Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells
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Tong Zhou1,*, Luke Erber1,*, Bing Liu2, Yankun Gao1, Hai-Bin Ruan2, Yue Chen1
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota at Twin Cities, Minneapolis, MN 55455, USA
2Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN 55455, USA
*These authors have contributed equally to this work
Yue Chen email: YueChen@umn.edu
Keywords: proline hydroxylation, oxygen-sensing, posttranslational modification, hypoxia, LCMS
Received: April 27, 2016 Accepted: September 25, 2016 Published: October 13, 2016
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.
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