Alternative translation initiation generates cytoplasmic sheep prion protein

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• Cytoplasmic localization of the prion protein (PrP) is a poorly understood phenomenon observed across species.
• Investigating the mechanisms behind aberrant PrP localization is crucial for understanding its cellular function and potential disease implications.

Purpose of the Study:

• To investigate the mechanisms underlying the cytoplasmic localization of sheep prion protein (PrP).
• To determine if N-terminal fragments of PrP contribute to its aberrant intracellular localization.
• To compare PrP processing in sheep and mouse models.

Main Methods:

• Expression of sheep prion protein (PrP) fused with green fluorescent protein (GFP) in N2a cells.
• Utilizing constructs with varied start codon contexts and mutations (e.g., Met17 to Thr).
• Western blotting to analyze protein glycosylation and fragment formation.
• Immunological detection of PrP fragments in sheep brain samples.

Main Results:

• Sheep PrP expressed with a wild-type sequence showed some N-terminal fragments (starting from Met17) localized intracellularly.
• Compromised Kozak sequences led to dispersed intracellular PrP fluorescence.
• N-terminal fragment formation was abolished when Met17 was mutated, confirming leaky ribosomal scanning as the cause.
• Similar processing was not observed for mouse PrP constructs.
• N-terminal PrP fragments were detected in sheep brain tissue.

Conclusions:

• Leaky ribosomal scanning and translation initiation at Met17 are responsible for the aberrant cytoplasmic localization of a fraction of sheep PrP.
• This processing mechanism is specific to sheep PrP and occurs in vivo.
• The findings provide insights into PrP cellular trafficking and processing.