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Abstract: TH-PO531

An In Situ Ascorbate Peroxidase Labeling in Secreted Exosomes Identifies Oxidative Stress-Induced Exosome Proteome Alteration in Renal Proximal Tubules

Session Information

  • Pathology and Lab Medicine
    November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pathology and Lab Medicine

  • 1700 Pathology and Lab Medicine

Authors

  • Kwon, Sang-Ho, Augusta University, Augusta, Georgia, United States
  • Lee, Byung Rho, Augusta University, Augusta, Georgia, United States
  • Oh, Sekyung, Catholic Kwandong University College of Medicine, Incheon, Korea (the Republic of)
  • Lee, Chang Min, Augusta University, Augusta, Georgia, United States
Background

The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids. Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts.

Methods

Here we report an in situ labeling approach for exosome cargo profiling, termed Exosome-Proxy APEX Labeling (EPAL), which bypasses the exosome isolation steps. In EPAL, proteins either in the exosome biogenesis vesicles in cells or in secreted exosomes in the conditioned medium can specifically be biotinylated by expressing a variant of the engineered ascorbic peroxidase APEX that is fused to an exosome cargo protein such as CD63.

Results

Mass spectrometry analysis of the proteins biotinylated in exosomes secreted by kidney proximal tubule-derived cells reveals that oxidative stress can induce an alteration in exosome protein contents, including accumulation of ribosomal proteins in exosomes.

Conclusion

In sum, our new method specifically labels exosome proteins with a genetic, chemical means in situ either in live cells or in secreted exosomes, thereby probing proteins packaged in exosomes and identifying where exosomes originate. This method might be useful to discover exosome-based biomarkers for kidney diseases.

Funding

  • NIDDK Support