title: Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells.
abstract: Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA.
authors.forename: Luke
authors.initials: L
authors.lastname: Erber
authors.affiliation: Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA.
authors.forename: Yankun
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
authors.forename: Hai-Bin
authors.initials: HB
authors.lastname: Ruan
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
mesh_terms: D018797:Cell Cycle Proteins; D015687:Cell Hypoxia; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006367:HeLa Cells; D006801:Humans; D006900:Hydroxylation; D008958:Models, Molecular; D009369:Neoplasms / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D011392:Proline; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins
keywords: LCMS; hypoxia; oxygen-sensing; posttranslational modification; proline hydroxylation