Mitochondrial sAC-cAMP-PKA Axis Modulates the ΔΨm-Dependent Control Coefficients of the Respiratory Chain Complexes: Evidence of Respirasome Plasticity.

The current view of the mitochondrial respiratory chain complexes I, III and IV foresees the occurrence of their assembly in supercomplexes, providing additional functional properties when compared with randomly colliding isolated complexes. According to the plasticity model, the two structural states of the respiratory chain may interconvert, influenced by the intracellular prevailing conditions. In previous studies, we suggested the mitochondrial membrane potential as a factor for controlling their dynamic balance. Here, we investigated if and how the cAMP/PKA-mediated signalling influences the aggregation state of the respiratory complexes. An analysis of the inhibitory titration profiles of the endogenous oxygen consumption rates in intact HepG2 cells with specific inhibitors of the respiratory complexes was performed to quantify, in the framework of the metabolic flux theory, the corresponding control coefficients. The attained results, pharmacologically inhibiting either PKA or sAC, indicated that the reversible phosphorylation of the respiratory chain complexes/supercomplexes influenced their assembly state in response to the membrane potential. This conclusion was supported by the scrutiny of the available structure of the CI/CIII2/CIV respirasome, enabling us to map several PKA-targeted serine residues exposed to the matrix side of the complexes I, III and IV at the contact interfaces of the three complexes.

Molecular chaperone function of three small heat-shock proteins from a model probiotic species.

Small heat-shock proteins (sHSP) are ubiquitous ATP-independent chaperones that prevent irreversible aggregation of heat-damaged denaturing proteins. Lactiplantibacillus plantarum is a widespread Gram-positive bacterium with probiotic claims and vast potential for agro-food, biotechnological and biomedical applications. L. plantarum possesses a family of three sHSP, which were previously demonstrated to be involved in its stress tolerance mechanisms. Here, the three L. plantarum sHSP were heterologously expressed, purified and shown to have a chaperone activity in vitro, measuring their capacity to suppress protein aggregation, as assayed spectrophotometrically by light scattering. Their anti-aggregative capacity was found to be differently influenced by pH. Differences were also found relative to their holdase function and their capacity to modulate liposome membrane fluidity, suggesting interplays between them and indicating diversified activities. This is the first study assessing the chaperone action of sHSP from a probiotic model. The different roles of the three sHSP can increase L. plantarum's capabilities to survive the various types of stress characterising the diverse habitats of this highly adaptable species. Reported evidence supports the interest in L. plantarum as one of the model species for bacteria that have three different sHSP-encoding genes in their genomes.

Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments.

BACKGROUND: The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues. METHODS: MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs. RESULTS: As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells. CONCLUSIONS: Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.

On the Origin of Hemoglobin Cooperativity under Non-equilibrium Conditions.

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of red blood cells through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium oxygen dissociation curve and show that under these conditions the true nature of hemoglobin cooperativity is revealed as emerging solely from the consecutive binding of oxygen to each one of the four subunits of hemoglobin until the entire tetramer is saturated. We call this form of cooperativity the sequential cooperativity of hemoglobin and define the simplest model that includes it as the minimalist model of hemoglobin. A single instantiation of this model accounts for ~70% of hemoglobin cooperativity under non-equilibrium conditions. The total cooperativity of hemoglobin can be viewed more correctly as the summation of two instantiations of the minimalist model (each one corresponding to a tetramer of low and high affinity for O2, respectively) in equilibrium with each other, as in the Monod-Wyman-Changeux model of hemoglobin. In addition to offering new insights on the nature of hemoglobin reaction with oxygen, the methodology described here for the determination of hemoglobin non-equilibrium oxygen dissociation curve provides a simple, fast, low-cost alternative to complex spectrophotometric methods, which is expected to be particularly valuable in regions where hemoglobinopathies are a significant public health problem, but where highly specialized laboratories capable of determining a traditional oxygen dissociation curve are not easily accessible.

Pathogenic DNM1L Variant (1085G>A) Linked to Infantile Progressive Neurological Disorder: Evidence of Maternal Transmission by Germline Mosaicism and Influence of a Contemporary in cis Variant (1535T>C).

Mitochondria are dynamic organelles undergoing continuous fusion and fission with Drp1, encoded by the DNM1L gene, required for mitochondrial fragmentation. DNM1L dominant pathogenic variants lead to progressive neurological disorders with early exitus. Herein we report on the case of a boy affected by epileptic encephalopathy carrying two heterozygous variants (in cis) of the DNM1L gene: a pathogenic variant (PV) c.1085G>A (p.Gly362Asp) accompanied with a variant of unknown significance (VUS) c.1535T>C (p.Ile512Thr). Amplicon sequencing of the mother's DNA revealed the presence of the PV and VUS in 5% of cells, with the remaining cells presenting only VUS. Functional investigations performed on the patient and his mother's cells unveiled altered mitochondrial respiratory chain activities, network architecture and Ca2+ homeostasis as compared with healthy unrelated subjects' samples. Modelling Drp1 harbouring the two variants, separately or in combination, resulted in structural changes as compared with Wt protein. Considering the clinical history of the mother, PV transmission by a maternal germline mosaicism mechanism is proposed. Altered Drp1 function leads to changes in the mitochondrial structure and bioenergetics as well as in Ca2+ homeostasis. The novel VUS might be a modifier that synergistically worsens the phenotype when associated with the PV.

TRAP1 enhances Warburg metabolism through modulation of PFK1 expression/activity and favors resistance to EGFR inhibitors in human colorectal carcinomas.

Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient-derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18 F-fluoro-2-deoxy-glucose (18 F-FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient-derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase-1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS-wild-type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity in vitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18 F-FDG PET uptake and poor response to cetuximab in first-line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism.

Mitochondrial calcium drives clock gene-dependent activation of pyruvate dehydrogenase and of oxidative phosphorylation.

Regulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In a previous study we showed that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as here documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork.

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

Nandrolone induces a stem cell-like phenotype in human hepatocarcinoma-derived cell line inhibiting mitochondrial respiratory activity.

Nandrolone is a testosterone analogue with anabolic properties commonly abused worldwide, recently utilized also as therapeutic agent in chronic diseases, cancer included. Here we investigated the impact of nandrolone on the metabolic phenotype in HepG2 cell line. The results attained show that pharmacological dosage of nandrolone, slowing cell growth, repressed mitochondrial respiration, inhibited the respiratory chain complexes I and III and enhanced mitochondrial reactive oxygen species (ROS) production. Intriguingly, nandrolone caused a significant increase of stemness-markers in both 2D and 3D cultures, which resulted to be CxIII-ROS dependent. Notably, nandrolone negatively affected differentiation both in healthy hematopoietic and mesenchymal stem cells. Finally, nandrolone administration in mice confirmed the up-regulation of stemness-markers in liver, spleen and kidney. Our observations show, for the first time, that chronic administration of nandrolone, favoring maintenance of stem cells in different tissues would represent a precondition that, in addition to multiple hits, might enhance risk of carcinogenesis raising warnings about its abuse and therapeutic utilization.

Parkin Mutation Affects Clock Gene-Dependent Energy Metabolism.

Growing evidence highlights a tight connection between circadian rhythms, molecular clockworks, and mitochondrial function. In particular, mitochondrial quality control and bioenergetics have been proven to undergo circadian oscillations driven by core clock genes. Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a selective loss of dopaminergic neurons. Almost half of the autosomal recessive forms of juvenile parkinsonism have been associated with mutations in the PARK2 gene coding for parkin, shown to be involved in mitophagy-mediated mitochondrial quality control. The aim of this study was to investigate, in fibroblasts from genetic PD patients carrying parkin mutations, the interplay between mitochondrial bioenergetics and the cell autonomous circadian clock. Using two different in vitro synchronization protocols, we demonstrated that normal fibroblasts displayed rhythmic oscillations of both mitochondrial respiration and glycolytic activity. Conversely, in fibroblasts obtained from PD patients, a severe damping of the bioenergetic oscillatory patterns was observed. Analysis of the core clock genes showed deregulation of their expression patterns in PD fibroblasts, which was confirmed in induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) derived thereof. The results from this study support a reciprocal interplay between the clockwork machinery and mitochondrial energy metabolism, point to a parkin-dependent mechanism of regulation, and unveil a hitherto unappreciated level of complexity in the pathophysiology of PD and eventually other neurodegenerative diseases.

The Circadian Clock Regulates Metabolic Phenotype Rewiring Via HKDC1 and Modulates Tumor Progression and Drug Response in Colorectal Cancer.

An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock-regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy.

Targeting Endoplasmic Reticulum and/or Mitochondrial Ca2+ Fluxes as Therapeutic Strategy for HCV Infection.

Chronic hepatitis C is characterized by metabolic disorders and by a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that can in the long term lead to liver cirrhosis and hepatocellular carcinoma. Several lines of evidence suggest that mitochondrial dysfunctions play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV) proteins also localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory and need to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems. In the past decade we have been proposing a temporal sequence of events in the HCV-infected cell whereby the primary alteration is localized at the mitochondria-associated ER membranes and causes release of Ca2+ from the ER, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting in decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis. Here we resume the major results provided by our group in the context of HCV-mediated alterations of the cellular inter-compartmental calcium flux homeostasis and present new evidence suggesting targeting of ER and/or mitochondrial calcium transporters as a novel therapeutic strategy.

Febrile temperature reprograms by redox-mediated signaling the mitochondrial metabolic phenotype in monocyte-derived dendritic cells.

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3h exposure at 39°C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca2+ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift.

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control.

Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles.

An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism.

Possible Mechanisms of Mercury Toxicity and Cancer Promotion: Involvement of Gap Junction Intercellular Communications and Inflammatory Cytokines.

A number of observations indicate that heavy metals are able to alter cellular metabolic pathways through induction of a prooxidative state. Nevertheless, the outcome of heavy metal-mediated effects in the development of human diseases is debated and needs further insights. Cancer is a well-established DNA mutation-linked disease; however, epigenetic events are perhaps more important and harmful than genetic alterations. Unfortunately, we do not have reliable screening methods to assess/validate the epigenetic (promoter) effects of a physical or a chemical agent. We propose a mechanism of action whereby mercury acts as a possible promoter carcinogen. In the present contribution, we resume our previous studies on mercury tested at concentrations comparable with its occurrence as environmental pollutant. It is shown that Hg(II) elicits a prooxidative state in keratinocytes linked to inhibition of gap junction-mediated intercellular communication and proinflammatory cytokine production. These combined effects may on one hand isolate cells from tissue-specific homeostasis promoting their proliferation and on the other hand tamper the immune system defense/surveillance checkmating the whole organism. Since Hg(II) is not a mutagenic/genotoxic compound directly affecting gene expression, in a broader sense, mercury might be an example of an epigenetic tumor promoter or, further expanding this concept, a "metagenetic" effector.

Clock-genes and mitochondrial respiratory activity: Evidence of a reciprocal interplay.

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. 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.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

The iron chelator deferasirox affects redox signalling in haematopoietic stem/progenitor cells.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (ß-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.