Cardiolipin-deficient cells have decreased levels of the iron-sulfur biogenesis protein frataxin.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays an important role in mitochondrial bioenergetics. Previous studies in the yeast model have indicated that CL is required for optimal iron homeostasis, which is disrupted by a mechanism not yet determined in the yeast CL mutant, crd1Δ. This finding has implications for the severe genetic disorder, Barth syndrome (BTHS), in which CL metabolism is perturbed because of mutations in the CL-remodeling enzyme, tafazzin. Here, we investigate the effects of tafazzin deficiency on iron homeostasis in the mouse myoblast model of BTHS tafazzin knockout (TAZ-KO) cells. Similarly to CL-deficient yeast cells, TAZ-KO cells exhibited elevated sensitivity to iron, as well as to H2O2, which was alleviated by the iron chelator deferoxamine. TAZ-KO cells exhibited increased expression of the iron exporter ferroportin and decreased expression of the iron importer transferrin receptor, likely reflecting a regulatory response to elevated mitochondrial iron. Reduced activities of mitochondrial iron-sulfur cluster enzymes suggested that the mechanism underlying perturbation of iron homeostasis was defective iron-sulfur biogenesis. We observed decreased levels of Yfh1/frataxin, an essential component of the iron-sulfur biogenesis machinery, in mitochondria from TAZ-KO mouse cells and in CL-deleted yeast crd1Δ cells, indicating that the role of CL in iron-sulfur biogenesis is highly conserved. Yeast crd1Δ cells exhibited decreased processing of the Yfh1 precursor upon import, which likely contributes to the iron homeostasis defects. Implications for understanding the pathogenesis of BTHS are discussed.

Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis.

Cytochrome c (Cyt c) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cyt c has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cyt c is tightly regulated by allosteric mechanisms, tissue-specific isoforms, and post-translational modifications (PTMs). Distinct residues of Cyt c are modified by PTMs, primarily phosphorylations, in a highly tissue-specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨm), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia-reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨm hyperpolarization, excessive ROS generation, and the release of Cyt c. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cyt c and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨm, limiting ROS generation. We examine the role of Cyt c PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.-Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis.

Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney 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). 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 Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

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.

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency.

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.

Aerobic Exercise Preconception and During Pregnancy Enhances Oxidative Capacity in the Hindlimb Muscles of Mice Offspring.

Liu, J, Lee, I, Feng, H-Z, Galen, SS, Hüttemann, PP, Perkins, GA, Jin, J-P, Hüttemann, M, and Malek, MH. Aerobic exercise preconception and during pregnancy enhances oxidative capacity in the hindlimb muscles of mice offspring. J Strength Cond Res 32(5): 1391-1403, 2018-Little is known about the effect of maternal exercise on offspring skeletal muscle health. The purpose of this study, therefore, was to determine whether maternal exercise (preconception and during pregnancy) alters offspring skeletal muscle capillarity and mitochondrial biogenesis. We hypothesized that offspring from exercised dams would have higher capillarity and mitochondrial density in the hindlimb muscles compared with offspring from sedentary dams. Female mice in the exercise condition had access to a running wheel in their individual cage 30 days before mating and throughout pregnancy, whereas the sedentary group did not have access to the running wheel before mating and during pregnancy. Male offspring from both groups were killed when they were 2 months old, and their tissues were analyzed. The results indicated no significant (p > 0.05) mean differences for capillarity density, capillarity-to-fiber ratio, or regulators of angiogenesis such as VEGF-A and TSP-1. Compared with offspring from sedentary dams, however, offspring from exercised dams had an increase in protein expression of myosin heavy chain type I (MHC I) (∼134%; p = 0.009), but no change in MHC II. For mitochondrial morphology, we found significant (all p-values ≤ 0.0124) increases in mitochondrial volume density (∼55%) and length (∼18%) as well as mitochondria per unit area (∼19%). For mitochondrial enzymes, there were also significant (all p-values ≤ 0.0058) increases in basal citrate synthase (∼79%) and cytochrome c oxidase activity (∼67%) in the nonoxidative muscle fibers as well as increases in basal (ATP) (∼52%). Last, there were also significant mean differences in protein expression for regulators (FIS1, Lon protease, and TFAM) of mitochondrial biogenesis. These findings suggest that maternal exercise before and during pregnancy enhances offspring skeletal muscle mitochondria functionality, but not capillarity.

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.

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.

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.

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 P(enh) 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.

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

Oxidative metabolism is needed for sustained skeletal muscle function. A key component of such metabolism is cytochrome c oxidase, the 13-subunit terminal complex of the mitochondrial electron transport chain. We used mice null for one of the two isoforms of Cox subunit 7a, heart/skeletal muscle-specific Cox7a1, to examine the cellular and functional responses of muscle adaptation in response to mitochondrial dysfunction. Specifically we determined if deletion of Cox7a1 would (1) limit exercise capacity, and (2) alter genes responsible for skeletal muscle capillarity and mitochondrial biogenesis. Sixteen male mice (Cox7a1 null mice, n = 8, and littermate controls, n = 8) performed incremental and run-to-exhaustion treadmill tests. The hindlimb muscles for both groups were analysed. The results indicated that capillary indices were reduced (by 30.7­44.9%) in the Cox7a1 null mice relative to controls. In addition, resting ATP levels and Cox specific activity were significantly reduced (>60%) in both glycolytic and oxidative muscle fibre types despite an increase in a major regulator of mitochondrial biogenesis, PGC-1ß. These changes in the skeletal muscle resulted in exercise intolerance for the Cox7a1 null mice. Thus, our data indicate that deletion of the Cox7a1 isoform results in reduced muscle bioenergetics and hindlimb capillarity, helping to explain the observed impairment of muscle structure and function.

