Tetracysteine-tagged prion protein allows discrimination between the native and converted forms

Area of Science:

• Biochemistry
• Neuroscience
• Molecular Biology

Background:

• Transmissible spongiform encephalopathies are characterized by prion protein (PrP) conformational conversion.
• Current methods using generic amyloid dyes are limited for live cell imaging.
• Developing specific probes for PrP misfolding is crucial for studying conformational diseases.

Purpose of the Study:

• To develop a novel, cell-based method for monitoring prion protein (PrP) misfolding.
• To synthesize and utilize membrane-permeable biarsenical reagents for PrP conformational studies.
• To investigate the impact of tetracysteine (TC) tag placement on murine PrP (mPrP) conversion.

Main Methods:

• Synthesis of membrane-permeable and impermeable biarsenical reagents.
• Introduction of tetracysteine (TC) tags at N-terminus, C-terminus, and helix 2-helix 3 loop of murine PrP (mPrP).
• Development of a quantitative, protease-free method using fluorescein arsenical helix binder to differentiate monomeric and fibrilized mPrP.

Main Results:

• TC-tagged mPrP at N-terminus and C-terminus supported fibril formation.
• TC tag insertion into the helix 2-helix 3 loop inhibited mPrP conversion.
• The developed method successfully detected TC-tagged mPrP on transfected cells, differentiating monomeric and fibrilized forms.

Conclusions:

• Novel biarsenical reagents and TC-tagged mPrP provide a sensitive method for monitoring PrP misfolding.
• The location of TC tags significantly influences PrP conformational conversion.
• This approach offers a valuable tool for in vivo imaging and studying conformational diseases like prion diseases.