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In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups
Ralf Lucassen1, Koren Nishina, Surachai Supattapone
1Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Abstract:
Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).
Insights
Prion protein (PrPSc) misfolding, the cause of fatal brain diseases, was studied using an in vitro amplification method. This research reveals that a thiol-containing factor is essential for the conformational change of PrP(C) to PrP(Sc).
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Transmissible spongiform encephalopathies are caused by prions, which are misfolded proteins (PrPSc).
- The exact molecular mechanism of how normal prion protein (PrPC) converts to the infectious PrPSc form is not fully understood.
- Understanding this conversion is crucial for developing therapeutic strategies against prion diseases.
Purpose of the Study:
- To investigate the molecular mechanism underlying the conformational change of PrPC to PrPSc.
- To utilize an adapted in vitro protein misfolding cyclic amplification (PMCA) technique for prion amplification.
- To identify factors involved in the PrPC to PrPSc conversion process.
Main Methods:
- Employed an in vitro PrPSc amplification technique adapted from PMCA.
- Mixed scrapie-infected brain homogenate with normal brain homogenate under nondenaturing conditions.
- Assessed the effect of pH and thiol-specific chemical agents (N-ethylmaleimide, PHMB, mersalyl acid) on PrPSc amplification.
Main Results:
- Successfully amplified PrPSc in vitro >10-fold without sonication.
- PrPSc amplification demonstrated species and strain specificity, dependent on time and temperature.
- Inhibition of amplification by thiol-specific agents indicated the requirement of a thiol-containing factor for PrPC to PrPSc conformational change.
Conclusions:
- The study provides the first evidence that a reactive chemical group, specifically a thiol-containing factor, is essential for the conformational conversion of PrPC to PrPSc.
- The developed in vitro amplification method is a valuable tool for studying prion propagation mechanisms.
- These findings offer new insights into the molecular basis of prion diseases and potential targets for intervention.