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.
Highlights
Proline hydroxylation (Hyp) is a key oxygensensing posttranslational modification that is dynamically modulated during tumor cell proliferation and angiogenesis [1,2,3,4,5,6]
The proline hydroxylation is catalyzed by prolyl hydroxylases which require oxygen as co-substrate and iron, alpha-ketoglutarate and ascorbate as co-factors
Advances in high resolution mass spectrometry led to the identification of an increasing number of proline hydroxylation targets [16,17,18,19,20,21,22]
Summary
Proline hydroxylation (Hyp) is a key oxygensensing posttranslational modification that is dynamically modulated during tumor cell proliferation and angiogenesis [1,2,3,4,5,6]. Recent biochemical studies with high resolution mass spectrometry have identified and validated a number of www.impactjournals.com/oncotarget other proline hydroxylation substrates including FOXO3a, Argonaute 2, ATF-4 and PKM [16,17,18,19,20,21,22], which revealed diverse HIF-independent oxygen-sensing activities in RNA interference and transcriptional regulation. These evidence suggest that the hypoxia-response mechanism mediated through proline hydroxylation regulates diverse cellular pathways and signaling processes in cells and may play a much wider role in regulating cellular physiology and protein functions
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