HaloTag Fusion Enables Dynamic Analysis of Prion Protein Biosynthesis, Turnover, and Misfolding

Understanding the dynamics of prion protein (PrP) biosynthesis, misfolding, and degradation is essential for elucidating prion disease pathogenesis and exploring therapeutic strategies. Here, we developed a HaloTag-based PrP fusion (PrP-Halo) that enables real-time analysis of PrP biosynthesis, conformational changes, and turnover in living cells. The HaloTag moiety allows specific, covalent labeling with fluorescent ligands, facilitating pulse-chase imaging and biochemical tracking of distinct PrP subpopulations. PrP-Halo maintains physiological localization, trafficking, and proteolytic processing, faithfully recapitulating the behavior of native PrP. Using this system, we captured early misfolding events induced by pathogenic mutations. Moreover, we show that PrP-Halo can be used to probe the mechanisms of action of PrP-lowering compounds, distinguishing their effects on newly synthesized versus cell-surface PrP. Together these findings establish PrP-Halo as a versatile and sensitive tool for dissecting the molecular and cellular mechanisms underlying PrP misfolding and turnover.