Regulation Of Cytochrome C Functions By Phosphorylation

The long term goal of my thesis research is to understand how tissue-specific phosphorylations on the small mitochondrial protein, cytochrome c (Cytc), regulate its functions, under both physiologically healthy and stressed conditions, and to identify the cell signaling pathways targeting Cytc. Cytc is a functionally diverse protein that carries electrons in the electron transport chain and plays a critical role in cellular apoptosis, two diverse pathways that maintain cellular health that are active under diverse conditions. Since Cytc plays a pivotal role in both these highly divergent pathways, regulation of the protein is very important—phosphorylation of the protein under physiological conditions hence implies a regulation by cell signaling pathways that have yet to be identified and studied. Previous work by our lab suggests the importance of reversible phosphorylation of Cytc in regulating its functions 1-4 . We hypothesize that under healthy conditions, phosphorylated Cytc partially inhibits mitochondrial respiration and maintains healthy mitochondrial membrane potential, preventing ROS generation, while cellular stress- mediated dephosphorylation leads to increased respiration and ROS generation, initiating apoptosis. To further test this hypothesis and to extend our understanding of Cytc phosphorylation on its functions, I conducted two studies. In the first study, I investigated the physiological phosphorylation status of Cytc in mammalian kidney tissues. To begin with, I purified bovine kidney Cytc in the presence of phosphatase inhibitors, identified threonine phosphorylation by immunoblot analysis, and determined threonine 28 1 phosphorylation by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (Nano/LC/ESI/MS/MS). To characterize the effect of Thr28 phosphorylation on Cytc functions, I mutated Thr28 to glutamate, a phosphomimetic mutation, and alanine, a nonphosphorylatable control. I went on to express and purify wild-type, the phosphomimetic mutant and the non- phosphorylatable mutant Cytc in bacterial cells. I also expressed and analyzed wild-type, the phosphomimetic mutant and the nonphosphorylatable mutant Cytc in mammalian cells to determine the effects of the Cytc mutations on the functions of the protein in vitro and on overall cellular metabolism and physiology, under healthy and stressed conditions. I also found that Thr28 phosphorylation is AMP kinase-mediated, and AMP kinase colocalizes with Cytc to the mitochondrial intermembrane space. Our data suggest that Thr28, conserved in mammalian Cytc, is an important regulatory site that leads to regulation of ETC flux via ‘controlled respiration,’ preventing m hyperpolarization, a known cause of ROS and trigger of apoptosis (discussed in Chapter 2, manuscript under preparation). In the second study, the phosphorylation status of Cytc in ischemic brain was investigated to determine if insulin-induced neuroprotection and inhibition of Cytc release in ischemic brain was associated with Cytc phosphorylation. We used an animal model of global brain ischemia, and found a 50% decreased death rate of CA1 hippocampal neurons after neuroprotective post-ischemic insulin administration as compared to untreated controls. The increased survival of CA1 neurons was correlated to inhibition of Cytc release from mitochondria into cytosol 24 hours post reperfusion, which in turn was mediated by Cytc phosphorylation on Tyr97. We thus propose that Cytc is phosphorylated by an insulin-dependent signaling pathway, and this may impede with 2 its release from mitochondria and its ability to induce apoptosis (discussed in Chapter 3, manuscript published in PLoS One, 2013 8(11):e78627)

Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation

Cytochrome c (cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2o2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis

Lysine 53 Acetylation of Cytochrome c in Prostate Cancer: Warburg Metabolism and Evasion of Apoptosis

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), supporting the Warburg effect. In contrast to WT, K53Q Cytc had significantly lower caspase-3 activity, suggesting that modification of Cytc K53 helps cancer cells evade apoptosis. Cardiolipin peroxidase activity, which is another proapoptotic function of the protein, was lower in acetylmimetic Cytc. Acetylmimetic Cytc also had a higher capacity to scavenge reactive oxygen species (ROS), another pro-survival feature. We discuss our experimental results in light of structural features of K53Q Cytc, which we crystallized at a resolution of 1.31 Å, together with molecular dynamics simulations. In conclusion, we propose that K53 acetylation of Cytc affects two hallmarks of cancer by regulating respiration and apoptosis in prostate cancer xenografts

Lysine 53 Acetylation of Cytochrome c in Prostate Cancer: Warburg Metabolism and Evasion of Apoptosis

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), supporting the Warburg effect. In contrast to WT, K53Q Cytc had significantly lower caspase-3 activity, suggesting that modification of Cytc K53 helps cancer cells evade apoptosis. Cardiolipin peroxidase activity, which is another proapoptotic function of the protein, was lower in acetylmimetic Cytc. Acetylmimetic Cytc also had a higher capacity to scavenge reactive oxygen species (ROS), another pro-survival feature. We discuss our experimental results in light of structural features of K53Q Cytc, which we crystallized at a resolution of 1.31 Å, together with molecular dynamics simulations. In conclusion, we propose that K53 acetylation of Cytc affects two hallmarks of cancer by regulating respiration and apoptosis in prostate cancer xenografts