Ablation of Atp5if1 impairs metabolic reprogramming and proliferation of T lymphocytes and compromises mouse survival.

T cells experience metabolic reprogramming to an enhanced glycolysis upon activation. Herein, we have investigated whether ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of mitochondrial ATP synthase, participates in rewiring T cells to a particular metabolic phenotype. We show that the activation of naive CD4+ T lymphocytes both in vitro and in vivo is accompanied by a sharp upregulation of IF1, which is expressed only in Th1 effector cells. T lymphocytes of conditional CD4+-IF1-knockout mice display impaired glucose uptake and flux through glycolysis, reducing the biogenesis of mitochondria and cellular proliferation after activation. Consequently, mice devoid of IF1 in T lymphocytes cannot mount an effective Th1 response against bacterial infection compromising their survival. Overall, we show that the inhibition of a fraction of ATP synthase by IF1 regulates metabolic reprogramming and functionality of T cells, highlighting the essential role of IF1 in adaptive immune responses.

Manipulating mitochondrial electron flow enhances tumor immunogenicity.

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

IF1 promotes oligomeric assemblies of sluggish ATP synthase and outlines the heterogeneity of the mitochondrial membrane potential.

The coexistence of two pools of ATP synthase in mitochondria has been largely neglected despite in vitro indications for the existence of reversible active/inactive state transitions in the F1-domain of the enzyme. Herein, using cells and mitochondria from mouse tissues, we demonstrate the existence in vivo of two pools of ATP synthase: one active, the other IF1-bound inactive. IF1 is required for oligomerization and inactivation of ATP synthase and for proper cristae formation. Immunoelectron microscopy shows the co-distribution of IF1 and ATP synthase, placing the inactive "sluggish" ATP synthase preferentially at cristae tips. The intramitochondrial distribution of IF1 correlates with cristae microdomains of high membrane potential, partially explaining its heterogeneous distribution. These findings support that IF1 is the in vivo regulator of the active/inactive state transitions of the ATP synthase and suggest that local regulation of IF1-ATP synthase interactions is essential to activate the sluggish ATP synthase.

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

The ATPase Inhibitory Factor 1 is a Tissue-Specific Physiological Regulator of the Structure and Function of Mitochondrial ATP Synthase: A Closer Look Into Neuronal Function.

The ATP synthase is an essential multifunctional enzyme complex of mitochondria that produces most of cellular ATP, shapes the structure of the inner membrane into cristae and regulates the signals that control cell fate or demise. The ATPase Inhibitory Factor 1 (IF1) functions in vivo as a physiological regulator of the ATP synthase and thereby controls mitochondrial structure and function, and the retrograde signaling pathways that reprogram nuclear gene expression. However, IF1 is not ubiquitously expressed in mammals, showing tissue-restricted expression in humans and mice and large expression differences between the two species in some tissues. Herein, we summarized key regulatory functions of IF1 for tissue homeostasis, with special emphasis on the deleterious effects that its genetic ablation in neurons has in learning. The development and characterization of tissue-specific mouse models with regulated expression of IF1 will be crucial to disentangle the contribution of the ATP synthase/IF1 axis in pathophysiology.

Analysis of the metabolic proteome of lung adenocarcinomas by reverse-phase protein arrays (RPPA) emphasizes mitochondria as targets for therapy.

Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.

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.

Reprogramming Oxidative Phosphorylation in Cancer: A Role for RNA-Binding Proteins.

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.

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.

Different mitochondrial genetic defects exhibit the same protein signature of metabolism in skeletal muscle of PEO and MELAS patients: A role for oxidative stress.

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.

The Role of Mitochondrial H+-ATP Synthase in Cancer.

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.

The mitochondrial ATP synthase is a shared drug target for aging and dementia.

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-F1 -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 ß (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.

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.