TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle.

It is controversial whether mitochondrial dysfunction in skeletal muscle is the cause or consequence of metabolic disorders. Herein, we demonstrate that in vivo inhibition of mitochondrial ATP synthase in muscle alters whole-body lipid homeostasis. Mice with restrained mitochondrial ATP synthase activity presented intrafiber lipid droplets, dysregulation of acyl-glycerides, and higher visceral adipose tissue deposits, poising these animals to insulin resistance. This mitochondrial energy crisis increases lactate production, prevents fatty acid ß-oxidation, and forces the catabolism of branched-chain amino acids (BCAA) to provide acetyl-CoA for de novo lipid synthesis. In turn, muscle accumulation of acetyl-CoA leads to acetylation-dependent inhibition of mitochondrial respiratory complex II enhancing oxidative phosphorylation dysfunction which results in augmented ROS production. By screening 702 FDA-approved drugs, we identified edaravone as a potent mitochondrial antioxidant and enhancer. Edaravone administration restored ROS and lipid homeostasis in skeletal muscle and reinstated insulin sensitivity. Our results suggest that muscular mitochondrial perturbations are causative of metabolic disorders and that edaravone is a potential treatment for these diseases.

The NADPH oxidase NOX4 regulates redox and metabolic homeostasis preventing HCC progression.

BACKGROUND AND AIMS: The NADPH oxidase NOX4 plays a tumor-suppressor function in HCC. Silencing NOX4 confers higher proliferative and migratory capacity to HCC cells and increases their in vivo tumorigenic potential in xenografts in mice. NOX4 gene deletions are frequent in HCC, correlating with higher tumor grade and worse recurrence-free and overall survival rates. However, despite the accumulating evidence of a protective regulatory role in HCC, the cellular processes governed by NOX4 are not yet understood. Accordingly, the aim of this work was to better understand the molecular mechanisms regulated by NOX4 in HCC in order to explain its tumor-suppressor action. APPROACH AND RESULTS: Experimental models: cell-based loss or gain of NOX4 function experiments, in vivo hepatocarcinogenesis induced by diethylnitrosamine in Nox4 -deficient mice, and analyses in human HCC samples. Methods include cellular and molecular biology analyses, proteomics, transcriptomics, and metabolomics, as well as histological and immunohistochemical analyses in tissues. Results identified MYC as being negatively regulated by NOX4. MYC mediated mitochondrial dynamics and a transcriptional program leading to increased oxidative metabolism, enhanced use of both glucose and fatty acids, and an overall higher energetic capacity and ATP level. NOX4 deletion induced a redox imbalance that augmented nuclear factor erythroid 2-related factor 2 (Nrf2) activity and was responsible for MYC up-regulation. CONCLUSIONS: Loss of NOX4 in HCC tumor cells induces metabolic reprogramming in a Nrf2/MYC-dependent manner to promote HCC progression.

Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition.

The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling.

Effective therapeutic strategies in a preclinical mouse model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a neuropathy that lacks effective therapy. CMT patients show degeneration of peripheral nerves, leading to muscle weakness and loss of proprioception. Loss of mitochondrial oxidative phosphorylation proteins and enzymes of the antioxidant response accompany degeneration of nerves in skin biopsies of CMT patients. Herein, we followed a drug-repurposing approach to find drugs in a Food and Drug Administration-approved library that could prevent development of CMT disease in the Gdap1-null mouse model. We found that the antibiotic florfenicol is a mitochondrial uncoupler that prevents the production of reactive oxygen species and activates respiration in human GDAP1-knockdown neuroblastoma cells and in dorsal root ganglion neurons of Gdap1-null mice. Treatment of CMT-affected Gdap1-null mice with florfenicol has no beneficial effect in the course of the disease. However, administration of florfenicol, or the antioxidant MitoQ, to pre-symptomatic GDAP1-null mice prevented weight gain and ameliorated the motor coordination deficiencies that developed in the Gdap1-null mice. Interestingly, both florfenicol and MitoQ halted the decay in mitochondrial and redox proteins in sciatic nerves of Gdap1-null mice, supporting that oxidative damage is implicated in the etiology of the neuropathy. These findings support the development of clinical trials for translation of these drugs for treatment of CMT patients.

