Deubiquitinases in macrophages infected with Salmonella enterica or Yersinia enterocolitica
Description
We used an activity-based proteomics screen of deubiquitinases in human macrophages to identify several deubiquitinases regulated in THP-1 derived macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica at 2 and 18 hours of infection.
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The THP-1 monocytes were grown in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum albumin and 1% combination pen/strep and differentiated to macrophages by 100 nM PMA for 48 hours on 10-cm dishes. The infections with Salmonella Typhimurium 12023 or Y. enterocolitica 8081 were performed using mid-log bacterial cultures for 2 and 18 hours. Cells were collected, washed in PBS, and frozen until further use. Cell pellets were resuspended in four pellet volumes of 150 mM NaCl, 20 mM MgCl2, 50 mM Tris [pH 7.5], 0.5% NP-40 containing 1 mM PMSF, 2 U/ml Turbo DNase, 20 U/ml RNase Cocktail (Ambion), and lysed for 30 minutes on ice. The samples were centrifuged for 15 min at 21,000 x g, and the supernatant was collected, and protein concentration measured. HA-Ubiquitin-Vinyl Sulfone probe was used for in vitro labeling reaction and added to samples at 1:100 probe:protein ratio . The samples were mixed with 100 µL washed and equilibrated EZview HA beads. The immunoprecipitation proceeded at 4°C for 2 hours, and samples on a rotator. The resin was then washed four times with 800 µL of 150 mM NaCl, 50 mM Tris [pH 7.5]. Finally, proteins were eluted by using 0.1% RapiGest SF (Waters), followed by boiling at 95°C for 5 minutes. Samples were then precipitated by 0.18 volumes of 100% TCA for 20 min on ice, spun for 30 min at 21,000 x g, and the precipitate was washed by 500 µL of -20°C acetone. The sample was spun again, acetone removed and precipitate was left to air dry for the tryptic digestion, and the samples were analyzed by LTQ Velos. Tandem mass spectra were extracted by Proteome Discoverer. Charge state deconvolution and deisotoping were performed. MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search human Uniprot database with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 30 PPM. Carbamidomethyl of cysteine was specified in Sequest (XCorr Only) and X! Tandem as a fixed modification. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version Scaffold 5.0.0) was used to validate peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped . The quantification was done using weighted spectral count where fold change was calculated between the proteins from infected samples compared to uninfected samples, and the samples were normalized to the overall spectral count. Fisher’s test was used to calculate the statistical significance of the findings.