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APEX2-Mediated Proximity Labeling Resolves the DDIT4-Interacting Proteome.

Marianna Naki1,2, Olga Gourdomichali1,3, Katerina Zonke1

  • 1Center of Basic Research, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.

International Journal of Molecular Sciences
|May 14, 2022
PubMed
Summary

DNA damage-inducible transcript 4 (DDIT4) protein interactions were mapped using proximity labeling. Stress increases DDIT4 partners, revealing moonlighting keratins and ribosomal proteins involved in cellular regulation.

Keywords:
APEX2DDIT4LC-MS/MSacute stressinteractomeproteomicsproximity labeling

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Area of Science:

  • Molecular Biology
  • Cellular Stress Response
  • Proteomics

Background:

  • DNA damage-inducible transcript 4 (DDIT4) is a stress-responsive protein that represses mTORC1 signaling.
  • DDIT4 regulates key cellular processes including metabolism, oxidative stress, and apoptosis.
  • The interacting partners and specific molecular functions of DDIT4 remain largely uncharacterized.

Purpose of the Study:

  • To identify proteins in the immediate vicinity of DDIT4 under unstressed and acute stress conditions.
  • To investigate the context-dependent nature of DDIT4's interacting proteome.
  • To elucidate the molecular functions of DDIT4-interacting proteins.

Main Methods:

  • Utilized enhanced ascorbate peroxidase 2 (APEX2) for proximity-dependent biotinylation of DDIT4.
  • Employed mass spectrometry to identify biotinylated proteins in situ.
  • Quantitatively compared DDIT4 interactomes under unstressed versus acute stress conditions.

Main Results:

  • DDIT4 exhibited approximately twice as many interaction partners during acute stress compared to unstressed conditions.
  • The identified proteomes were quantitatively distinct but functionally similar across conditions.
  • Moonlighting keratins and ribosomal proteins were dominant interactors, possessing known non-canonical stress roles.

Conclusions:

  • DDIT4 interacts with moonlighting keratins and ribosomal proteins, suggesting novel regulatory mechanisms.
  • Keratins modulate mTORC1 signaling, while ribosomal proteins influence translation and cell fate.
  • These findings reveal potentially distinct molecular functions of DDIT4 and open avenues for further research.