Publications

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    ABSTRACT: Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as the ideal trigger of spatio-temporally complex intracellular Ca2+ signals. However, the identity of the intracellular Ca2+ store(s) recruited by NAADP, which may include either the endolysosomal (EL) or the endoplasmic reticulum (ER) Ca2+ pools, is still elusive. Here, we show that the Ca2+ response to NAADP was suppressed by interfering with either EL or ER Ca2+ sequestration. The measurement of EL and ER Ca2+ levels by using selectively targeted aequorin unveiled that the preventing ER Ca2+ storage also affected ER Ca2+ loading and viceversa. This indicates that a functional Ca2+-mediated cross-talk exists at the EL-ER interface and exerts profound implications for the study of NAADP-induced Ca2+ signals. Extreme caution is warranted when dissecting NAADP targets by pharmacologically inhibiting EL and/or the ER Ca2+ pools. Moreover, Ca2+ transfer between these compartments might be essential to regulate vital Ca2+-dependent processes in both organelles.
    Cell Calcium. 01/2015;
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    ABSTRACT: Significance: in all cells, the endoplasmic reticulum (ER) and mitochondria are physically connected to form junctions termed mitochondria-associated membranes (MAMs). This subcellular compartment is under intense investigation because it represents a "hot spot" for the intracellular signaling of important pathways, including the synthesis of cholesterol and phospholipids, calcium homeostasis and reactive oxygen species (ROS) generation and activity. Recent advances: the advanced methods currently used to study this fascinating intracellular microdomain in detail have enabled the identification of the molecular composition of MAMs and their involvement within different physiopathological contexts. Critical issues: here we review the knowledge regarding i) MAMs composition in terms of protein composition, ii) the relationship between MAMs and ROS, iii) the involvement of MAMs in cell death programs with particular emphasis within the tumor context, iv) the emerging role of MAMs during inflammation and v) the key role of MAMs alterations in selected neurological disorders. Future directions: whether alterations in MAMs represent a response to the disease pathogenesis or directly contribute to the disease has not yet been unequivocally established. In any case, the signaling at the MAMs represent a promising pharmacological target for several important human diseases.
    Antioxidants & redox signaling. 01/2015;
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    Simone Patergnani, Paolo Pinton
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    ABSTRACT: Mitochondria are highly dynamic organelles, with a morphology ranging from small roundish elements to large interconnected networks. This fine architecture has a significant impact on mitochondrial homeostasis, and mitochondrial morphology is highly connected to specific cellular process. Autophagy is a catabolic process in which cell constituents, including proteins and organelles, are delivered to the lysosomal compartment for degradation. Autophagy has multiple physiological functions and recent advances have demonstrated that this process is linked to different human diseases, such as cancer and neurodegenerative disorders.In particular, it has been found that autophagy is a key determinant for the life span of mitochondria through a particularly fine-tuned mechanism called mitophagy, a selective form of autophagy, which ensures the preservation of healthy mitochondria through the removal of damaged or superfluous mitochondria. Mitophagy has been found to be altered in several pathologies and aberrant or excessive levels of this process are found in common human disorders. Thus, the measurement of the mitophagy levels is of fundamental relevance to elucidate the molecular mechanism of this process and, most importantly, its role in cellular homeostasis and disease.In this review, we will provide an overview of the current methods used to measure mitophagic levels, with particular emphasis on the techniques based on fluorescent probes.
    Methods in Molecular Biology 01/2015; 1241:181-94. · 1.29 Impact Factor
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    ABSTRACT: The adaptor protein p66Shc links membrane receptors to intracellular signalling pathways and has the potential to respond to energy status changes and regulate mitogenic signalling. Initially reported to mediate growth signals in normal and cancer cells, p66Shc has also been recognized as a pro-apoptotic protein involved in the cellular response to oxidative stress. Moreover, it is a key element in processes such as cancer cell proliferation, tumor progression, metastasis and metabolic reprogramming. Recent findings on the role of p66Shc in the above-mentioned processes have been obtained through the use of various tumor cell types, including prostate, breast, ovarian, lung, colon, skin and thyroid cancer cells. Interestingly, the impact of p66Shc on the proliferation rate was mainly observed in prostate tumors, while its impact on metastasis was mainly found in breast cancers. In this review, we summarize the current knowledge about the possible roles of p66Shc in different cancers.
