4.5 Article

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence

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ELSEVIER
DOI: 10.1016/j.bbagen.2019.129481

Keywords

Urate hydroperoxide; Cell surface PDI; Thiol oxidation; HUVEC

Funding

  1. Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [CEPID Redoxoma 2013/07937-8, 2011/18106-4]
  2. Conselho Nacional de Pesquisa Tecnologica (CNPq) [472105/2012-4]
  3. Coordenacao de Aperfeicoamento de Pessoal (CAPES) [PROEX 1888/2016]

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Background: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. Methods: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. Results: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 x 10(3) M-1 s(-1). Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. Conclusions: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. General significance: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

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