Exploiting the passenger ACO1-deficiency arising from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

Tissue-specific expression and post-transcriptional regulation of the ATPase inhibitory factor 1 (IF1) in human and mouse tissues.

The ATPase inhibitory factor 1 (IF1) is an intrinsically disordered protein that regulates the activity of the mitochondrial ATP synthase. Phosphorylation of S39 in IF1 prevents it from binding to the enzyme and thus abolishes its inhibitory activity. Dysregulation of IF1 is linked to different human diseases, providing a relevant biomarker of cancer progression. However, the tissue content of IF1 relative to the abundance of the ATP synthase is unknown. In this study, we characterized the tissue-specific expression of IF1 in human and mouse tissues and quantitated the content of IF1 and of ATP synthase. We found relevant differences in IF1 expression between human and mouse tissues and found that in high-energy-demanding tissues, the molar content of IF1 exceeds that of the ATP synthase. In these tissues, a fraction of IF1 is bound to the enzyme, and the other fraction is phosphorylated and hence is unable to bind the enzyme. Post-transcriptional control accounts for most of the regulated expression of IF1, especially in mouse heart, where IF1 mRNA translation is repressed by the leucine-rich pentatricopeptide repeat containing protein. Overall, these findings enlighten the cellular biology of IF1 and pave the way to development of additional models that address its role in pathophysiology.-Esparza-Moltó, P. B., Nuevo-Tapioles, C., Chamorro, M., Nájera, L., Torresano, L., Santacatterina, F., Cuezva, J. M. Tissue-specific expression and post-transcriptional regulation of the ATPase inhibitory factor 1 (IF1) in human and mouse tissues.

The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and proliferative response.

Recent findings indicate that prevalent human carcinomas overexpress the mitochondrial ATPase Inhibitory Factor 1 (IF1). Overexpression of IF1 inhibits the synthase activity of the mitochondrial H(+)-ATP synthase and plays a crucial role in metabolic adaptation of cancer cells to enhanced aerobic glycolysis. Herein, we demonstrate that IF1 overexpression in colon cancer cells triggers mitochondrial hyperpolarization and the subsequent production of superoxide radical, a reactive oxygen species (ROS). ROS are required to promote the transcriptional activation of the NFκB pathway via phosphorylation-dependent IκBα degradation. Activation of NFκB results in a cellular adaptive response that includes proliferation and Bcl-xL mediated resistance to drug-induced cell death. Quenching the mitochondrial production of ROS prevents the activation of NFκB and abolishes the IF1-mediated cellular adaptive response. Overall, our findings provide evidence linking the activity of a mitochondrial protein with retrograde signaling to the nucleus to promote cellular proliferation and survival.

Changes in the Turnover of the Cellular Proteome during Metabolic Reprogramming: A Role for mtROS in Proteostasis.

The role played by protein turnover in metabolic reprogramming is unknown. Herein, using a SILAC approach, we have studied the changes in the half-life of 266 proteins of energy metabolism and of translation during the metabolic switch induced by the prolyl hydroxylases inhibitor dimethyloxalylglycine (DMOG). DMOG induces HIF-1α expression and triggers the activation of glycolysis and the concurrent inhibition of mitochondrial respiration in colon cancer cells. Changes in the activity of energy provision pathways correlated with increased turnover rates of glycolytic enzymes and the stabilization of mitochondrial proteins. Moreover, reprogramming also stabilized the proteins of translation. The partial DMOG-mediated arrest of the synthesis of mitochondrial and translation proteins results from the inhibition of the mTORC1/p70SK/S6 signaling pathway. In contrast, DMOG stimulated the synthesis of glycolytic enzymes, emphasizing the opposite and differential regulation of the two pathways of energy provision. Addition of MitoQ, a mitochondrial reactive oxygen species (mtROS) scavenger, stabilized the turnover of cellular proteins similarly as when protein degradation is inhibited with leupeptin, a serine-protease inhibitor. Overall, the results show that the higher the activity of a pathway the lower is the half-life of the proteins involved and suggest a role for mtROS in cellular proteostasis. Data are available via ProteomeXchange with identifier PXD013482.

