases, deformed templating involves a forced conversion in which there is a mismatch between the template and the substrate and/or the templating environment, typically a recombinant PrP amyloid, adept at converting recombinant PrP under denaturing conditions (e.g., presence of chaotropic agents), encountering a glycosylated, GPI-anchored PrPC substrate under physiological conversion conditions. Deformed templating is characterized by emergence of intermediate conformers that exhibit biochemical characteristics that are intermediate between those of the initial PrP amyloid and the final PrPSc conformers. Here, we took advantage of the recent elucidation of the structure of a PrP amyloid by cryo-EM and the availability of a physically plausible atomistic model of PrPSc that we have recently proposed. Using modeling and Molecular Dynamics (MD) approaches, we built a complete molecular modelization of deformed templating, including an atomistic model of a glycosylated intermediate conformer and a modified model of PrPSc. Among other unanticipated outcomes, our results show that fully glycosylated PrP can be stacked in-register, and how 4-rung ?-solenoid (4R?S) PrP architectures can share key structural motifs with parallel-in register intermolecular sheet (PIRIBS) PrP amyloids. Our results shed light on the mechanisms of prion replication.

SUBMITTER: Spagnolli G

PROVIDER: S-EPMC7573312 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Modeling PrPSc Generation Through Deformed Templating.

Spagnolli Giovanni G Rigoli Marta M Novi Inverardi Giovanni G Codeseira Yaiza B YB Biasini Emiliano E Requena Jesús R JR

Frontiers in bioengineering and biotechnology 20201006

Deformed templating is the process by which self-replicating protein conformations with a given cross-β folding pattern can seed formation of an alternative self-replicating state with different cross-β folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a bona fide infectious conformer, PrPSc through deformed templating. The process can take many rounds of replication (if taking place in vitro) or even several passages of the evol ...[more]

PMID: 33123520

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Project description:Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)-sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.

Project description:Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκβα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.

Dataset Information

Modeling PrPSc Generation Through Deformed Templating.

Publications

Modeling PrP<sup>Sc</sup> Generation Through Deformed Templating.

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