Profiling Protein Citrullination in Extracellular Vesicles by Single-Molecule Detection Using Direct Stochastic...

Area of Science:

• Biochemistry
• Cell Biology
• Biomarkers

Background:

• Extracellular vesicles (EVs) play key roles in cell communication and serve as pathological biomarkers.
• Post-translational citrullination of proteins is implicated in various diseases, but their specific localization within EVs remains unclear.

Purpose of the Study:

• To investigate the localization of citrullinated proteins within specific extracellular vesicle (EV) subtypes.
• To determine if citrullinated proteins are exported as intraluminal cargo or reside on the EV surface.

Main Methods:

• Utilized dSTORM super-resolution microscopy to co-localize citrullinated proteins (pan-Cit) and citrullinated histone H3 (CitH3) with EV subtypes.
• Captured EVs using tetraspanin trio (TT) and phosphatidylserine (PS) markers.
• Analyzed permeabilized and non-permeabilized EVs using a Bayesian framework.

Main Results:

• Confirmed the presence of pan-Cit and CitH3 as both intraluminal cargo and surface proteins on EVs.
• Detected higher levels of citrullinated proteins in permeabilized EVs compared to non-permeabilized ones.
• Observed differential localization: pan-Cit was higher in TT-bound EVs, while CitH3 was higher in PS-bound EVs.

Conclusions:

• This research expands the understanding of EV-associated post-translational modifications.
• The findings highlight the potential of the EV citrullinome for developing novel liquid biopsy tools for cancer detection.