Jenney Liu

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.

Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive element

Subunit 4 of cytochrome c oxidase (CcO) is a nuclear‐encoded regulatory subunit of the terminal complex of the mitochondrial electron transport chain. We have recently discovered an isoform of CcO 4 (CcO4‐2) which is specific to lung and trachea, and is induced after birth. The role of CcO as the major cellular oxygen consumer, and the lung‐specific expression of CcO4‐2, led us to investigate CcO4‐2 gene regulation. We cloned the CcO4‐2 promoter regions of cow, rat and mouse and compared them with the human promoter. Promoter activity is localized within a 118‐bp proximal region of the human promoter and is stimulated by hypoxia, reaching a maximum (threefold) under 4% oxygen compared with normoxia. CcO4‐2 oxygen responsiveness was assigned by mutagenesis to a novel promoter element (5′‐GGACGTTCCCACG‐3′) that lies within a 24‐bp region that is 79% conserved in all four species. This element is able to bind protein, and competition experiments revealed that, within the element, the four core bases 5′‐TCNCA‐3′ are obligatory for transcription factor binding. CcO isolated from lung showed a 2.5‐fold increased maximal turnover compared with liver CcO. We propose that CcO4‐2 expression in highly oxygenated lung and trachea protects these tissues from oxidative damage by accelerating the last step in the electron transport chain, leading to a decrease in available electrons for free radical formation.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung‐specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2‐knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2‐knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2‐knockout mice, lung COX activity and cellular ATP levels were significantly reduced (—50 and — 29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot‐Leyden crystals. In addition, there was an interesting sex‐specific phenotype, in which the knockout females showed reduced lean mass (—12%), reduced total oxygen consumption rate (—8%), improved glucose tolerance, and reduced grip force (—14%) compared to wild‐type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long‐term lung pathology develops in the knockout mice due to impairment of energy‐costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.—Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar‐Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus‐Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2‐knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. FASEB J. 26, 3916–3930 (2012). www.fasebj.org

Inhibition of N-Methyl-d-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

Background: NMDA receptor hyperactivity results in mitochondrial dysfunction in neurons promoting neurodegenerative disorders. Results: Short polyarginine peptides target mitochondria to promote neuronal survival. Conclusion: Short polyarginine peptides reduce mitochondrial respiration, membrane hyperpolarization, and generation of reactive oxygen species. Significance: Treatment with polyarginine has the potential to minimize neuronal damage resulting from stroke or traumatic brain injury and may be therapeutic to ameliorate multiple sclerosis and Parkinson disease. It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons.

Deletion of heart-type cytochrome c oxidase subunit 7a1 impairs skeletal muscle angiogenesis and oxidative phosphorylation.

•  Cytochrome c oxidase (Cox) is the proposed rate‐limiting enzyme of aerobic metabolism in mammals. •  This study examined a mouse model that has a heart/skeletal muscle‐specific isoform of Cox deleted and represents a mild form of mitochondrial myopathy. •  The results indicated that endurance capacity was severely limited in the knockout animals compared with controls. •  The observed exercise intolerance was accompanied by decreased capillarity in the hindlimb muscle as well as reduced energy production. •  The results suggest that mild mitochondrial myopathy results in adverse structural and functions changes in muscle bioenergetics.

Theophylline treatment improves mitochondrial function after upper cervical spinal cord hemisection.

The importance of mitochondria in spinal cord injury has mainly been attributed to their participation in apoptosis at the site of injury. But another aspect of mitochondrial function is the generation of more than 90% of cellular energy in the form of ATP, mediated by the oxidative phosphorylation (OxPhos) process. Cytochrome c oxidase (CcO) is a central OxPhos component and changes in its activity reflect changes in energy demand. A recent study suggests that respiratory muscle function in chronic obstructive pulmonary disease (COPD) patients is compromised via alterations in mitochondrial function. In an animal model of cervical spinal cord hemisection (C2HS) respiratory dysfunction, we have shown that theophylline improves respiratory function. In the present study, we tested the hypothesis that theophylline improves respiratory function at the cellular level via improved mitochondrial function in the C2HS model. We demonstrate that CcO activity was significantly (33%) increased in the spinal cord adjacent to the site of injury (C3-C5), and that administration of theophylline (20mg/kg 3x daily orally) after C2HS leads to an even more pronounced increase in CcO activity of 62% compared to sham-operated animals. These results are paralleled by a significant increase in cellular ATP levels (51% in the hemidiaphragm ipsilateral to the hemisection). We conclude that C2HS increases energy demand and activates mitochondrial respiration, and that theophylline treatment improves energy levels through activation of the mitochondrial OxPhos process to provide energy for tissue repair and functional recovery after paralysis in the C2HS model.

COX7AR is a Stress-inducible Mitochondrial COX Subunit that Promotes Breast Cancer Malignancy.

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in regulating mitochondrial energy production and cell survival. COX subunit VIIa polypeptide 2-like protein (COX7AR) is a novel COX subunit that was recently found to be involved in mitochondrial supercomplex assembly and mitochondrial respiration activity. Here, we report that COX7AR is expressed in high energy-demanding tissues, such as brain, heart, liver, and aggressive forms of human breast cancer cells. Under cellular stress that stimulates energy metabolism, COX7AR is induced and incorporated into the mitochondrial COX complex. Functionally, COX7AR promotes cellular energy production in human mammary epithelial cells. Gain- and loss-of-function analysis demonstrates that COX7AR is required for human breast cancer cells to maintain higher rates of proliferation, clone formation, and invasion. In summary, our study revealed that COX7AR is a stress-inducible mitochondrial COX subunit that facilitates human breast cancer malignancy. These findings have important implications in the understanding and treatment of human breast cancer and the diseases associated with mitochondrial energy metabolism.

Epicatechin Stimulates Mitochondrial Activity and Selectively Sensitizes Cancer Cells to Radiation

Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.

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 isolated from kidneys is phosphorylated on Thr28, leading to a partial 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 superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central 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 kidney, 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 ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury

The interaction of light with biological tissue has been successfully utilized for multiple therapeutic purposes. Previous studies have suggested that near infrared light (NIR) enhances the activity of mitochondria by increasing cytochrome c oxidase (COX) activity, which we confirmed for 810 nm NIR. In contrast, scanning the NIR spectrum between 700 nm and 1000 nm revealed two NIR wavelengths (750 nm and 950 nm) that reduced the activity of isolated COX. COX-inhibitory wavelengths reduced mitochondrial respiration, reduced the mitochondrial membrane potential (ΔΨm), attenuated mitochondrial superoxide production, and attenuated neuronal death following oxygen glucose deprivation, whereas NIR that activates COX provided no benefit. We evaluated COX-inhibitory NIR as a potential therapy for cerebral reperfusion injury using a rat model of global brain ischemia. Untreated animals demonstrated an 86% loss of neurons in the CA1 hippocampus post-reperfusion whereas inhibitory NIR groups were robustly protected, with neuronal loss ranging from 11% to 35%. Moreover, neurologic function, assessed by radial arm maze performance, was preserved at control levels in rats treated with a combination of both COX-inhibitory NIR wavelengths. Taken together, our data suggest that COX-inhibitory NIR may be a viable non-pharmacologic and noninvasive therapy for the treatment of cerebral reperfusion injury.