Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.
Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE foll...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2018-01-01
|
| Series: | PLoS Pathogens |
| Online Access: | http://europepmc.org/articles/PMC5809102?pdf=render |
| _version_ | 1828379071131680768 |
|---|---|
| author | Alejandro M Sevillano Natalia Fernández-Borges Neelam Younas Fei Wang Saioa R Elezgarai Susana Bravo Ester Vázquez-Fernández Isaac Rosa Hasier Eraña David Gil Sonia Veiga Enric Vidal Melissa L Erickson-Beltran Esteban Guitián Christopher J Silva Romolo Nonno Jiyan Ma Joaquín Castilla Jesús R Requena |
| author_facet | Alejandro M Sevillano Natalia Fernández-Borges Neelam Younas Fei Wang Saioa R Elezgarai Susana Bravo Ester Vázquez-Fernández Isaac Rosa Hasier Eraña David Gil Sonia Veiga Enric Vidal Melissa L Erickson-Beltran Esteban Guitián Christopher J Silva Romolo Nonno Jiyan Ma Joaquín Castilla Jesús R Requena |
| author_sort | Alejandro M Sevillano |
| collection | DOAJ |
| description | Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc. |
| first_indexed | 2024-12-10T03:34:02Z |
| format | Article |
| id | doaj.art-a5a65062a45e40f184b776930d9e28d0 |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-12-10T03:34:02Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-a5a65062a45e40f184b776930d9e28d02022-12-22T02:03:45ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742018-01-01141e100679710.1371/journal.ppat.1006797Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.Alejandro M SevillanoNatalia Fernández-BorgesNeelam YounasFei WangSaioa R ElezgaraiSusana BravoEster Vázquez-FernándezIsaac RosaHasier ErañaDavid GilSonia VeigaEnric VidalMelissa L Erickson-BeltranEsteban GuitiánChristopher J SilvaRomolo NonnoJiyan MaJoaquín CastillaJesús R RequenaVery solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.http://europepmc.org/articles/PMC5809102?pdf=render |
| spellingShingle | Alejandro M Sevillano Natalia Fernández-Borges Neelam Younas Fei Wang Saioa R Elezgarai Susana Bravo Ester Vázquez-Fernández Isaac Rosa Hasier Eraña David Gil Sonia Veiga Enric Vidal Melissa L Erickson-Beltran Esteban Guitián Christopher J Silva Romolo Nonno Jiyan Ma Joaquín Castilla Jesús R Requena Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. PLoS Pathogens |
| title | Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. |
| title_full | Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. |
| title_fullStr | Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. |
| title_full_unstemmed | Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. |
| title_short | Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis. |
| title_sort | recombinant prpsc shares structural features with brain derived prpsc insights from limited proteolysis |
| url | http://europepmc.org/articles/PMC5809102?pdf=render |
| work_keys_str_mv | AT alejandromsevillano recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT nataliafernandezborges recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT neelamyounas recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT feiwang recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT saioarelezgarai recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT susanabravo recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT estervazquezfernandez recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT isaacrosa recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT hasiererana recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT davidgil recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT soniaveiga recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT enricvidal recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT melissalericksonbeltran recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT estebanguitian recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT christopherjsilva recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT romolononno recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT jiyanma recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT joaquincastilla recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis AT jesusrrequena recombinantprpscsharesstructuralfeatureswithbrainderivedprpscinsightsfromlimitedproteolysis |