Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Area of Science:

• Biochemistry
• Molecular Biology
• Veterinary Science

Background:

• Sheep prion diseases, like scrapie, are linked to specific genetic variations in the prion protein (PrP).
• Polymorphisms at PrP positions 136, 154, and 171 determine scrapie susceptibility, with VRQ conferring high susceptibility and ARR conferring resistance.

Purpose of the Study:

• To investigate the molecular mechanisms underlying scrapie susceptibility and resistance in sheep.
• To analyze the unfolding pathways of different sheep PrP variants using differential scanning calorimetry across a range of pH levels.

Main Methods:

• Differential scanning calorimetry (DSC) was employed to study the thermal unfolding of recombinant sheep PrP variants.
• Analysis of unfolding intermediates, including their stability, activation energy, and secondary structures, was performed.
• Thioflavin T fluorescence and electron microscopy were used to characterize amyloid properties of refolded intermediates.

Main Results:

• Sheep PrP variants exhibit pH-dependent unfolding pathways, forming stable intermediates at pH <4.5 and >6.0.
• Scrapie susceptibility variants (VRQ, ARQ) show higher thermal stability and activation energy for intermediate formation compared to resistance variants (AHQ, ARR).
• Unfolding intermediates of resistance variants (ARR) have random coil structures, while susceptibility variants (VRQ, ARQ) show beta-sheet structures, with higher formation rates at pH 4.0 for VRQ and ARQ.

Conclusions:

• Sheep PrP exhibits genetically modulated plasticity, influencing its stability and transconformation rates.
• Thermodynamic stability and transconformation rates of PrP variants provide a molecular basis for sheep predisposition to scrapie.
• The structural differences in unfolding intermediates correlate with scrapie susceptibility phenotypes.