Santos Gérsika Bitencourt, Gonzalez-Perilli Lucia, Mastrogiovanni Mauricio, Aicardo Adrián, Cerdeira Cláudio Daniel, Trostchansky Andrés, Brigagão Maísa Ribeiro Pereira Lima
Department of Biochemistry (DBq), Institute of Biomedical Sciences, Federal University of Alfenas (UNIFAL-MG), Alfenas, Brazil.
Biochemistry Department, Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Chem Biol Interact. 2017 Jun 25;272:117-124. doi: 10.1016/j.cbi.2017.05.015. Epub 2017 May 19.
Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol.
In the presence of Tempol (5-120 μM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC of 22.9 ± 10.8 μM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a'active site. More specifically, Cys was modified by Tempol.
We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.
炎症过程产生的氧化应激是细胞和组织损伤的严重原因。Tempol是一种具有超氧化物歧化酶样活性的高效抗氧化剂。本文旨在探讨氮氧化物Tempol对蛋白质二硫键异构酶（PDI）的抑制作用，PDI是一种重要的氧化还原伴侣，其活性位点含有半胱氨酸-甘氨酸-组氨酸-半胱氨酸（CXXC）基序。
在Tempol（5-120μM）存在的情况下，PDI的还原酶活性在体外和活化的小鼠中性粒细胞中均受到可逆影响，IC为22.9±10.8μM。通过胰岛素法和曙红-谷胱甘肽（E-GSH）荧光形成法证实了抑制活性。通过电子顺磁共振（EPR）和质谱法测定了Tempol与该酶结合的能力。在PDI存在的情况下，EPR Tempol信号降低，而当PDI硫醇先前用N-乙基马来酰亚胺（NEM）阻断时，该信号保持不变。当分析总蛋白时，发现有1个和4个Tempol分子与该蛋白结合。然而，仅发现1个分子在a'活性位点与PDI共价结合。更具体地说，半胱氨酸被Tempol修饰。
我们已经表明，氮氧化物Tempol通过与蛋白质结构的半胱氨酸400共价结合而作为PDI的抑制剂。由于PDI与吞噬细胞的NADPH氧化酶复合物的组装相关联，这些发现揭示了Tempol的一种新作用，其在该蛋白参与的病理过程中为靶向PDI敲低的治疗干预提供了潜在的临床应用。
