Nitration of protein tyrosine residues by the pro-oxidant peroxynitrite is a prevalent, functionally significant post- translation modification leading to either a gain or loss of protein function and alterations in cellular activity. We have demonstrated nitration of many placental proteins including p53, taurine transporter, P2X4 receptor and p38MAP kinase and that the extent of nitration is increased in pregnancies complicated by preeclampsia. The oxidative stress of pregnancy is increased further in preeclampsia where we identified increased placental expression of NADPH oxidase (NOX) enzyme isoforms that produce superoxide and increased eNOS expression in vascular endothelial cells and syncytiotrophoblast. In preeclampsia there is mitochondrial dysfunction and increased trophoblast apoptosis, via downregulation of anti-apoptotic proteins including Bcl-2 and up-regulation of pro-apoptotic p53. The specific mitochondrial NOS isoform, mtNOS, generates NO which at low concentrations inactivates the hemoprotein cytochrome c oxidase to regulate mitochondrial respiration and cellular function. At higher concentrations NO forms peroxynitrite which damages mitochondria causing release of cytochrome c and apoptosis. Nitration of mitochondrial proteins is a reversible process dependent on oxygen concentration hence the hypoxic environment or ischemia/reperfusion of the preeclamptic placenta may cause protein nitration and altered cellular respiration and placental function. This proposal will study the role of protein nitration in regulation of placental mitochondrial function and trophoblast apoptosis utilizing ex vivo and in vitro approaches in human placental tissue and trophoblast cell culture. We will describe the nitroproteome in isolated heavy (syncytiotrophoblast) and light (cytotrophoblast) mitochondrial fractions, comparing tissues from normal pregnancies with mild late onset or severe early onset preeclampsia. Expression of nitrated mitochondrial proteins will be related to activity of mitochondrial enzyme complexes and apoptotic molecules. The involvement of the mtNOS, eNOS and iNOS isoforms, NOX enzymes and manganese superoxide dismutase in nitration and the role of hypoxia and hypoxia/reoxygenation on protein nitration and the apoptotic cascade (P53, caspase 3 and Bcl-2) will be investigated using pharmacologic, knockdown by siRNA and overexpression approaches in trophoblast cell culture.
Program Narrative Preeclampsia is a major problem in obstetrics affecting 7% of pregnancies and being the leading cause of fetal growth restriction, indicated preterm delivery and maternal death particularly with early onset severe disease. The maternal syndrome of preeclampsia appears to be linked to inadequate trophoblast invasion, placental hypoxia and oxidative stress and increased trophoblast apoptosis and shedding into the maternal vasculature. This proposal will test the hypothesis that oxidative and nitrative stress occur in placental mitochondria leading to protein nitration, a covalent modification that alters protein and thus mitochondrial and placental function which is critical to the development of preeclampsia.
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