Fulvio Santacatterina

Expression, regulation and clinical relevance of the ATPase inhibitory factor 1 in human cancers.

Recent findings in colon cancer cells indicate that inhibition of the mitochondrial H+-adenosine triphosphate (ATP) synthase by the ATPase inhibitory factor 1 (IF1) promotes aerobic glycolysis and a reactive oxygen species (ROS)-mediated signal that enhances proliferation and cell survival. Herein, we have studied the expression, biological relevance, mechanism of regulation and potential clinical impact of IF1 in some prevalent human carcinomas. We show that IF1 is highly overexpressed in most (>90%) of the colon (n=64), lung (n=30), breast (n=129) and ovarian (n=10) carcinomas studied as assessed by different approaches in independent cohorts of cancer patients. The expression of IF1 in the corresponding normal tissues is negligible. By contrast, the endometrium, stomach and kidney show high expression of IF1 in the normal tissue revealing subtle differences by carcinogenesis. The overexpression of IF1 also promotes the activation of aerobic glycolysis and a concurrent ROS signal in mitochondria of the lung, breast and ovarian cancer cells mimicking the activity of oligomycin. IF1-mediated ROS signaling activates cell-type specific adaptive responses aimed at preventing death in these cell lines. Remarkably, regulation of IF1 expression in the colon, lung, breast and ovarian carcinomas is exerted at post-transcriptional levels. We demonstrate that IF1 is a short-lived protein (t1/2 ∼100 min) strongly implicating translation and/or protein stabilization as main drivers of metabolic reprogramming and cell survival in these human cancers. Analysis of tumor expression of IF1 in cohorts of breast and colon cancer patients revealed its relevance as a predictive marker for clinical outcome, emphasizing the high potential of IF1 as therapeutic target.

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.

Degradation of IF1 controls energy metabolism during osteogenic differentiation of stem cells

Differentiation of human mesenchymal stem cells (hMSCs) requires the rewiring of energy metabolism. Herein, we demonstrate that the ATPase inhibitory factor 1 (IF1) is expressed in hMSCs and in prostate and colon stem cells but is not expressed in the differentiated cells. IF1 inhibits oxidative phosphorylation and regulates the activity of aerobic glycolysis in hMSCs. Silencing of IF1 in hMSCs mimics the metabolic changes observed in osteocytes and accelerates cellular differentiation. Activation of IF1 degradation acts as the switch that regulates energy metabolism during differentiation. We conclude that IF1 is a stemness marker important for maintaining the quiescence state.

Down-regulation of oxidative phosphorylation in the liver by expression of the ATPase inhibitory factor 1 induces a tumor-promoter metabolic state

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.

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

BackgroundMuscle 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.MethodsHerein, 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, β-oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies.ResultsThe 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.ConclusionsWe 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.

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

BackgroundMetabolic 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.MethodsA 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).ResultsWhen 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.ConclusionsExpression 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.

Plasma metabolome and skin proteins in Charcot-Marie-Tooth 1A patients

Objective Charcot-Marie-Tooth 1A (CMT1A) disease is the most common inherited neuropathy that lacks of therapy and of molecular markers to assess disease severity. Herein, we have pursued the identification of potential biomarkers in plasma samples and skin biopsies that could define the phenotype of CMT1A patients at mild (Mi), moderate (Mo) and severe (Se) stages of disease as assessed by the CMT neuropathy score to contribute to the understanding of CMT pathophysiology and eventually inform of the severity of the disease. Methods We have used: (i) a high-throughput untargeted metabolomic approach of plasma samples in a cohort of 42 CMT1A patients and 15 healthy controls (CRL) using ultrahigh liquid chromatography coupled to mass spectrometry and (ii) reverse phase protein microarrays to quantitate the expression of some proteins of energy metabolism and of the antioxidant response in skin biopsies of a cohort of 70 CMT1A patients and 13 healthy controls. Results The metabolomic approach identified 194 metabolites with significant differences among the four groups (Mi, Mo, Se, CRL) of samples. A multivariate Linear Discriminant Analysis model using 12 metabolites afforded the correct classification of the samples. These metabolites indicate an increase in protein catabolism and the mobilization of membrane lipids involved in signaling inflammation with severity of CMT1A. A concurrent depletion of leucine, which is required for the biogenesis of the muscle, is also observed in the patients. Protein expression in skin biopsies indicates early loss of mitochondrial and antioxidant proteins in patients’ biopsies. Conclusion The findings indicate that CMT1A disease is associated with a metabolic state resembling inflammation and sarcopenia suggesting that it might represent a potential target to prevent the nerve and muscle wasting phenotype in these patients. The observed changes in metabolites could be useful as potential biomarkers of CMT1A disease after appropriate validation in future longitudinal studies.

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., Näjera, 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. FASEB J. 33, 1836–1851 (2019). www.fasebj.org

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

Abstract 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. Graphical abstract Summary of the alterations found in protein expression of metabolic and antioxidant enzymes in muscle biopsies of two independent cohorts of PEO and MELAS patients using reverse phase protein arrays. Genetic alterations are identified by red stars on nDNA and mtDNA. Red and green colors respectively denote decreased and enhanced expression of the indicated enzymes. Oxidative damage is depicted in a black background. Figure. No caption available. HighlightsQuantification of metabolic proteins in muscle of MDs patients by RPPA.Biopsies of PEO and MELAS patients portray the same proteome of metabolism.Four protein biomarkers discriminate healthy from pathological samples.Down‐regulation of SOD2 and catalase are hallmarks of PEO and MELAS samples.Oxidative damage occurs despite the induction of enzymes of the antioxidant response.