Comparison of denatured and non-denatured FIH interactomes
Hypoxia occurs when tissue or cellular oxygen demand exceeds its supply and is a frequent condition in health and disease. Metazoans have developed a regulatory system that allows them to sense changes in oxygen levels in their microenvironment and adapt to them. The asparagine hydroxylase factor inhibiting HIF (FIH) regulates the transcriptional activity of the hypoxia-inducible factor (HIF), the master regulator of the cellular adaptive response to hypoxia [1, 2]. We recently identified the deubiquitinating enzyme ovarian tumour domain containing, ubiquitin aldehyde binding protein 1 (OTUB1) as novel bona fide FIH target protein with the hydroxylation occurring at the asparagine residue N22 [3, 4]. Surprisingly, in a follow-up investigation we observed a SDS-PAGE resistant interaction between FIH and OTUB1, resulting in a FIH-OTUB1 heterodimer (HD). Further analysis of the FIH-OTUB1 HD demonstrated that it is not linked by a disulfide bond, oxyester or thioester but likely through an amide bond. Interestingly, mutation of the hydroxylation acceptor site N22 in OTUB1 and genetic and pharmacologic inhibition of FIH prevented the formation of the heterodimer, demonstrating that HD formation is a consequence of FIH activity. To investigate if FIH-dependent stable protein complex formation was exclusive for OTUB1, we carried out a mass spectrometry (MS)-based FIH interactome analyses with denatured and non-denatured cell lysates (with or without SDS treatment and boiling). The results indicate the existence of further novel denaturing condition resistant protein complexes. 1. Mahon, P.C., K. Hirota, and G.L. Semenza, FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes & development, 2001. 15(20): p. 2675-86. 2. Lando, D., et al., FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes & development, 2002. 16(12): p. 1466-71. 3. Scholz, C.C., et al., Regulation of IL-1beta-induced NF-kappaB by hydroxylases links key hypoxic and inflammatory signaling pathways. Proc Natl Acad Sci U S A, 2013. 110(46): p. 18490-5. 4. Scholz, C.C., et al., FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1. PLoS biology, 2016. 14(1): p. e1002347.
Sample Processing Protocol
HEK293 cell were transiently transfected with pEGFP or pFIH-V5 for 48 h and subsequently lysed with “lysis buffer” without (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1% Triton-X100; “-SDS”) or with 1% SDS (“+SDS”). 0.75 U/µl benzonase was added to all samples. Samples containing SDS were boiled for 10 min at 95°C, samples without SDS were left on ice. Following 1:10 dilution with lysis buffer without SDS, proteins were immunoprecipitated with anti-V5 agarose for 1 hour at 4°C. After washing the beads twice with lysis buffer (without SDS) and twice with “washing buffer” (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM MgCl2), the beads were resuspended in 45 µl “digestion buffer” (10 mM Tris, 2 mM CaCl2, pH 8.2) and the proteins were on-bead digested using 5 µl of Sequencing Grade Trypsin (100 ng/µl in 10 mM HCl, Promega). The digestion was carried out in a microwave instrument (Discover System, CEM Corporation) for 30 min at 5 W and 60°C. The supernatants were transferred into new tubes and the beads were additionally digested for 3 h at room temperature. The beads were washed with 100 µl TFA-buffer (0.1% TFA, 10mM Tris, 2 mM CaCl2), the supernatants were collected and combined with the previously collected supernatants. The samples were dried in the SpeedVac (Thermo Fisher Scientific), resolubilized in 25 µl of 3% acetonitrile, 0.1% Formic acid spiked with iRT peptides (Biognosys) and centrifuged at max speed (20,000 g) for 10 minutes. Twenty microliters were transferred into liquid chromatography-mass spectrometry (LC-MS) vials. Mass spectrometry analysis was performed on a Q Exactive mass spectrometer coupled to a Nano Easy 1000 liquid chromatography system (Thermo Fisher Scientific). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. For each sample, 4 μL of peptides were loaded on a commercial Acclaim PepMapTM Trap Column (75 µm x 20 mm, Thermo Fisher Scientific) followed by a PepMapTM RSLC C18 Snail Column (75 μm × 500 mm, Thermo Fisher Scientific). The peptides were eluted at a flow rate of 300 nL/min by a gradient from 5 to 22% B in 79 min, 32% B in 11 min and 95% B in 10 min. Samples were acquired in a randomized order. The mass spectrometer was operated in data-dependent mode (DDA), acquiring a full-scan MS spectra (300−1,700 m/z) at a resolution of 70,000 at 200 m/z after accumulation to a target value of 3,000,000, followed by HCD (higher-energy collision dissociation) fragmentation on the twelve most intense signals per cycle. HCD spectra were acquired at a resolution of 35,000 using a normalized collision energy of 25 and a maximum injection time of 120 ms. The automatic gain control (AGC) was set to 50,000 ions. Charge state screening was enabled and singly and unassigned charge states were rejected. Only precursors with intensity above 8,300 were selected for MS/MS (2% underfill ratio). Precursor masses previously selected for MS/MS measurement were excluded from further selection for 30 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.1010 and 445.1200.
Data Processing Protocol
The acquired raw mass spectrometry MS data were processed by MaxQuant (version 1.6.1; ), followed by protein identification using the integrated Andromeda search engine . Spectra were searched against a UniProt Homo Sapiens (taxonomy 9606) reference proteome (canonical version from 2016-12-09), concatenated to its reversed decoyed fasta database and common protein contaminants. Carbamidomethylation of cysteine was set as fixed, while methionine oxidation and N-terminal protein acetylation were set as variable modifications. MaxQuant Orbitrap default search settings were used. Enzyme specificity was set to trypsin/P. For Label-Free-Quantification, MaxQuant default settings were applied. In the MaxQuant experimental design template, the biological and biochemical replicates were grouped into non-adjacent fractions, to allow match-between-runs within but not between conditions. Each file was treated as a separate experiment to obtain individual quantitative values. Protein fold changes were computed based on intensity values reported in the proteinGroups.txt file. A set of functions implemented in the R package SRMService  was used to filter for proteins with 2 or more peptides, with quantification in at least 4 samples, and to normalize the data with a modified robust z-score transformation and to compute p-values using the t-test with pooled variance. 1. Cox, J. and M. Mann, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature biotechnology, 2008. 26(12): p. 1367-72. 2. Cox, J., et al., Andromeda: a peptide search engine integrated into the MaxQuant environment. Journal of proteome research, 2011. 10(4): p. 1794-805. 3. Wolski, W., Grossmann, J., Panse, C., SRMService - R-Package to Report Quantitative Mass Spectrometry Data. 2018. http://github.com/protViz/SRMService.
Pickel C, Günter J, Ruiz-Serrano A, Spielmann P, Fabrizio JA, Wolski W, Peet DJ, Wenger RH, Scholz CC. Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1. Redox Biol. 2019 26:101265 PubMed: 31299612