Valproate inhibits mitochondrial bioenergetics and increases glycolysis in Saccharomyces cerevisiae.

The widely used mood stabilizer valproate (VPA) causes perturbation of energy metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. To gain insight into these mechanisms, we determined the effects of VPA on energy metabolism in yeast. VPA treatment increased levels of glycolytic intermediates, increased expression of glycolysis genes, and increased ethanol production. Increased glycolysis was likely a response to perturbation of mitochondrial function, as reflected in decreased membrane potential and oxygen consumption. Interestingly, yeast, mouse liver, and isolated bovine cytochrome c oxidase were directly inhibited by the drug, while activities of other oxidative phosphorylation complexes (III and V) were not affected. These findings have implications for mechanisms of therapeutic action and toxicity.

Decreased membrane cholesterol in liver mitochondria of the point mutation mouse model of juvenile Niemann-Pick C1, Npc1nmf164.

It has long been known that there is decreased mitochondrial function in several tissues of Niemann-Pick C1 model mice and cultured cells. These defects contribute to the accumulation of Reactive Oxygen Species (ROS) and tissue damage. It is also well established that there is increased unesterified cholesterol, stored in late endosomes/lysosomes, in many tissues in mutant humans, mouse models, and mutant cultured cells. Using a mouse model with an NPC1 point mutation that is more typical of the most common form of the disease, and highly purified liver mitochondria, we find markedly decreased mitochondrial membrane cholesterol. This is compared to previous reports of increased mitochondrial membrane cholesterol. We also find that, although in wild-type or heterozygous mitochondria cytochrome c oxidase (COX) activity decreases with age as expected, surprisingly, COX activity in homozygous mutant mice improves with age. COX activity is less than half of wild-type amounts in young mutant mice but later reaches wild-type levels while total liver cholesterol is decreasing. Mutant mice also contain a decreased number of mitochondria that are morphologically abnormal. We suggest that the decreased mitochondrial membrane cholesterol is causative for the mitochondrial energy defects. In addition, we find that the mitochondrial stress regulator protein MNRR1 can stimulate NPC1 synthesis and is deficient in mutant mouse livers. Furthermore, the age curve of MNRR1 deficiency paralleled levels of total cholesterol. The role of such altered mitochondria in initiating the abnormal autophagy and neuroinflammation found in NPC1 mouse models is discussed.

Cytochrome c phosphorylation: Control of mitochondrial electron transport chain flux and apoptosis.

Cytochrome c (Cytc)1is a cellular life and death decision molecule that regulates cellular energy supply and apoptosis through tissue specific post-translational modifications. Cytc is an electron carrier in the mitochondrial electron transport chain (ETC) and thus central for aerobic energy production. Under conditions of cellular stress, Cytc release from the mitochondria is a committing step for apoptosis, leading to apoptosome formation, caspase activation, and cell death. Recently, Cytc was shown to be a target of cellular signaling pathways that regulate the functions of Cytc by tissue-specific phosphorylations. So far five phosphorylation sites of Cytc have been mapped and functionally characterized, Tyr97, Tyr48, Thr28, Ser47, and Thr58. All five phosphorylations partially inhibit respiration, which we propose results in optimal intermediate mitochondrial membrane potentials and low ROS production under normal conditions. Four of the phosphorylations result in inhibition of the apoptotic functions of Cytc, suggesting a cytoprotective role for phosphorylated Cytc. Interestingly, these phosphorylations are lost during stress conditions such as ischemia. This results in maximal ETC flux during reperfusion, mitochondrial membrane potential hyperpolarization, excessive ROS generation, and apoptosis. We here present a new model proposing that the electron transfer from Cytc to cytochrome c oxidase is the rate-limiting step of the ETC, which is regulated via post-translational modifications of Cytc. This regulation may be dysfunctional in disease conditions such as ischemia-reperfusion injury and neurodegenerative disorders through increased ROS, or cancer, where post-translational modifications on Cytc may provide a mechanism to evade apoptosis.

EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming.

The molecular mechanisms driving metastatic progression in triple-negative breast cancer (TNBC) patients are poorly understood. In this study, we demonstrate that epidermal growth factor-like 9 (EGFL9) is significantly upregulated in basal-like breast cancer cells and associated with metastatic progression in breast tumor samples. Functionally, EGFL9 is both necessary and sufficient to enhance cancer cell migration and invasion, as well as distant metastasis. Mechanistically, we demonstrate that EGFL9 binds cMET, activating cMET-mediated downstream signaling. EGFL9 and cMET co-localize at both the cell membrane and within the mitochondria. We further identify an interaction between EGFL9 and the cytochrome c oxidase (COX) assembly factor COA3. Consequently, EGFL9 regulates COX activity and modulates cell metabolism, promoting a Warburg-like metabolic phenotype. Finally, we show that combined pharmacological inhibition of cMET and glycolysis reverses EGFL9-driven stemness. Our results identify EGFL9 as a therapeutic target for combating metastatic progression in TNBC.

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.

Novel Pyrrolo[3,2-d]pyrimidine Compounds Target Mitochondrial and Cytosolic One-carbon Metabolism with Broad-spectrum Antitumor Efficacy.

Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, ß-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure-activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

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.

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

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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.