    European Journal of Clinical Investigation 01/2015; 45(s1). · 3.37 Impact Factor
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    ABSTRACT: The ATP receptor P2X7 (P2X7R or P2RX7) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with post-mortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly compared to wild-type (wt) mice. Intratumoral IL-1ß and VEGF release were drastically reduced and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone-marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells (DCs) from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an anti-tumor immune response, and to restrict tumor growth and metastatic diffusion. Copyright © 2014, American Association for Cancer Research.
    Cancer Research 12/2014; · 9.28 Impact Factor
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    ABSTRACT: One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.
    Oncotarget 12/2014; · 6.63 Impact Factor
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    ABSTRACT: Selenoprotein N (SEPN1), is a broadly expressed resident protein of the endoplasmic reticulum (ER) whose loss-of-function inexplicably leads to human muscle disease. We found that SEPN1 levels parallel those of endoplamic reticulum oxidoreductin 1 (ERO1), an ER protein thiol oxidase, and that SEPN1's redox activity defends the ER from ERO1-generated peroxides. Moreover, we have defined the redox-regulated interactome of SEPN1 and identified the ER calcium import SERCA2 pump as a redox-partner of SEPN1. SEPN1 enhances SERCA2 activity by reducing luminal cysteines that are hyperoxidised by ERO1-generated peroxides. Cells lacking SEPN1 are hypersensitive to ERO1 overexpression and conspicuously defective in ER calcium re-uptake. After being muscle-transduced with an adeno-associated virus driving ERO1α SEPN1 knockout mice unmasks a myopathy that resembles the dense core disease due to human mutations in SEPN1, whereas the combined attenuation of ERO1α and SEPN1 enhances cell fitness. These observations reveal the involvement of SEPN1 in ER redox and calcium homeostasis and that an ERO1 inhibitor, restoring redox-dependent calcium homeostasis, may ameliorate the myopathy of SEPN1 deficiency. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Human Molecular Genetics 12/2014; · 6.68 Impact Factor
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    ABSTRACT: 2 Equipe 11 labelis ee par la Ligue Nationale contre le cancer, A lavian and colleagues recently pro-vided further evidence in support of the notion that the c subunit of the mito-chondrial F 1 F O ATP synthase constitutes the long-sought pore-forming unit of the supramolecular complex responsible for the so-called 'mitochondrial permeability transition' (MPT). Besides shedding new light on the molecular mechanisms that underlie the MPT, these findings corrob-orate the notion that several components of the cell death machinery, including cytochrome c and the F 1 F O ATP syn-thase, mediate critical metabolic activities. The term 'mitochondrial permeability transition' (MPT) is generally employed to indicate an abrupt increase of the perme-ability of the inner mitochondrial mem-brane (IMM) to small solutes. This results not only in the immediate dissipation of the mitochondrial transmembrane poten-tial (Dc m), and hence in the arrest of mito-chondrial ATP synthesis, but also in a profound ionic imbalance that provokes the osmotic breakdown of the organelle.
    Cell cycle (Georgetown, Tex.) 11/2014; 13(17):2666--2670. · 5.01 Impact Factor
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    ABSTRACT: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare autosomal dominant inherited intraepidermal blistering genodermatosis. Mutations in the ATP2C1 gene encoding for the Golgi secretory pathway Ca(2+) /Mn(2+) -ATPasi protein 1 (SPCA1) affect the processing of desmosomal components and the epidermal suprabasal cell-cell adhesion by deregulating the keratinocyte cytosolic Ca(2+) concentration. We report the unexpected, dramatic, and persistent clinical improvement of the skin lesions of a patient affected with longstanding HHD with daily intake of a solution containing magnesium chloride hexahydrate (MgCl2 ). We investigated the effect of MgCl2 on the intracellular Ca(2+) homeostasis and on the activity of particular Ca(2+) -effectors in HeLa cells transfected with chimeric aequorins (cytAEQ, mtAEQ, erAEQ and GoAEQ) targeted to different subcellular compartments (cytosol, mitochondria, endoplasmic reticulum, and Golgi, respectively). Experimental investigations on HeLa cells showed the effect of MgCl2 on the function of Ca(2+) -extrusor systems, resulting in increased cytosolic and mitochondrial Ca(2+) levels, without altering the mechanisms of intraluminal Ca(2+) -filling and Ca(2+) -release of stores. Based on our clinical observation and experimental results, it can be hypothesized that MgCl2 could act as an inhibitor of the Ca(2+) -extruding activity in keratinocytes favoring intracellular Ca(2+) -disponibility and Ca(2+) -dependent mechanisms in desmosome assembly. This may represent the molecular basis of the good response of the HHD clinical features with MgCl2 solution in the patient described. © 2014 The International Society of Dermatology.
