APEX2-Mediated Proximity Labeling Resolves the DDIT4-Interacting Proteome

Abstract

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins’ molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings.

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“…Interestingly, among them, DDIT3 , DDIT4 , CDKN1A , SRXN1 , PXDN , SPHK1 , CRYAB , DUSP1 , DUSP8 , CHAC1 , and GADD45A genes were involved in multiple pathways associated with apoptosis, oxidative stress, DNA damage, and cell cycle. As indicators of cellular oxidative stress, DNA damage-inducible transcripts 3 and 4 ( DDIT3 and DDIT4 ) play essential roles in responding to oxidative stress and inducing cell cycle arrest and apoptosis. , CDKN1A is a biomarker for DNA damage . In this study, DDIT3 , DDIT4 , and CDKN1A were significantly upregulated in all DBP exposure groups, demonstrating that DBP induced oxidative stress and DNA damage.…”

“…As indicators of cellular oxidative stress, DNA damage-inducible transcripts 3 and 4 (DDIT3 and DDIT4) play essential roles in responding to oxidative stress and inducing cell cycle arrest and apoptosis. 46,47 CDKN1A is a biomarker for DNA damage. 48 In this study, DDIT3, DDIT4, and CDKN1A were significantly upregulated in all DBP exposure groups, demonstrating that DBP induced oxidative stress and DNA damage.…”