Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Transmissible spongiform encephalopathies (TSEs) are linked to infectious prion proteins (PrPSc) that form stable, protease-resistant amyloid fibrils.
• PrPSc degradation is challenging due to its stability and infectious nature, necessitating safe in vitro models.
• Bovine spongiform encephalopathy (BSE) prions show some degradability by keratinase and other proteases under specific conditions.

Purpose of the Study:

• To develop and characterize a safe, nonpathogenic surrogate protein for studying prion aggregate degradation.
• To investigate the degradation of yeast prion-like protein Sup35NM aggregates using keratinase and proteinase K.
• To provide insights into optimizing prion degradation mechanisms for TSE research.

Main Methods:

• Cloning, overexpression in E. coli, and purification of the yeast prion-like protein Sup35NM.
• Characterization of Sup35NM aggregation and deaggregation using electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting.
• Comparative study of Sup35NM aggregate degradation by keratinase and proteinase K under various conditions.

Main Results:

• Sup35NM was successfully purified and characterized as a suitable model for prion-like protein aggregation.
• The study compared the degradation efficiency of keratinase and proteinase K on Sup35NM aggregates.
• Established conditions for the degradation of prion-like protein aggregates.

Conclusions:

• The yeast prion-like protein Sup35NM serves as a safe and effective model for studying prion degradation.
• Understanding the degradation of Sup35NM provides a foundation for optimizing prion inactivation strategies.
• This research contributes to safer laboratory practices when investigating transmissible spongiform encephalopathies.