Icksoo Lee

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.

Regulation of oxidative phosphorylation, the mitochondrial membrane potential, and their role in human disease

Thirty years after Peter Mitchell was awarded the Nobel Prize for the chemiosmotic hypothesis, which links the mitochondrial membrane potential generated by the proton pumps of the electron transport chain to ATP production by ATP synthase, the molecular players involved once again attract attention. This is so because medical research increasingly recognizes mitochondrial dysfunction as a major factor in the pathology of numerous human diseases, including diabetes, cancer, neurodegenerative diseases, and ischemia reperfusion injury. We propose a model linking mitochondrial oxidative phosphorylation (OxPhos) to human disease, through a lack of energy, excessive free radical production, or a combination of both. We discuss the regulation of OxPhos by cell signaling pathways as a main regulatory mechanism in higher organisms, which in turn determines the magnitude of the mitochondrial membrane potential: if too low, ATP production cannot meet demand, and if too high, free radicals are produced. This model is presented in light of the recently emerging understanding of mechanisms that regulate mammalian cytochrome c oxidase and its substrate cytochrome c as representative enzymes for the entire OxPhos system.

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation.

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase

Mitochondrial oxidative phosphorylation provides most cellular energy. As part of this process, cytochrome c oxidase (CcO) pumps protons across the inner mitochondrial membrane, contributing to the generation of the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. During acute inflammation, as in sepsis, aerobic metabolism appears to malfunction and switches to glycolytic energy production. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) has been shown to play a central role in inflammation. We hypothesized that TNFα-triggered cell signaling targets CcO, which is a central enzyme of the aerobic energy metabolism and can be regulated through phosphorylation. Using total bovine and murine hepatocyte homogenates TNFα treatment led to an ∼60% reduction in CcO activity. In contrast, there was no direct effect of TNFα on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFα-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFα treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was ∼50 and 70% inhibited at high cytochrome c concentrations in the presence of allosteric activator ADP and inhibitor ATP, respectively. CcO phosphorylation occurs on tyrosine 304 as demonstrated with a phosphoepitope-specific antibody. Furthermore, the mitochondrial membrane potential was decreased in H2.35 cells in response to TNFα. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFα-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients.

Epidermal Growth Factor Receptor Translocation to the Mitochondria: REGULATION AND EFFECT*

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

New control of mitochondrial membrane potential and ROS formation--a hypothesis.

Abstract A new control of mitochondrial membrane potential ΔΨm and formation of reactive oxygen species (ROS) is presented, based on allosteric ATPinhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100 120 mV), the ATP/ADP ratio will also be maximal at this ΔΨm (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADPratio keeps ΔΨm low. In contrast, the known respiratory control leads to an inhibition of respiration only at high ΔΨm values (150 200 mV) which cause ROS formation. ATPinhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that stress hormones which increase intracellular [Ca 2+ ] also increase ΔΨm and ROS formation, which promote degenerative diseases and accelerate aging.

TXNIP Links Innate Host Defense Mechanisms to Oxidative Stress and Inflammation in Retinal Muller Glia under Chronic Hyperglycemia: Implications for Diabetic Retinopathy

Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1β) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-α and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

Phosphorylation of Mammalian Cytochrome c and Cytochrome c Oxidase in the Regulation of Cell Destiny: Respiration, Apoptosis, and Human Disease

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

TLR4-Mediated AKT Activation Is MyD88/TRIF Dependent and Critical for Induction of Oxidative Phosphorylation and Mitochondrial Transcription Factor A in Murine Macrophages

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis, and function requires coordinated expression of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam) and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory responses, but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase that plays an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence we investigated the role of LPS-mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macrophages (B6-MCL) and bone marrow-derived macrophages. We show that LPS challenge led to increased expression of T-fam and COX subunits I and IV in a time-dependent manner through early phosphorylation of the PI3K/AKT pathway. PI3K/AKT pathway inhibitors abrogated LPS-mediated T-fam and COX induction. Lack of induction was associated with decreased ATP production, increased proinflammatory cytokines (TNF-α), NO production, and cell death. The TLR4-mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β. Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.