Correction to: Mitochondrial H+-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance.

Owing to an oversight, the authors omitted to note that Dr Taub is a co-founder of and equity holder in Cardero Therapeutics.

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Regulation of the H+-ATP synthase by IF1: a role in mitohormesis.

The mitochondrial H+-ATP synthase is a primary hub of cellular homeostasis by providing the energy required to sustain cellular activity and regulating the production of signaling molecules that reprogram nuclear activity needed for adaption to changing cues. Herein, we summarize findings regarding the regulation of the activity of the H+-ATP synthase by its physiological inhibitor, the ATPase inhibitory factor 1 (IF1) and their functional role in cellular homeostasis. First, we outline the structure and the main molecular mechanisms that regulate the activity of the enzyme. Next, we describe the molecular biology of IF1 and summarize the regulation of IF1 expression and activity as an inhibitor of the H+-ATP synthase emphasizing the role of IF1 as a main driver of energy rewiring and cellular signaling in cancer. Findings in transgenic mice in vivo indicate that the overexpression of IF1 is sufficient to reprogram energy metabolism to an enhanced glycolysis and activate reactive oxygen species (ROS)-dependent signaling pathways that promote cell survival. These findings are placed in the context of mitohormesis, a program in which a mild mitochondrial stress triggers adaptive cytoprotective mechanisms that improve lifespan. In this regard, we emphasize the role played by the H+-ATP synthase in modulating signaling pathways that activate the mitohormetic response, namely ATP, ROS and target of rapamycin (TOR). Overall, we aim to highlight the relevant role of the H+-ATP synthase and of IF1 in cellular physiology and the need of additional studies to decipher their contributions to aging and age-related diseases.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Mitochondrial H+-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance.

AIMS/HYPOTHESIS: Mitochondria are important regulators of the metabolic phenotype in type 2 diabetes. A key factor in mitochondrial physiology is the H+-ATP synthase. The expression and activity of its physiological inhibitor, ATPase inhibitory factor 1 (IF1), controls tissue homeostasis, metabolic reprogramming and signalling. We aimed to characterise the putative role of IF1 in mediating skeletal muscle metabolism in obesity and diabetes. METHODS: We examined the 'mitochondrial signature' of obesity and type 2 diabetes in a cohort of 100 metabolically characterised human skeletal muscle biopsy samples. The expression and activity of H+-ATP synthase, IF1 and key mitochondrial proteins were characterised, including their association with BMI, fasting plasma insulin, fasting plasma glucose and HOMA-IR. IF1 was also overexpressed in primary cultures of human myotubes derived from the same biopsies to unveil the possible role played by the pathological inhibition of the H+-ATP synthase in skeletal muscle. RESULTS: The results indicate that type 2 diabetes and obesity act via different mechanisms to impair H+-ATP synthase activity in human skeletal muscle (76% reduction in its catalytic subunit vs 280% increase in IF1 expression, respectively) and unveil a new pathway by which IF1 influences lipid metabolism. Mechanistically, IF1 altered cellular levels of α-ketoglutarate and L-carnitine metabolism in the myotubes of obese (84% of control) and diabetic (76% of control) individuals, leading to limited ß-oxidation of fatty acids (60% of control) and their cytosolic accumulation (164% of control). These events led to enhanced release of TNF-α (10 ± 2 pg/ml, 27 ± 5 pg/ml and 35 ± 4 pg/ml in control, obese and type 2 diabetic participants, respectively), which probably contributes to an insulin resistant phenotype. CONCLUSIONS/INTERPRETATION: Overall, our data highlight IF1 as a novel regulator of lipid metabolism and metabolic disorders, and a possible target for therapeutic intervention.

The ATPase Inhibitory Factor 1 (IF1): A master regulator of energy metabolism and of cell survival.

