Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

Zhou, Tong; Erber, Luke; Liu, Bing; Gao, Yankun; Ruan, Hai Bin; Chen, Yue

doi:10.18632/oncotarget.12632

Cited by 29 publications

(39 citation statements)

References 89 publications

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“…Plasmids, siRNAs, and Peptides-pFlag-CMV2-Brd4 (1-1362) was a gift from Eric Verdin (Addgene plasmid # 22304, Watertown, Massachusetts). The plasmid containing flag-BRD4 P536A has been previously described (32). c-myc promoter luciferase vector was a gift of Bert Vogelstein (Addgene plasmid #16601).…”

Section: Methodsmentioning

confidence: 99%

“…Through proteomics-based screening, we previously identified Bromo-domain containing protein 4 (Brd4) as a new Hyp target with a high modification stoichiometry (ϳ60%) at an evolutionarily conserved site P536 (supplemental Fig. S1) (32). In the present study, we applied label-free targeted proteomics analysis with parallel reaction monitoring (PRM) to identify the regulatory enzyme of Brd4 proline hydroxylation and then performed quantitative interactome analysis to reveal the functional significance of the modification on Brd4-mediated transcriptional pathways.…”

Section: Targeted and Interactome Proteomics Revealed The Role Of Phdmentioning

confidence: 99%

“…To measure the change in proline hydroxylation on PHD2 knockdown, 293T cells stably expressing control shRNA and PHD2 shRNA were generated as previously described (31). Cells were lysed and BRD4 was immunoprecipitated using either anti-flag agarose beads or with 1 g BRD4 antibody (Millipore) and protein A/G beads as previously described (32). Protein was separated with SDS-PAGE, stained with Coomassie blue (National Diagnostics, Atlanta, Georgia) and digested with trypsin as previously described (34).…”

Section: Generation Of Stable Knockdown Cell Lines-mentioning

confidence: 99%

“…Tryptic peptides were analyzed by LC-MS/MS as previously described (32). Peptides were injected into the nano-LC Proxeon (Easy-nLC 1000, Thermo Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific).…”

Section: Generation Of Stable Knockdown Cell Lines-mentioning

confidence: 99%

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Targeted and Interactome Proteomics Revealed the Role of PHD2 in Regulating BRD4 Proline Hydroxylation

Erber¹,

Luo²,

Chen³

2019

Molecular & Cellular Proteomics

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Targeted quantification assays using MS-based parallel-reaction monitoring and biochemical analysis revealed PHD2 as a key regulatory enzyme of BRD4 proline hydroxylation. Quantitative MS-based interactome and biochemical analyses revealed the functional significance of the proline hydroxylation modification pathway in BRD4-mediated protein-protein interactions and transcriptional activation. Taken together, these results provided mechanistic insights into the oxygen-dependent modification of BRD4 and revealed new roles of the proline hydroxylation pathway in regulating BRD4-mediated protein-protein interaction and gene expression.

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Section: Methodsmentioning

confidence: 99%

Section: Targeted and Interactome Proteomics Revealed The Role Of Phdmentioning

confidence: 99%

Section: Generation Of Stable Knockdown Cell Lines-mentioning

confidence: 99%

Section: Generation Of Stable Knockdown Cell Lines-mentioning

confidence: 99%

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Targeted and Interactome Proteomics Revealed the Role of PHD2 in Regulating BRD4 Proline Hydroxylation

Erber¹,

Luo²,

Chen³

2019

Molecular & Cellular Proteomics

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“…Here, we evaluate methods considering separately collagen and signalling examples as well as single proteins versus high throughput MS experiments. The majority of new hydroxylated prolines (Hyp) come from two MS experiments, one on HeLa cells and another based on a large experiment made on multiple tissues and samples, recently published and not yet available in public databases when predictors were trained (23)(24)(25). The number of MS hydroxylated sites is comparable to the entire SwissProt database.…”

Section: Introductionmentioning

confidence: 99%

Assessing predictors for new post translational modification sites: a case study on hydroxylation

