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Abstract
Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IκB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological “substrate trapping” together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the ∼300 ARD-containing species in the human proteome.
Highlights
Post-translational hydroxylation has been considered an unusual modification on intracellular proteins
These experiments demonstrated that exposure to DMOG (1 mM for a period of 16 h) was sufficient to enhance the capture of factor inhibiting HIF (FIH)-associated species as revealed by Coomassie Blue staining (Fig. 1B)
A Mascot (DDA), ProteinLynx Global Server (PLGS) (MSE). b Derived from sample treated with DMOG. c Derived from untreated sample. d Bait protein used as internal standard for an equal ratio of heavy to light peptides. e FIH interaction of this protein confirmed in this study. f Shown previously to be on FIH substrate [4]. g Including peptide/protein probabilities of Ͻ50% as calculated by PLGS
Summary
Proteomics Screens and SILAC Protocol—Human embryonic kidney (HEK) 293 cells stably expressing SPA-tagged FIH (Sequential Peptide Affinity tag; 3ϫ FLAG epitope tag, tobacco etch virus protease site, and calmodulin binding peptide [11]) were used in proteomics screens for FIH-co-precipitating proteins. For the analysis of MSE-derived SILAC data, PLGS (version 2.2.5) was used to search against the UniProtKB/Swiss-Prot database (release 55) with the following parameters: peptide tolerance, 15 ppm; fragment tolerance, 0.015 Da; carbamidomethylation as a fixed modification; and [13C]Lys (ϩ6 Da) and [13C,15N]Arg (ϩ10 Da) as variable modifications. DDA-derived MS/MS spectra (peak lists) were searched against the UniProtKB/Swiss-Prot database using either PLGS as described or alternatively using Mascot version 2.2 (Matrix Science) with the following parameters: peptide tolerance, 0.2 Da; 13C ϭ 1; fragment tolerance, 0.1 Da; missed cleavages, 2; instrument type, ESI-Q-TOF; variable modifications, carbamidomethylation, methionine/asparagine oxidation, and for SILAC data label Lys ϩ6 Da and Arg ϩ10 Da. All database searches were restricted to human species because of the complexity of the searches when combined with multiple modifications. Samples were eluted in ammonium hydroxide and either resolved by SDS-PAGE or desalted by methanol/chloroform precipitation prior to tryptic digestion as described previously [17]
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