Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm
| Autor: | Aishwarya Sriraman, Satish K. Nemani, Camilo Duque Velásquez, Leonardo M. Cortez, Holger Wille, YongLiang Wang, Valerie L. Sim, Debbie McKenzie |
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| Rok vydání: | 2021 |
| Předmět: |
Glycosylation
PrPSc Proteins animal diseases Glycobiology Biochemistry Prion Diseases Animal Diseases Photometry chemistry.chemical_compound Mice 0302 clinical medicine Medical Conditions Cricetinae Zoonoses Medicine and Health Sciences Fractionation Biology (General) Post-Translational Modification chemistry.chemical_classification Mammals 0303 health sciences Strain (chemistry) biology Chemistry Monomers Eukaryota Phenotype Separation Processes Animal Prion Diseases Infectious Diseases Physical Sciences Vertebrates Hamsters Research Article Proteases QH301-705.5 Prions Immunology Immunoblotting Molecular Probe Techniques Research and Analysis Methods Microbiology Rodents 03 medical and health sciences Virology Genetics Animals Protein Structure Quaternary Molecular Biology Techniques Molecular Biology 030304 developmental biology Organisms Biology and Life Sciences Proteins RC581-607 Proteinase K Polymer Chemistry Protein tertiary structure Dynamic Light Scattering nervous system diseases nervous system Amniotes Biophysics biology.protein Hydrodynamics Parasitology Protein quaternary structure Immunologic diseases. Allergy Glycoprotein Zoology 030217 neurology & neurosurgery |
| Zdroj: | PLoS Pathogens PLoS Pathogens, Vol 17, Iss 6, p e1009703 (2021) |
| ISSN: | 1553-7374 |
| Popis: | Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype. Author summary Prion diseases are neurodegenerative diseases that include bovine spongiform encephalopathy (BSE or mad cow disease) in cattle, chronic wasting disease (CWD) in cervids and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are caused by self-propagated misfolding and aggregation of the naturally occurring prion protein. Variations in the structure of prion aggregates are associated with distinct disease phenotypes, but how this prion structural heterogeneity translates into clinical presentation has been difficult to determine, largely because it is technically difficult to isolate and characterize the full range of prion structures from prion-infected brain. Here, we overcame this challenge by using a versatile fractionation technique, one that is strikingly unexplored in neurodegenerative research, and present the most detailed description, to date, of strain-specific prion subpopulations. We found that prion quaternary structure was a major contributor to structural heterogeneity. We also discovered that all prion strains studied underwent a significant structural change resulting in two distinctive subpopulations whose proportions correlated with the strain phenotype. Our work provides new insights into the molecular basis of prion strain variation and is a proof of concept that can be applied to other protein misfolding neurodegenerative disorders. |
| Databáze: | OpenAIRE |
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