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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

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/δ2/2) 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 30 Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 30UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ2/2 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, MCIN/AEI/10.13039/501100011033 Grants PID2019-108349RB-100 (to J.J.S.-E. and A.C.) and SAF2016-79792R (to J.J.S.-E. and A.C.)

Significance: Cancer is a major disease imposing high personal and economic burden draining large part of National Health Care and Research budgets worldwide. In the last decade, research in cancer has underscored the reprogramming of metabolism to an enhanced aerobic glycolysis as a major trait of the cancer phenotype with great potential for targeted therapy. Recent Advances: Mitochondria are essential organelles in metabolic reprogramming for controlling the production of biological energy through oxidative phosphorylation (OXPHOS) and the supply of metabolic precursors that sustain proliferation. In addition, mitochondria are critical hubs that integrate different signaling pathways that control cellular metabolism and cell fate. The mitochondrial ATP synthase plays a fundamental role in OXPHOS and cellular signaling. Critical Issues: This review overviews mitochondrial metabolism and OXPHOS, and the major changes reported in the expression and function of mitochondrial proteins of OXPHOS in oncogenesis and in cellular differentiation. We summarize the prominent role that RNA-binding proteins (RNABPs) play in the sorting and localized translation of nuclear-encoded mRNAs that help define the mitochondrial cell-type-specific phenotype. Moreover, we emphasize the mechanisms that contribute to restrain the activity and expression of the mitochondrial ATP synthase in carcinomas, and illustrate that the dysregulation of proteins that control energy metabolism correlates with patients' survival. Future Directions: Future research should elucidate the mechanisms and RNABPs that promote the specific alterations of the mitochondrial phenotype in carcinomas arising from different tissues with the final aim of developing new therapeutic strategies to treat cancer.

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., Graphical Abstract Graphical Abstract

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 gene (ACO1), 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., Instituto de Salud Carlos III (ACCI-CIBERER-17); Spanish Ministerio de Economía y Competitividad (SAF2015-70561-R; MINECO/FEDER, EU); Spanish Ministerio de Ciencia, Innovación y Universidades (RTI2018-093330-B-I00; MCIU/FEDER, EU); Universidad Autónoma de Madrid, Spain (B2017/BMD-3778; LINFOMAS-CM); Spanish Association Against Cancer (AECC, 2018; PROYE18054PIRI); Fundación Ramón Areces (CIVP19S7917); Institutional grants from Fundación Ramón Areces and Banco de Santander

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. ©, MINECO (SAF2013-41945-R and SAF2016-75916-R), CIBERER-ISCIII, and Fundación Ramón Areces, Spain, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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., This study explores the role of ATPase inhibitory factor 1 (IF-1) in neuronal function, revealing that IF-1-mediated changes in ATP synthase modulate the production of mitochondrial reactive oxygen species, and that these changes influence synaptic transmission and cognition.

© The Author(s) 2021., SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function., This work was supported by grants from ISCIII-MCUI (FIS PI19/00934 and CIBERONCCB16/12/00352), Junta de Castilla y Leon (SA264P18-UIC 076), and Ramon Areces Foundation (CIVP19A5942). The research was co-financed by FEDER funds.

© 2020 by the authors., Background: Tumor-associated macrophages (TAMs) play a crucial role in suppressing the immunosurveillance function of the immune system that prevents tumor growth. Indeed, macrophages can also be targeted by different chemotherapeutic agents improving the action over immune checkpoints to fight cancer. Here we describe the effect of trabectedin and lurbinectedin on human macrophage cell viability and function. Methods: Blood monocytes from healthy donors were differentiated into macrophages and exposed to different stimuli promoting functional polarization and differentiation into tumor-associated macrophages. Cells were challenged with the chemotherapeutic drugs and the effects on cell viability and function were analyzed. Results: Human macrophages exhibit at least two different profiles in response to these drugs. One-fourth of the blood donors assayed (164 individuals) were extremely sensitive to trabectedin and lurbinectedin, which promoted apoptotic cell death. Macrophages from other individuals retained viability but responded to the drugs increasing reactive oxygen production and showing a rapid intracellular calcium rise and a loss of mitochondrial oxygen consumption. Cell-membrane exposure of programmed-death ligand 1 (PD-L1) significantly decreased after treatment with therapeutic doses of these drugs, including changes in the gene expression profile of hypoxia-inducible factor 1 alpha (HIF-1α)-dependent genes, among other. Conclusions: The results provide evidence of additional onco-therapeutic actions for these drugs., This research was supported by the Ministerio Economía, Industria y Competitividad (SAF2017-82436-R/RTC2017-6283-1 and PID2019-108674RB-100), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222), PharmaMar (contract 164249), Fundación Ramón Areces (CIUP18A3864), Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional.

