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PHD2 is a major regulatory enzyme that targets hypoxia-inducible factor-1α (HIF-1α). Under normoxia, PHD2-mediated proline hydroxylation promotes the binding of pVHL binding towards HIF-1α that allows its polyubiquitylation and subsequent degradation, while under hypoxia, stabilized HIF-1α translocates to the nuclear and induces the transcriptional activation of over 100 hypoxia-response genes. Recent studies showed that other than HIF-αs, PHD2 interacts and targets other proteins in an oxygen-dependent or independent manner. Given the critical role of PHD2 in oxygen-sensing and metabolism, it is important to comprehensively characterize PHD2 interactome and identify potentially novel PHD2 enzyme targets. In this study, we applied crosslinking-technology for quantitative analysis of PHD2 interactome. Methods: We engineered HeLa cells that stably express empty vector (control) and HTBH-tagged PHD2. To identify PHD2 interactome, cells were lysed and proteins were subject to streptavidin-based purification to pulldown PHD2 and interacting proteins. After incubation, the beads were resuspending in 1 mM DSSO MS-cleavable crosslinker for one hour. Proteins were then reduced and alkylated on beads followed by overnight tryptic digestion. Peptides were desalted and subjected to LCMS analysis with MS3-based fragmentation of crosslinked peptides. To identify PHD2-HIF1A interacting sites, cells were transfected with HIF1A plasmids prior to LCMS analysis of crosslinked peptides. Prolyl hydroxylase inhibitor, DMOG , was applied to cell treatment to improve PHD2 interactome analysis. Results: We performed quadruplicate analysis of the PHD2 interactome analysis for control cells and cells expressing HTBH-tagged PHD2 with or without DMOG treatment. We identified a total of 2236 proteins including 18 known PHD2 interacting proteins. Using label-free quantification, we quantified 1639 proteins in all treatment conditions. Among these proteins, 96 proteins were identified to be significantly enriched in PHD2 expressing cells and 93 proteins were significantly in DMOG treated cells expressing PHD2. Our result confirmed the enrichment of HIF1A in analysis of PHD2 expressing cells as expected. We also identified six PHD2-HIF1A interacting sites, providing the first direct mapping of PHD2-HIF1A interaction in solution. Protein complex enrichment identified COPS9 signalsome complex (CSN), a critical deneddylase complex for Cullin E3 ligase, as significantly enriched for PHD2 interaction. To confirm this finding, we performed Co-IP analysis with HTBH-tagged PHD2 cells. To demonstrate that the interaction was not specific to the cell line, we performed Co-IP analysis in 293T cells as well as using HA-Flag-tagged CSN6 for reciprocal IP. These data clearly demonstrated the interaction of PHD2 and CSN protein complex. Finally, we found that inhibition of PHD2 activity with DMOG led to significantly decreased neddylation on Cullin Ring E3 ligases, linking PHD2 activity with CSN complex-mediated deneddylation pathway. Conclusions: Summary of this project, through engineering the stable cell line, DSSO MS cleavable crosslinker application, we developed the streamline workflow to identify PHD2 interactome. With this workflow, we identified the CSN complex as the novel PHD2 interactor, and the interaction with PHD2 enhances the deneddylation activity of CSN complex, resulting in the deneddylation of Cullin Ring E3 ligases. The deneddylated Cullin Ring E3 ligase is inactivated and unable to ubiquitinate its downstream substrates. We appreciated the Center for Mass Spectrometry and Proteomics and the Masonic Cancer Center MS service of UMN for instrument access and support. We also thank the Doctoral Dissertation Fellowships(DDF) for supporting this project as well as members from the Chen lab and Huang Lab for helpful discussions and critical comments for this study.
Ouyang et al. (Fri,) studied this question.