Siddhesh Aras

Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2

Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded by both mitochondrial and nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 and the tissue-specific (predominantly lung) isoform 2 (COX4I2). COX4I2 renders lung COX about 2-fold more active compared with liver COX, which lacks COX4I2. We previously identified a highly conserved 13-bp sequence in the proximal promoter of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at a 4% oxygen concentration. Here, we have identified three transcription factors that bind this conserved ORE, namely recombination signal sequence–binding protein Jκ (RBPJ), coiled-coil-helix-coiled-coil-helix domain 2 (CHCHD2) and CXXC finger protein 5 (CXXC5). We demonstrate that RBPJ and CHCHD2 function towards activating the ORE at 4% oxygen, whereas CXXC5 functions as an inhibitor. To validate results derived from cultured cells, we show using RNA interference a similar effect of these transcription factors in the gene regulation of COX4I2 in primary pulmonary arterial smooth muscle cells. Depending on the oxygen tension, a concerted action of the three transcription factors regulates the expression of COX4I2 that, as we discuss, could augment both COX activity and its ability to cope with altered cellular energy requirements.

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism.

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

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

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing

Rationale: Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. Objectives: To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Methods and Results: Isolated ventilated and perfused lungs from Cox4i2−/− mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2−/− mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2−/− PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2−/− PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2−/− PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Conclusions: Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Loss of ABHD5 promotes the aggressiveness of prostate cancer cells

The accumulation of neutral lipids in intracellular lipid droplets has been associated with the formation and progression of many cancers, including prostate cancer (PCa). Alpha-beta Hydrolase Domain Containing 5 (ABHD5) is a key regulator of intracellular neutral lipids that has been recently identified as a tumor suppressor in colorectal cancer, yet its potential role in PCa has not been investigated. Through mining publicly accessible PCa gene expression datasets, we found that ABHD5 gene expression is markedly decreased in metastatic castration-resistant PCa (mCRPC) samples. We further demonstrated that RNAi-mediated ABHD5 silencing promotes, whereas ectopic ABHD5 overexpression inhibits, the invasion and proliferation of PCa cells. Mechanistically, we found that ABHD5 knockdown induces epithelial to mesenchymal transition, increasing aerobic glycolysis by upregulating the glycolytic enzymes hexokinase 2 and phosphofrucokinase, while decreasing mitochondrial respiration by downregulating respiratory chain complexes I and III. Interestingly, knockdown of ATGL, the best-known molecular target of ABHD5, impeded the proliferation and invasion, suggesting an ATGL-independent role of ABHD5 in modulating PCa aggressiveness. Collectively, these results provide evidence that ABHD5 acts as a metabolic tumor suppressor in PCa that prevents EMT and the Warburg effect, and indicates that ABHD5 is a potential therapeutic target against mCRPC, the deadly aggressive PCa.

Abl2 kinase phosphorylates Bi-organellar regulator MNRR1 in mitochondria, stimulating respiration

We previously showed that MNRR1 (Mitochondrial Nuclear Retrograde Regulator 1, also CHCHD2) functions in two subcellular compartments, displaying a different function in each. In the mitochondria it is a stress regulator of respiration that binds to cytochrome c oxidase (COX) whereas in the nucleus it is a transactivator of COX4I2 and other hypoxia-stimulated genes. We now show that binding of MNRR1 to COX is promoted by phosphorylation at tyrosine-99 and that this interaction stimulates respiration. We show that phosphorylation of MNRR1 takes place in mitochondria and is mediated by Abl2 kinase (ARG). A family with Charcot-Marie-Tooth disease type 1A with an exaggerated phenotype harbors a Q112H mutation in MNRR1, located in a domain that is necessary for transcriptional activation by MNRR1. Furthermore, the mutation causes the protein to function suboptimally in the mitochondria in response to cellular stress. The Q112H mutation hinders the ability of the protein to interact with Abl kinase, leading to defective tyrosine phosphorylation and a resultant defect in respiration.