    International journal of dermatology 11/2014; · 1.23 Impact Factor
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    Massimo Bonora, Paolo Pinton
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    ABSTRACT: Since its discovery in the 1970s, the mitochondrial permeability transition (MPT) has been proposed to be a strategic regulator of cell death. Intense research efforts have focused on elucidating the molecular components of the MPT because this knowledge may help to better understand and treat various pathologies ranging from neurodegenerative and cardiac diseases to cancer. In the case of cancer, several studies have revealed alterations in the activity of the mitochondrial permeability transition pore (mPTP) and have determined its regulatory mechanism; these studies have also suggested that suppression of the activity of the mPTP, rather than its inactivation, commonly occurs in solid neoplasms. This review focuses on the most recent advances in understanding mPTP regulation in cancer and highlights the ability of the mPTP to impede the mechanisms of cell death.
    Frontiers in Oncology 11/2014; 4:302.
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    ABSTRACT: Structural fat grafting utilizes the centrifugation of liposuction aspirates to create a graded density of adipose tissue. This study was performed to qualitatively investigate the effects of centrifugation on stem cells present in adipose tissue. Liposuction aspirates were obtained from healthy donors and either not centrifuged or centrifuged at 1,800 rpm for 3 minutes. The obtained fat volumes were divided into three layers and then analyzed. The results demonstrate that centrifugation induces a different distribution of stem cells in the three layers. The high-density layer displays the highest expression of mesenchymal stem cell and endothelial markers. The low-density layer exhibits an enrichment of multipotent stem cells. We conclude that appropriate centrifugation concentrates stem cells. This finding may influence the clinical practice of liposuction aspirate centrifugation and enhance graft uptake.
    PLoS ONE 11/2014; 9(11):e110796. · 3.53 Impact Factor
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    ABSTRACT: Renal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20–40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma.
    FEBS Open Bio. 11/2014;
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    ABSTRACT: Paroxysmal nonkinesigenic dyskinesia (PNKD) is a rare movement disorder characterized by sudden attacks of involuntary movements. Familial PNKD is an autosomal dominant trait, caused by mutations in the myofibrillogenesis regulator 1 (MR-1) gene on chromosome 2q35. Three different mutations have been described; all of them reside in the N-terminal region common to isoforms L and S, that has been suggested to code for a mitochondrial targeting sequence, necessary for the correct sub-cellular localization of the protein into mitochondria. We report on four patients of the same family, affected by PNKD. Skin fibroblasts were used to analysed oxygen consumption and to measure mitochondrial matrix calcium response after agonist stimulation. Mitotracker-based visualization was also used to assess fragmentation of the mitochondrial network. the paroxysmal movements were dystonic in two patients and dystonic/choreiform in the other ones; in three cases the symptoms started in one limb and then generalized, while in one case remained focal. Three had a very early onset, within the first two years of life. The frequency of episodes showed a great variability, ranging from 2 times a day to 3 times a year, while the duration of the attacks ranged from 2 min to 1,5 h, always with sudden onset and end and complete recover in between. All affected subjects harbored a heterozygous C to T substitution in MR-1, causing an Ala9Val amino acid change in the N-terminal region. A significant reduction of oxygen consumption and altered calcium homeostasis were found in mutant fibroblasts compared to controls, while no difference was detected in mitochondrial network. The data on reduced oxygen consumption and altered calcium homeostasis obtained on mutant fibroblasts are the first evidences, in physiological conditions, of a mitochondrial dysfunction in PNKD. Copyright © 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
    European journal of paediatric neurology: EJPN: official journal of the European Paediatric Neurology Society 10/2014; · 2.01 Impact Factor
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    Massimo Bonora, Paolo Pinton
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    ABSTRACT: Mitochondrial F1/F0 ATP synthase is one of the most intensively studied enzymatic complexes because of the nature of its molecular machinery and the role that it plays in regulating a cell's life in terms of ATP synthesis. In recent years, it has been proposed that this molecular complex could play an unexpected role in cell death, especially in the mitochondrial permeability transition pore (mPTP).