In this contribution we summarize most of the findings reported for the molecular and cellular biology of the physiological inhibitor of the mitochondrial H(+)-ATP synthase, the engine of oxidative phosphorylation (OXPHOS) and gate of cell death. We first describe the structure and major mechanisms and molecules that regulate the activity of the ATP synthase placing the ATPase Inhibitory Factor 1 (IF1) as a major determinant in the regulation of the activity of the ATP synthase and hence of OXPHOS. Next, we summarize the post-transcriptional mechanisms that regulate the expression of IF1 and emphasize, in addition to the regulation afforded by the protonation state of histidine residues, that the activity of IF1 as an inhibitor of the ATP synthase is also regulated by phosphorylation of a serine residue. Phosphorylation of S39 in IF1 by the action of a mitochondrial cAMP-dependent protein kinase A hampers its interaction with the ATP synthase, i.e., only dephosphorylated IF1 interacts with the enzyme. Upon IF1 interaction with the ATP synthase both the synthetic and hydrolytic activities of the engine of OXPHOS are inhibited. These findings are further placed into the physiological context to stress the emerging roles played by IF1 in metabolic reprogramming in cancer, in hypoxia and in cellular differentiation. We review also the implication of IF1 in other cellular situations that involve the malfunctioning of mitochondria. Special emphasis is given to the role of IF1 as driver of the generation of a reactive oxygen species signal that, emanating from mitochondria, is able to reprogram the nucleus of the cell to confer by various signaling pathways a cell-death resistant phenotype against oxidative stress. Overall, our intention is to highlight the urgent need of further investigations in the molecular and cellular biology of IF1 and of its target, the ATP synthase, to unveil new therapeutic strategies in human pathology. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis.

BACKGROUND: Metabolic alterations play a role in the development of inflammatory myopathies (IMs). Herein, we have investigated through a multiplex assay whether proteins of energy metabolism could provide biomarkers of IMs. METHODS: A cohort of thirty-two muscle biopsies and forty plasma samples comprising polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (sIBM) and control donors was interrogated with monoclonal antibodies against proteins of energy metabolism using reverse phase protein microarrays (RPPA). RESULTS: When compared to controls the expression of the proteins is not significantly affected in the muscle of PM patients. However, the expression of ß-actin is significantly increased in DM and sIBM in consistence with muscle and fiber regeneration. Concurrently, the expression of some proteins involved in glucose metabolism displayed a significant reduction in muscle of sIBM suggesting a repression of glycolytic metabolism in these patients. In contrasts to these findings, the expression of the glycolytic pyruvate kinase isoform M2 (PKM2) and of the mitochondrial ATPase Inhibitor Factor 1 (IF1) and Hsp60 were significantly augmented in DM when compared to other IMs in accordance with a metabolic shift prone to cancer development. PKM2 alone or in combination with other biomarkers allowed the discrimination of control and IMs with very high (>95%) sensitivity and specificity. Unfortunately, plasma levels of PKM2 were not significantly altered in DM patients to recommend its use as a non-invasive biomarker of the disease. CONCLUSIONS: Expression of proteins of energy metabolism in muscle enabled discrimination of patients with IMs. RPPA identified the glycolysis promoting PKM2 and IF1 proteins as specific biomarkers of dermatomyositis, providing a biochemical link of this IM with oncogenesis.

Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells.

Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activity.

RNA-binding proteins regulate cell respiration and coenzyme Q biosynthesis by post-transcriptional regulation of COQ7.