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.

Oxidative phosphorylation (OXPHOS) generates ROS as a byproduct of mitochondrial complex I activity. ROS-detoxifying enzymes are made available through the activation of their antioxidant response elements (ARE) in their gene promoters. NRF2 binds to AREs and induces this anti-oxidant response. We show that cells from multiple origins performing OXPHOS induced NRF2 expression and its transcriptional activity. The NRF2 promoter contains MEF2 binding sites and the MAPK ERK5 induced MEF2-dependent NRF2 expression. Blocking OXPHOS in a mouse model decreased Erk5 and Nrf2 expression. Furthermore, fibroblasts derived from patients with mitochondrial disorders also showed low expression of ERK5 and NRF2 mRNAs. Notably, in cells lacking functional mitochondrial complex I activity OXPHOS did not induce ERK5 expression and failed to generate this anti-oxidant response. Complex I activity induces ERK5 expression through fumarate accumulation. Eukaryotic cells have evolved a genetic program to prevent oxidative stress directly linked to OXPHOS and not requiring ROS., Higher Education Commission, Pakistan (AK) and fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche (MESR) (DNV)

Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases., Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (FIS 12/439, RETICS RD12/009 RIER, and Miguel Servet program CP13/00014 to GC) cofinanced by European Regional Development Fund (FEDER), and by the Comunidad de Madrid (RAPHYME-CM S2010/BMD2350)

© 2020 by the authors., Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic glycolysis (Warburg effect), influencing the response to therapies. We have analyzed the expression of metabolic markers (β-F1-ATPase/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio, pyruvate kinase M2 (PKM2), oxygen consume ratio, and lactate extracellular production) in the resistance to PDT of mouse BCC cell lines (named ASZ and CSZ, heterozygous for ptch1). We have also evaluated the ability of metformin (Metf), an antidiabetic type II compound that acts through inhibition of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway to sensitize resistant cells to PDT. The results obtained indicated that resistant cells showed an aerobic glycolysis metabolism. The treatment with Metf induced arrest in the G0/G1 phase and a reduction in the lactate extracellular production in all cell lines. The addition of Metf to MAL-PDT improved the cytotoxic effect on parental and resistant cells, which was not dependent on the PS protoporphyrin IX (PpIX) production. After Metf + MAL-PDT treatment, activation of pAMPK was detected, suppressing the mTOR pathway in most of the cells. Enhanced PDT-response with Metf was also observed in ASZ tumors. In conclusion, Metf increased the response to MAL-PDT in murine BCC cells resistant to PDT with aerobic glycolysis., This research was funded by Spanish grants from Instituto de Salud Carlos III MINECO and Feder Funds (FIS PI15/00974 and PI18/00708) and Ministerio de Ciencia, Innovación y Universidades (SAF2016-75916-R).

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 β‐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., Inhibition of ATP synthase in the muscle alters whole‐body lipid metabolism and induces insulin resistance in mice, two phenotypes that can be reverted by edavarone treatment.

Genomics has contributed to the treatment of a fraction of cancer patients. However, there is a need to profile the proteins that define the phenotype of cancer and its pathogenesis. The reprogramming of metabolism is a major trait of the cancer phenotype with great potential for prognosis and targeted therapy. This review overviews the major changes reported in the steady-state levels of proteins of metabolism in primary carcinomas, paying attention to those enzymes that correlate with patients' survival. The upregulation of enzymes of glycolysis, pentose phosphate pathway, lipogenesis, glutaminolysis and the antioxidant defense is concurrent with the downregulation of mitochondrial proteins involved in oxidative phosphorylation, emphasizing the potential of mitochondrial metabolism as a promising therapeutic target in cancer. We stress that high-throughput quantitative expression profiling of differentially expressed proteins in large cohorts of carcinomas paired with normal tissues will accelerate translation of metabolism to a successful personalized medicine in cancer.

