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Date of Award
Molecular Biology and Genetics
Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain (ETC) and plays a key role in apoptosis. Our lab has previously shown that tissue-specific phosphorylations of Cytc in the heart (Y97), liver (Y48), and kidney (T28, T58) play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E) along with recombinant unphosphorylated WT and S47A Cytc as controls. We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic S47E Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase (COX). S47-phosphorylated and phosphomimetic Cytc also showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. The effect of S47-phosphorylation was also studied in fibroblast cell lines stably expressing S47E phosphomimetic Cytc, unphosphorylated WT, or S47A Cytc. Our results show that S47E Cytc results in partial inhibition of mitochondrial respiration corresponding with lower mitochondrial membrane potentials (ΔΨm) and reduced reactive oxygen species (ROS) production. When exposed to an oxygen-glucose deprivation/reoxygenation (OGD/R) model simulating ischemia/reperfusion injury, the Cytc S47E phosphomimetic cell line showed minimal ROS generation compared to the unphosphorylated WT Cytc cell line. Consequently, the S47E Cytc cell line also resulted in significantly lower cell death upon exposure to OGD/R and H2O2 treatment, confirming the cytoprotective role of S47 phosphorylation of Cytc. Finally, we propose that pro-survival kinase Akt (protein kinase B) is a likely mediator of the S47 phosphorylation of Cytc in the brain. PI3K/Akt inhibitor wortmannin abolished S47 phosphorylation of Cytc, compared to Akt activator SC79. Overall, our results suggest that loss of S47 phosphorylation of Cytc during brain ischemia drives reperfusion injury through maximal ETC flux, ΔΨm hyperpolarization, and ROS-triggered cell death.
Kalpage, Hasini, "Serine 47 (s47) Phosphorylation Of Cytochrome C: Implications For Ischemia/reperfusion Injury" (2021). Wayne State University Dissertations. 3443.