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain carrying electrons from complexes I and II to complex III and it is an intrinsic component of the respirasome. CoQ concentration is highly regulated in cells in order to adapt the metabolism of the cell to challenges of nutrient availability and stress stimuli. At least 10 proteins have been shown to be required for CoQ biosynthesis in a multi-peptide complex and COQ7 is a central regulatory factor of this pathway. We found that the first 765 bp of the 3'-untranslated region (UTR) of COQ7 mRNA contains cis-acting elements of interaction with RNA-binding proteins (RBPs) HuR and hnRNP C1/C2. Binding of hnRNP C1/C2 to COQ7 mRNA was found to require the presence of HuR, and hnRNP C1/C2 silencing appeared to stabilize COQ7 mRNA modestly. By contrast, lowering HuR levels by silencing or depriving cells of serum destabilized and reduced the half-life of COQ7 mRNA, thereby reducing COQ7 protein and CoQ biosynthesis rate. Accordingly, HuR knockdown decreased oxygen consumption rate and mitochondrial production of ATP, and increased lactate levels. Taken together, our results indicate that a reduction in COQ7 mRNA levels by HuR depletion causes mitochondrial dysfunction and a switch toward an enhanced aerobic glycolysis, the characteristic phenotype exhibited by primary deficiency of CoQ10. Thus HuR contributes to efficient oxidative phosphorylation by regulating of CoQ10 biosynthesis.

The H(+)-ATP synthase: a gate to ROS-mediated cell death or cell survival.

Cellular oxidative stress results from the increased generation of reactive oxygen species and/or the dysfunction of the antioxidant systems. Most intracellular reactive oxygen species derive from superoxide radical although the majority of the biological effects of reactive oxygen species are mediated by hydrogen peroxide. In this contribution we overview the major cellular sites of reactive oxygen species production, with special emphasis in the mitochondrial pathways. Reactive oxygen species regulate signaling pathways involved in promoting survival and cell death, proliferation, metabolic regulation, the activation of the antioxidant response, the control of iron metabolism and Ca(2+) signaling. The reversible oxidation of cysteines in transducers of reactive oxygen species is the primary mechanism of regulation of the activity of these proteins. Next, we present the mitochondrial H(+)-ATP synthase as a core hub in energy and cell death regulation, defining both the rate of energy metabolism and the reactive oxygen species-mediated cell death in response to chemotherapy. Two main mechanisms that affect the expression and activity of the H(+)-ATP synthase down-regulate oxidative phosphorylation in prevalent human carcinomas. In this context, we emphasize the prominent role played by the ATPase Inhibitory Factor 1 in human carcinogenesis as an inhibitor of the H(+)-ATP synthase activity and a mediator of cell survival. The ATPase Inhibitory Factor 1 promotes metabolic rewiring to an enhanced aerobic glycolysis and the subsequent production of mitochondrial reactive oxygen species. The generated reactive oxygen species are able to reprogram the nucleus to support tumor development by arresting cell death. Overall, we discuss the cross-talk between reactive oxygen species signaling and mitochondrial function that is crucial in determining the cellular fate. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Quantitative analysis of proteins of metabolism by reverse phase protein microarrays identifies potential biomarkers of rare neuromuscular diseases.

BACKGROUND: Muscle diseases have been associated with changes in the expression of proteins involved in energy metabolism. To this aim we have developed a number of monoclonal antibodies against proteins of energy metabolism. METHODS: Herein, we have used Reverse Phase Protein Microarrays (RPMA), a high throughput technique, to investigate quantitative changes in protein expression with the aim of identifying potential biomarkers in rare neuromuscular diseases. A cohort of 73 muscle biopsies that included samples from patients diagnosed of Duchenne (DMD), Becker (BMD), symptomatic forms of DMD and BMD in female carriers (Xp21 Carriers), Limb Girdle Muscular Dystrophy Type 2C (LGMD2C), neuronal ceroid lipofuscinosis (NCL), glycogenosis type V (Mc Ardle disease), isolated mitochondrial complex I deficiency, intensive care unit myopathy and control donors were investigated. The nineteen proteins of energy metabolism studied included members of the mitochondrial oxidation of pyruvate, the tricarboxylic acid cycle, ß-oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies. RESULTS: The results indicate that the phenotype of energy metabolism offers potential biomarkers that could be implemented to refine the understanding of the biological principles of rare diseases and, eventually, the management of these patients. CONCLUSIONS: We suggest that some biomarkers of energy metabolism could be translated into the clinics to contribute to the improvement of the clinical handling of patients affected by rare diseases.