The cellular stress proteins CHCHD10 and MNRR1 (CHCHD2): Partners in mitochondrial and nuclear function and dysfunction

Coiled-coil-helix-coiled-coil-helix domain–containing 10 (CHCHD10) and CHCHD2 (MNRR1) are homologous proteins with 58% sequence identity and belong to the twin CX9C family of proteins that mediate cellular stress responses. Despite the identification of several neurodegeneration-associated mutations in the CHCHD10 gene, few studies have assessed its physiological role. Here, we investigated CHCHD10s function as a regulator of oxidative phosphorylation in the mitochondria and the nucleus. We show that CHCHD10 copurifies with cytochrome c oxidase (COX) and up-regulates COX activity by serving as a scaffolding protein required for MNRR1 phosphorylation, mediated by ARG (ABL proto-oncogene 2, nonreceptor tyrosine kinase (ABL2)). The CHCHD10 gene was maximally transcribed in cultured cells at 8% oxygen, unlike MNRR1, which was maximally expressed at 4%, suggesting a fine-tuned oxygen-sensing system that adapts to the varying oxygen concentrations in the human body under physiological conditions. We show that nuclear CHCHD10 protein down-regulates the expression of genes harboring the oxygen-responsive element (ORE) in their promoters by interacting with and augmenting the activity of the largely uncharacterized transcriptional repressor CXXC finger protein 5 (CXXC5). We further show that two genetic CHCHD10 disease variants, G66V and P80L, in the mitochondria exhibit faulty interactions with MNRR1 and COX, reducing respiration and increasing reactive oxygen species (ROS), and in the nucleus abrogating transcriptional repression of ORE-containing genes. Our results reveal that CHCHD10 positively regulates mitochondrial respiration and contributes to transcriptional repression of ORE-containing genes in the nucleus, and that genetic CHCHD10 variants are impaired in these activities.

Serine catabolism is essential to maintain mitochondrial respiration in mammalian cells

Mitochondrial respiratory complex assembly requires the one-carbon unit generated from serine catabolism. Breakdown of serine by the enzyme serine hydroxymethyltransferase (SHMT) produces glycine and one-carbon (1C) units. These serine catabolites provide important metabolic intermediates for the synthesis of nucleotides, as well as methyl groups for biosynthetic and regulatory methylation reactions. Recently, it has been shown that serine catabolism is required for efficient cellular respiration. Using CRISPR-Cas9 gene editing, we demonstrate that the mitochondrial SHMT enzyme, SHMT2, is essential to maintain cellular respiration, the main process through which mammalian cells acquire energy. We show that SHMT2 is required for the assembly of Complex I of the respiratory chain. Furthermore, supplementation of formate, a bona fide 1C donor, restores Complex I assembly in the absence of SHMT2. Thus, provision of 1C units by mitochondrial serine catabolism is critical for cellular respiration, at least in part by influencing the assembly of the respiratory apparatus.

MNRR1, a Biorganellar Regulator of Mitochondria

The central role of energy metabolism in cellular activities is becoming widely recognized. However, there are many gaps in our knowledge of the mechanisms by which mitochondria evaluate their status and call upon the nucleus to make adjustments. Recently, a protein family consisting of twin CX9C proteins has been shown to play a role in human pathophysiology. We focus here on two family members, the isoforms CHCHD2 (renamed MNRR1) and CHCHD10. The better studied isoform, MNRR1, has the unusual property of functioning in both the mitochondria and the nucleus and of having a different function in each. In the mitochondria, it functions by binding to cytochrome c oxidase (COX), which stimulates respiration. Its binding to COX is promoted by tyrosine-99 phosphorylation, carried out by ABL2 kinase (ARG). In the nucleus, MNRR1 binds to a novel promoter element in COX4I2 and itself, increasing transcription at 4% oxygen. We discuss mutations in both MNRR1 and CHCHD10 found in a number of chronic, mostly neurodegenerative, diseases. Finally, we propose a model of a graded response to hypoxic and oxidative stresses, mediated under different oxygen tensions by CHCHD10, MNRR1, and HIF1, which operate at intermediate and very low oxygen concentrations, respectively.