Piovesan

Hatos

Minervini

et al. 2020

Preprint

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Post-translational modification (PTM) sites have become popular for predictor development. However, with the exception of phosphorylation and a handful of other examples, PTMs suffer from a limited number of available training examples and their sparsity in protein sequences. Here, proline hydroxylation is taken as an example to compare different methods and evaluate their performance on new experimentally determined sites. As a proxy for an effective experimental design, predictors require both high specificity and sensitivity. However, the self-reported performance is often not indicative of prediction quality and detection of new sites is not guaranteed. We have benchmarked seven published hydroxylation site predictors on two newly constructed independent datasets. The self-reported performance widely overestimates the real accuracy measured on independent datasets. No predictor performs better than random on new examples, indicating the refined models are not sufficiently general to detect new sites. The number of false positives is high and precision low, in particular for non-collagen proteins whose motifs are not conserved. In short, existing predictors for hydroxylation sites do not appear to generalize to new data. Caution is advised when dealing with PTM predictors in the absence of independent evaluations, in particular for unique specific sites such as those involved in signalling. Author SummaryMachine learning methods are extensively used by biologists to design and interpret experiments. Predictors which take the only sequence as input are of particular interest due to the large amount of sequence data available and self-reported performance is often very high. In this work, we evaluated post-translational modification (PTM) predictors for hydroxylation sites and found that they perform no better than random, in strong contrast to performances reported in the original publications. PTMs are chemical amino acids alterations providing the cell with conditional mechanisms to fine tune protein function, thereby regulating complex biological processes such as signalling and cell cycle. Hydroxylation sites are a good PTM test case due to the availability of a range of predictors and an abundance of newly experimentally detected modification sites. Poor performances in our results highlight the overlooked problem of predicting PTMs when best practices are not followed and training data are likely incomplete. Experimentalists should be careful when using PTM predictors blindly and more independent assessments are needed to separate the wheat from the chaff in the field.

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A Quantitative Chemical Proteomics Approach for Site‐specific Stoichiometry Analysis of Ubiquitination

Evers

Luo

et al. 2018

Angewandte Chemie

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Stoichiometric analysis of post-translational modifications is an emerging strategy for absolute quantification of the fractional abundance of the modification. Herein, aquantitative chemicalp roteomic workflowf or stoichiometric analysis of ubiquitination is reported, named isotopically balanced quantification of ubiquitination (IBAQ-Ub). The strategy utilizes an ew amine-reactive chemical tag (AcGG-NHS) that is structurally homologous to the GG remnant of ubiquitin on modified lysine after trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and secondary stable isotopic labeling.T he strategy is highly robust, sensitive,a nd accurate with aw ide dynamic range using either protein standards or complex cell lysates.Thus,this work provides an efficient chemicalp roteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways.Ubiquitination (Ub) is an essential pathway in eukaryotic cells that controls the signaling flux in diverse biological processes. [1] Thecountering activities of the elegant E1-E2-E3 enzymatic cascade and the deubiquitinases (DUBs) determine the physiological stoichiometry of ubiquitination at the site-specific level. [1a,b] Advances in the quantitative proteomics,s uch as stable isotope labeling by/with amino acids in cell culture (SILAC) and isobaric tagging, [2] have enabled system-wide discoveries of Ub dynamics during signaling processes. [3] More recently,a bsolute quantification of ubiquitination has been achieved using spike-in stable-isotope labeled synthetic peptide standards (UB-AQUA) [4] or recombinant proteins (PSAQ), [5] which allowed targeted analysis of polyubiquitin linkages in ER stress,D NA damage response,m itophagy,a nd in vitro enzyme activities. [4, 6] Stoichiometry analysis is an emerging approach to measure the fractional abundance of post-translational modifications (PTMs). [6b, 7] It allows the quantitative comparison of PTM abundance between different sites on the same,o r different, target proteins.S ystematic analysis of PTM stoichiometries does not have to rely on the synthesis of in vitro isotopically labeled standards and therefore,p otentially enables global untargeted discoveries of modification abundance at the physiological levels.R ecent advances in quantitative proteomics have enabled stoichiometric analysis of phosphorylation, lysine acetylation, and succinylation on ag lobal scale. [7a-c,f-h,j-n] Despite these advances,a ccurate and site-specific stoichiometric analysis of ubiquitination has been challenging.I nt his study,t he development of an efficient chemical-based quantitative proteomic approach (termed IBAQ-Ub) is reported, which enables the determination of the absolute site-specific stoichiometry of ubiquitination.Following our previously successful experience in stoichiometry analysis of lysine acetylation, [7k] we designed an isotopically balanced quantification strategy for stoichiometry analy...

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Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

Cited by 29 publications

References 89 publications

Targeted and Interactome Proteomics Revealed the Role of PHD2 in Regulating BRD4 Proline Hydroxylation

Targeted and Interactome Proteomics Revealed the Role of PHD2 in Regulating BRD4 Proline Hydroxylation

Assessing predictors for new post translational modification sites: a case study on hydroxylation

A Quantitative Chemical Proteomics Approach for Site‐specific Stoichiometry Analysis of Ubiquitination

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