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

Abstract

Background: Protein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes.

Results: The FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification.

Conclusion: The TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

Keywords: Biotin labeling; FOXO1; TurboID; U251 cells; hnRNPK.

Fig. 1

Construction of the overexpression plasmid and TurboID labeling flowchart. (A) Diagram of functional elements of the FOXO1-TurboID overexpression plasmid. (B) Biotin marker experimental workflow

Fig. 2

Validation of cell line stable transfection. (A) Fluorescence image of 293T cells highly expressing pLenti-CMV-EGFP. (B) Fluorescence image of U251 cells highly expressing pLenti-CMV-EGFP. (C) Validation of FOXO1 protein expression. NC, U251 cells; OE, cells with stable overexpression of FOXO1-TurboID

Fig. 3

Biotin labeling and silver staining. (A) Biotin labeling was assessed at different time points. 1, 0 min; 2, 10 min; 3, 1 h; 4, 3 h; 5, 6 h; 6, 12 h; 7, 24 h. (B) Protein identification by silver staining. M, protein ladder; 1, Blank Strip; 2, EGFP-U251 cells; 3, FOXO1-U251 cells; 3, LPS-treated FOXO1-U251 cells

Fig. 4

Analysis of FOXO1 proximates. (A) Enriched proximate proteins of the FOXO1 group based on cellular components. (B) Enriched proximate proteins of the FOXO1 group based on molecular function. (C) Enriched proximate proteins of the FOXO1 group based on biological process. (D) Enriched proximate proteins of the FOXO1 group based on KEGG analysis. (E) Enriched proximate proteins of the LPS-treated group based on cellular components. (F) Enriched proximate proteins of the LPS-treated group based on molecular function. (G) Enriched proximate proteins of the LPS-treated group based on biological process. (H) Enriched proximate proteins of the LPS-treated group based on KEGG analysis

Fig. 5

Validation of the interacting proteins. (A) Western blotting bands after immunoprecipitation. (B) Immunofluorescence image of the interaction between FOXO1 and hnRNPK in FOXO1-U251 cells. (C) Immunofluorescence image of the interaction between FOXO1 and RBM14 in FOXO1-U251 cells