Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Preclinical neuroblastoma models for a pharmacological study of a new MYCN oncogene inhibitor

Background. Neuroblastoma is the most deadly solid tumor of childhood. In the 25% of cases it is associated with MYCN amplification (MA), resulting in the disregulation of several genes involved in cancer progression, chemotherapy resistance and poor prognosis causing the disregulation of several genes involved in cancer progression and chemotherapy resistance and resulting in a poor prognosis. Moreover, in this contest, therapy-related p53 mutations are frequently found in relapsed cases conferring an even stronger aggressiveness. For this reason, the actual therapy requires new antitumor molecules. Therefore, rapid, accurate, and reproducible preclinical models are needed to evaluate the evolution of the different subtypes and the efficacy of new pharmacological strategies. Procedures. We report the real-time tumorigenesis of MA Neuroblastoma mouse models: transgenic TH-MYCN mice and orthotopic xenograft models with either p53wt or p53mut, by non-invasive micro PET and bioluminescent imaging, respectively. Characterization of MYCN amplification and expression was performed on every collected sample. We tested the efficacy of a new MYCN inhibitor in vitro and in vivo. Results. MicroPET in TH-MYCN mice permitted the identification of Neuroblastoma at an early stage and offered a sensitive method to follow metabolic progression of tumors. The MA orthotopic model harboring multitherapy-related p53 mutations showed a shorter latency and progression and a stronger aggressiveness respect to the p53wt model. The presence of MA and overexpression was confirmed in each model and we saw a better survival in the TH-MYCN homozigous mice treated with the inhibitor. Conclusions. The mouse models obtained show characteristics of non-invasiveness, rapidity and sensitivity that make them suitable for the in vivo preclinical study of MA-NB. In particular, our firstly reported p53mut BLI xenograft orthotopic mouse model offers the possibility to evaluate the role of multitherapy-related p53 mutations and to validate new p53 independent therapies for this highly aggressive Neuroblastoma subtype. Moreover, we have shown potential clinical suitability of an antigene strategy through its cellular and molecular activity, ability to specifically inhibit transcription and in vivo efficacy with no evidence of toxicity.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs: Toward a Personalized Therapy

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.