Mitochondrial metabolism has emerged as a promising target against the mechanisms of tumor growth. Herein, we have screened an FDA-approved library to identify drugs that inhibit mitochondrial respiration. The β1-blocker nebivolol specifically hinders oxidative phosphorylation in cancer cells by concertedly inhibiting Complex I and ATP synthase activities. Complex I inhibition is mediated by interfering the phosphorylation of NDUFS7. Inhibition of the ATP synthase is exerted by the overexpression and binding of the ATPase Inhibitory Factor 1 (IF1) to the enzyme. Remarkably, nebivolol also arrests tumor angiogenesis by arresting endothelial cell proliferation. Altogether, targeting mitochondria and angiogenesis triggers a metabolic and oxidative stress crisis that restricts the growth of colon and breast carcinomas. Nebivolol holds great promise to be repurposed for the treatment of cancer patients., FPI-MINECO and Fondo Social Europeo. L.F. received support from the Ramón y Cajal Program (RyC-2013-13693). The work was supported by grants from MINECO (SAF2016-75916-R and PID2019-108674RB-100), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces

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.

Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.

Background Binding of the programmed death-1 (PD-1) receptor to its ligands (PD-L1/2) transduces inhibitory signals that promote exhaustion of activated T cells. Blockade of the PD-1 pathway is widely used for cancer treatment, yet the inhibitory signals transduced by PD-1 in T cells remain elusive. Methods Expression profiles of human CD8+ T cells in resting, activated (CD3 + CD28) and PD-1-stimulated cells (CD3 + CD28 + PD-L1-Fc) conditions were evaluated by RNA-seq. Bioinformatic analyses were used to identify signaling pathways differentially regulated in PD-1-stimulated cells. Metabolic analyses were performed with SeaHorse technology, and mitochondrial ultrastructure was determined by transmission electron microscopy. PD-1-regulated mitochondrial genes were silenced using short-hairpin RNA in primary cells. Blue native gel electrophoresis was used to determine respiratory supercomplex assembly. Results PD-1 engagement in human CD8+ T cells triggers a specific, progressive genetic program different from that found in resting cells. Gene ontology identified metabolic processes, including glycolysis and oxidative phosphorylation (OXPHOS), as the main pathways targeted by PD-1. We observed severe functional and structural alterations in the mitochondria of PD-1-stimulated cells, including a reduction in the number and length of mitochondrial cristae. These cristae alterations were associated with reduced expression of CHCHD3 and CHCHD10, two proteins that form part of the mitochondrial contact site and cristae organizing system (MICOS). Although PD-1-stimulated cells showed severe cristae alterations, assembly of respiratory supercomplexes was unexpectedly greater in these cells than in activated T cells. CHCHD3 silencing in primary CD8+ T cells recapitulated some effects induced by PD-1 stimulation, including reduced mitochondrial polarization and interferon-γ production following T cell activation with anti-CD3 and -CD28 activating antibodies. Conclusions Our results suggest that mitochondria are the main targets of PD-1 inhibitory activity. PD-1 reprograms CD8+ T cell metabolism for efficient use of fatty acid oxidation; this mitochondrial phenotype might explain the long-lived phenotype of PD-1-engaged T cells. Electronic supplementary material The online version of this article (10.1186/s40425-019-0628-7) contains supplementary material, which is available to authorized users.

The ATPase Inhibitory Factor 1 (IF1) is an inhibitor of the mitochondrial H+-ATP synthase that regulates the activity of both oxidative phosphorylation (OXPHOS) and cell death. Here, we have developed transgenic Tet-On and Tet-Off mice that express a mutant active form of hIF1 in the hepatocytes to restrain OXPHOS in the liver to investigate the relevance of mitochondrial activity in hepatocarcinogenesis. The expression of hIF1 promotes the inhibition of OXPHOS in both Tet-On and Tet- Off mouse models and induces a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK. Expression of the transgene significantly augmented proliferation and apoptotic resistance of carcinoma cells, which contributed to an enhanced diethylnitrosamine-induced liver carcinogenesis. Moreover, the expression of hIF1 also diminished acetaminophen-induced apoptosis, which is unrelated to differences in permeability transition pore opening. Mechanistically, cell survival in hIF1-preconditioned hepatocytes results from a nuclear factor-erythroid 2-related factor (Nrf2)-guided antioxidant response. The results emphasize in vivo that a metabolic phenotype with a restrained OXPHOS in the liver is prone to the development of cancer, MINECO (SAF2013-41945-R to JMC and SAF2012-33243 to AMV), CIBERER-ISCIII to JMC and CIBERDEM-ISCIII to AMV, Comunidad de Madrid (S2011/BMD-2402 to JMC and S2010/BMD-2423 to AMV), Fundación Ramón Areces (FRA)

© 2015 Elsevier B.V. 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., Instituto de Salud Carlos III (FIS 09/00185). G. Ferrín was supported by the Networked Biomedical Research Center Hepatic and Digestive Diseases (CIBEREHD)

