Petr Pecina

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.

Phosphomimetic Substitution of Cytochrome c Tyrosine 48 Decreases Respiration and Binding to Cardiolipin and Abolishes Ability to Trigger Downstream Caspase Activation

Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

Chapter 11 Isolation of Regulatory‐Competent, Phosphorylated Cytochrome c Oxidase

The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation.

A SUGGESTED ROLE FOR MITOCHONDRIA IN NOONAN SYNDROME

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.

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

Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations.

The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LS(COX)). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32-54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LS(COX) cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LS(COX) cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I-III₂-IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I-III₂ supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85-140kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.

Cytochrome c is tyrosine 97 phosphorylated by neuroprotective insulin treatment

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Mice deleted for heart-type cytochrome c oxidase subunit 7a1 develop dilated cardiomyopathy

Subunit 7a of mouse cytochrome c oxidase (Cox) displays a contractile muscle-specific isoform, Cox7a1, that is the major cardiac form. To gain insight into the role of this isoform, we have produced a new knockout mouse line that lacks Cox7a1. We show that homozygous and heterozygous Cox7a1 knockout mice, although viable, have reduced Cox activity and develop a dilated cardiomyopathy at 6 weeks of age. Surprisingly, the cardiomyopathy improves and stabilizes by 6 months of age. Cox7a1 knockout mice incorporate more of the liver-type isoform Cox7a2 into the cardiac Cox holoenzyme and, also surprisingly, have higher tissue ATP levels.

Mitochondrial ATP synthasome: Expression and structural interaction of its components

Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.