Dataset Information

: Remodelling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO

ABSTRACT: Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein

SUBMITTER: Prerana Wagle

LAB HEAD: Stephan Baldus

PROVIDER: PXD033079 | Pride | 2024-05-23

REPOSITORIES: Pride

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			Action	DRS
	Q2_ColID_121_ProjID_2408_01.raw	Raw
	Q2_ColID_121_ProjID_2408_02.raw	Raw
	Q2_ColID_121_ProjID_2408_03.raw	Raw
	Q2_ColID_121_ProjID_2408_04.raw	Raw
	Q2_ColID_121_ProjID_2408_05.raw	Raw

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Publications

Remodeling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO.

Zheng Ruiyuan R Moynahan Kyle K Georgomanolis Theodoros T Pavlenko Egor E Geissen Simon S Mizi Athanasia A Grimm Simon S Nemade Harshal H Rehimi Rizwan R Bastigkeit Jil J Lackmann Jan-Wilm JW Adam Matti M Rada-Iglesias Alvaro A Nuernberg Peter P Klinke Anna A Poepsel Simon S Baldus Stephan S Papantonis Argyris A Kargapolova Yulia Y

iScience 20240112 2

Myeloperoxidase (MPO) is an enzyme that functions in host defense. MPO is released into the vascular lumen by neutrophils during inflammation and may adhere and subsequently penetrate endothelial cells (ECs) coating vascular walls. We show that MPO enters the nucleus of ECs and binds chromatin independently of its enzymatic activity. MPO drives chromatin decondensation at its binding sites and enhances condensation at neighboring regions. It binds loci relevant for endothelial-to-mesenchymal tra ...[more]

PMID: 38322992

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