A major challenge in mitochondrial diseases (MDs) is the identification of biomarkers that could inform of the mechanisms involved in the phenotypic expression of genetic defects. Herein, we have investigated the protein signature of metabolism and of the antioxidant response in muscle biopsies of clinically and genetically diagnosed patients with Progressive External Ophthalmoplegia due to single large-scale (PEO-sD) or multiple (PEO-mD) deletions of mtDNA and Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episode (MELAS) syndrome, and healthy donors. A high-throughput immunoassay technique that quantitates the expression of relevant proteins of glycolysis, glycogenolysis, pentose phosphate pathway, oxidative phosphorylation, pyruvate and fatty acid oxidation, tricarboxylic acid cycle and the antioxidant response in two large independent and retrospectively collected cohorts of PEO-sD, PEO-mD and MELAS patients revealed that despite the heterogeneity of the genetic alterations, the three MDs showed the same metabolic signatures in both cohorts of patients, which were highly divergent from those of healthy individuals. Linear Discriminant Analysis and Support Vector Machine classifier provided a minimum of four biomarkers to discriminate healthy from pathological samples. Regardless of the induction of a large number of enzymes involved in ameliorating oxidative stress, the down-regulation of mitochondrial superoxide dismutase (SOD2) and catalase expression favored the accumulation of oxidative damage in patients’ proteins. Down-regulation of SOD2 and catalase expression in MD patients is not due to relevant changes in the availability of their mRNAs, suggesting that oxidative stress regulates the expression of the two enzymes post-transcriptionally. We suggest that SOD2 and catalase could provide specific targets to improve the detoxification of reactive oxygen species that affects muscle proteins in these patients.

Cancer cells reprogram energy metabolism by boosting aerobic glycolysis as a main pathway for the provision of metabolic energy and of precursors for anabolic purposes. Accordingly, the relative expression of the catalytic subunit of the mitochondrial H-ATP synthase-the core hub of oxidative phosphorylation-is downregulated in human carcinomas when compared with its expression in normal tissues. Moreover, some prevalent carcinomas also upregulate the ATPase inhibitory factor 1 (IF1), which is the physiological inhibitor of the H-ATP synthase. IF1 overexpression, both in cells in culture and in tissue-specific mouse models, is sufficient to reprogram energy metabolism to an enhanced glycolysis by limiting ATP production by the H-ATP synthase. Furthermore, the IF1-mediated inhibition of the H-ATP synthase promotes the production of mitochondrial ROS (mtROS). mtROS modulate signaling pathways favoring cellular proliferation and invasion, the activation of antioxidant defenses, resistance to cell death, and modulation of the tissue immune response, favoring the acquisition of several cancer traits. Consistently, IF1 expression is an independent marker of cancer prognosis. By contrast, inhibition of the H-ATP synthase by α-ketoglutarate and the oncometabolite 2-hydroxyglutarate, reduces mTOR signaling, suppresses cancer cell growth, and contributes to lifespan extension in several model organisms. Hence, the H-ATP synthase appears as a conserved hub in mitochondria-to-nucleus signaling controlling cell fate. Unraveling the molecular mechanisms responsible for IF1 upregulation in cancer and the signaling cascades that are modulated by the H-ATP synthase are of utmost interest to decipher the metabolic and redox circuits contributing to cancer origin and progression., MINECO (SAF2016-75916-R), CIBERER-ISCIII, and Fundación Ramón Areces, Spain.

Aging is a major driving force underlying dementia, such as that caused by Alzheimer's disease (AD). While the idea of targeting aging as a therapeutic strategy is not new, it remains unclear how closely aging and age-associated diseases are coupled at the molecular level. Here, we discover a novel molecular link between aging and dementia through the identification of the molecular target for the AD drug candidate J147. J147 was developed using a series of phenotypic screening assays mimicking disease toxicities associated with the aging brain. We have previously demonstrated the therapeutic efficacy of J147 in several mouse models of AD. Here, we identify the mitochondrial α-F-ATP synthase (ATP5A) as a target for J147. By targeting ATP synthase, J147 causes an increase in intracellular calcium leading to sustained calcium/calmodulin-dependent protein kinase kinase β (CAMKK2)-dependent activation of the AMPK/mTOR pathway, a canonical longevity mechanism. Accordingly, modulation of mitochondrial processes by J147 prevents age-associated drift of the hippocampal transcriptome and plasma metabolome in mice and extends lifespan in drosophila. Our results link aging and age-associated dementia through ATP synthase, a molecular drug target that can potentially be exploited for the suppression of both. These findings demonstrate that novel screens for new AD drug candidates identify compounds that act on established aging pathways, suggesting an unexpectedly close molecular relationship between the two., NIH R01AG046153 (D.S) and NIH/NIA SBIR 2R44AG033427 (K.F. and E.R.), the Nomis Foundation (AC), AI104034 and the Della Thome Foundation (PM), Bundy Foundation (DD), the Hewitt Foundation (JG), the Paul F. Glenn Center for Aging Research at the Salk Institute (JG)

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 β-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.

Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model. Keywords: Petiveria alliacea, Breast cancer, β-F1-ATPase, Respiration, ATP depletion

Increasing evidences show that the ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of the ATP synthase, is overexpressed in a large number of carcinomas contributing to metabolic reprogramming and cancer progression. Herein, we show that in contrast to the findings in other carcinomas, the overexpression of IF1 in a cohort of colorectal carcinomas (CRC) predicts less chances of disease recurrence, IF1 being an independent predictor of survival. Bioinformatic and gene expression analyses of the transcriptome of colon cancer cells with differential expression of IF1 indicate that cells overexpressing IF1 display a less aggressive behavior than IF1 silenced (shIF1) cells. Proteomic and functional in vitro migration and invasion assays confirmed the higher tumorigenic potential of shIF1 cells. Moreover, shIF1 cells have increased in vivo metastatic potential. The higher metastatic potential of shIF1 cells relies on increased cFLIP-mediated resistance to undergo anoikis after cell detachment. Furthermore, tumor spheroids of shIF1 cells have an increased ability to escape from immune surveillance by NK cells. Altogether, the results reveal that the overexpression of IF1 acts as a tumor suppressor in CRC with an important anti-metastatic role, thus supporting IF1 as a potential therapeutic target in CRC., Ministerio de Ciencia, Innovación y Universidades (SAF2013-41945-R, SAF2016-75916-R and SAF2016-75452-R), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces, Spain

Partial suppression of mitochondrial oxidative phosphorylation and the concurrent activation of aerobic glycolysis is a hallmark of proliferating cancer cells. Overexpression of the ATPase inhibitory factor 1 (IF1), an in vivo inhibitor of the mitochondrial ATP synthase, is observed in most prevalent human carcinomas favoring metabolic rewiring to an enhanced glycolysis and cancer progression. Consistently, a high expression of IF1 in hepatocarcinomas and in carcinomas of the lung, bladder, and stomach and in gliomas is a biomarker of bad patient prognosis. In contrast to these findings, we have previously reported that a high expression level of IF1 in breast carcinomas is indicative of less chance to develop metastatic disease. This finding is especially relevant in the bad prognosis group of patients bearing triple-negative breast carcinomas. To investigate the molecular mechanisms that underlie the differential behavior of IF1 in breast cancer progression, we have developed the triple-negative BT549 breast cancer cell line that overexpresses IF1 stably. When compared to controls, IF1-cells partially shut down respiration and enhance aerobic glycolysis. Transcriptomic analysis suggested that migration and invasion were specifically inhibited in IF1-overexpressing breast cancer cells. Analysis of gene expression by qPCR and western blotting indicate that IF1 overexpression supports the maintenance of components of the extracellular matrix (ECM) and E-cadherin concurrently with the downregulation of components and signaling pathways involved in epithelial to mesenchymal transition. The overexpression of IF1 in breast cancer cells has no effect in the rates of cellular proliferation and in the cell death response to staurosporine and hydrogen peroxide. However, the overexpression of IF1 significantly diminishes the ability of the cells to grow in soft agar and to migrate and invade when compared to control cells. Overall, the results indicate that IF1 overexpression despite favoring a metabolic phenotype prone to cancer progression in the specific case of breast cancer cells also promotes the maintenance of the ECM impeding metastatic disease. These findings hence provide a mechanistic explanation to the better prognosis of breast cancer patients bearing tumors with high expression level of IF1., CN-T and IM-R were supported by pre-doctoral FPI-MEC and JAE-CSIC fellowships, respectively. This work was supported by grants from the Ministerio de Economía y Competitividad (SAF2013-41945-R; SAF2016-75916-R), Comunidad Madrid (S2011/BMD-2402), and Fundación Ramón Areces 2015, Spain.

[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 β-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., FS was supported by a pre-doctoral fellowship from FPI-UAM Spain. The work was supported by Grants from the Ministerio de Economía y Competitividad (SAF2013-41945-R; SAF2016-75916-R), Fundación Ramón Areces (FRA), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Fundación CELLEX and Comunidad de Madrid (S2011/BMD-2402), Spain. The CBMSO receives an institutional grant from FRA.

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.

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.

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