Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil; Heart Institute (Incor), University of São Paulo School of Medicine, São Paulo, Brazil.
Biochim Biophys Acta Gen Subj. 2020 Mar;1864(3):129481. doi: 10.1016/j.bbagen.2019.129481. Epub 2019 Nov 14.
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.
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.
Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 10 M s. 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.
Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence.
These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.
细胞外表面蛋白二硫键异构酶 A1（PDI）参与血小板聚集、血栓形成和血管重塑。PDI 与客户蛋白进行氧化还原交换，因此其被细胞外分子氧化可能会改变蛋白质功能和细胞反应。在这项研究中，我们研究了尿酸过氧化物对 PDI 的氧化作用，尿酸过氧化物是一种新描述的氧化剂，通过过氧化物酶氧化尿酸产生，可能在血管炎症中发挥作用。
通过 LC-MS/MS 和停流技术评估尿酸过氧化物对 PDI 氧化的氨基酸特异性和动力学。使用不可渗透的烷化试剂鉴定 HUVECs 中细胞表面 PDI 和其他巯基蛋白的氧化。使用特定的 LC-MS/MS 技术评估细胞内 GSH 和 GSSG 的氧化。通过结晶紫染色、细胞显微镜和 LDH 活性分别评估细胞黏附、脱落和活力。
尿酸过氧化物特异性氧化重组 PDI 催化位点的半胱氨酸残基，速率常数为 6×10 M s。HUVECs 与尿酸过氧化物孵育导致细胞表面 PDI 和其他未鉴定的细胞表面巯基蛋白氧化。尿酸过氧化物以及其他氧化剂、巯基烷化剂和 PDI 抑制剂都损害了细胞对纤维连接蛋白包被平板的黏附。尿酸过氧化物不影响细胞活力，但显著降低了 GSH/GSSG 比值。
我们的结果表明，尿酸过氧化物影响 PDI 和其他细胞表面蛋白的巯基氧化，从而损害细胞黏附。
这些发现有助于更好地理解尿酸影响内皮细胞功能和血管内稳态的机制。
