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Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology.

Yanting Su1, Yuanyuan Guo2, Jieyu Guo2

  • 1School of Basic Medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xianning, Hubei, 437000, China.

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Summary

Researchers used TurboID proximity labeling to identify proteins interacting with forkhead box transcription factor 1 (FOXO1). This method identified 325 potential FOXO1-interacting proteins, advancing the study of FOXO1

Keywords:
Biotin labelingFOXO1TurboIDU251 cellshnRNPK

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Protein-protein interactions (PPIs) are crucial for cellular functions.
  • TurboID offers enhanced catalytic efficiency for rapid proximity labeling compared to BioID and APEX.
  • Forkhead box transcription factor 1 (FOXO1) is a key regulator in cellular processes like proliferation, apoptosis, and metabolism, and is implicated in various diseases.

Purpose of the Study:

  • To identify novel proteins interacting with FOXO1 using the TurboID proximity labeling technique.
  • To establish a FOXO1-overexpressing cell line for interaction studies.
  • To investigate the FOXO1 regulatory network in U251 astrocytes.

Main Methods:

  • Construction of a U251 astrocyte cell line stably expressing a FOXO1-TurboID fusion gene.
  • Biotin labeling using TurboID followed by label-free mass spectrometry to identify interacting proteins.
  • Immunoprecipitation and immunofluorescence assays for validation of identified interacting proteins (hnRNPK and RBM14).

Main Results:

  • Identification of 325 potential FOXO1-interacting proteins.
  • 176 proteins were identified in the FOXO1 overexpression group and 227 in the Lipopolysaccharide-treated group.
  • hnRNPK and RBM14 were validated as FOXO1-interacting proteins.

Conclusions:

  • The TurboID technique successfully identified a significant number of FOXO1-interacting proteins.
  • This study provides a foundation for further investigation into FOXO1's functions and its associated regulatory networks.
  • The findings contribute to understanding FOXO1's role in physiological and pathological processes.