    Mitochondrion 10/2014; · 3.52 Impact Factor
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    Angela Bononi, Paolo Pinton
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    ABSTRACT: The tumor suppressor PTEN is a key regulator of a plethora of cellular processes that are crucial in cancer development. Through its lipid phosphatase activity PTEN suppresses the PI3K/AKT pathway to govern cell proliferation, growth, migration, energy metabolism and death. The repertoire of roles fulfilled by PTEN has recently been expanded to include crucial functions in the nucleus, where it favors genomic stability and restrains cell cycle progression, as well as protein phosphatase dependent activity at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), where PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and regulates Ca(2+) release from the ER and sensitivity to apoptosis. Indeed, PTEN is present in definite subcellular locations where it performs distinct functions acting on specific effectors. In this review, we summarize recent advantages in methods to study PTEN subcellular localization and the distinct biological functions of PTEN in different cellular compartments. A deeper understanding of PTEN's compartmentalized-functions will guide the rational design of novel therapies.
    Methods 10/2014; · 3.22 Impact Factor
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    Saverio Marchi, Paolo Pinton
    Frontiers in Oncology 10/2014; 4:276.
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    Guy A Rutter, Paolo Pinton
    Diabetes 10/2014; 63(10):3163-5. · 7.90 Impact Factor
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    ABSTRACT: Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as /`accidental cell death/' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. /`Regulated cell death/' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects i
    Cell death and differentiation 09/2014; · 8.24 Impact Factor
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    ABSTRACT: STAT3 mediates cytokine and growth factor receptor signalling, becoming transcriptionally active upon tyrosine 705 phosphorylation (Y-P). Constitutively Y-P STAT3 is observed in many tumors that become addicted to its activity, and STAT3 transcriptional activation is required for tumor transformation downstream of several oncogenes. We have recently demonstrated that constitutively active STAT3 drives a metabolic switch towards aerobic glycolysis through the transcriptional induction of Hif-1α and the down-regulation of mitochondrial activity, in both MEF cells expressing constitutively active STAT3 (Stat3C/C) and STAT3-addicted tumor cells. This novel metabolic function is likely involved in mediating pre-oncogenic features in the primary Stat3C/C MEFs such as resistance to apoptosis and senescence and rapid proliferation. Moreover, it strongly contributes to the ability of primary Stat3C/C MEFs to undergo malignant transformation upon spontaneous immortalization, a feature that may explain the well known causative link between STAT3 constitutive activity and tumor transformation under chronic inflammatory conditions. Taken together with the recently uncovered role of STAT3 in regulating energy metabolism from within the mitochondrion when phosphorylated on Ser 727, these data place STAT3 at the center of a hub regulating energy metabolism under different conditions, in most cases promoting cell survival, proliferation and malignant transformation even though with distinct mechanisms.
    Cancers. 09/2014; 6(3):1579-1596.
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    Saverio Marchi, Paolo Pinton
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    ABSTRACT: In this issue of the Biochemical Journal, Wu et al. describe the characterization of new low-affinity Ca 2 + indicators for monitoring Ca 2 + levels in both the ER (endoplasmic reticulum) and mitochondria. In contrast with other ER Ca 2 + sensors, these indicators emit in the red part of the spectrum, allowing for their use in combination with GFP-based probes and expanding the genetically encoded tool kit for the measurement of Ca 2 + inside organelles. Key words: aequorin, Ca 2 + measurement, endoplasmic reticulum, fluorescent Ca 2 + indicator, GCaMP, mitochondrion. Since the identification of the intense Ca 2 + connection that occurs between the ER (endoplasmic reticulum) and mitochondria, several researchers began to focus their efforts on developing accurate approaches to measure Ca 2 + in the two organelles. However, it was not long before they realized that this would not be so easy and that the generation of a Ca 2 + probe with minimal drawbacks would involve travelling a long and winding road. Indeed, monitoring Ca 2 + dynamics is considered extremely challenging, especially for the ER due to the intrinsically dynamic nature of the organelle, the complexity of the ER architecture and the difficulty of targeting sensors into the ER lumen in a specific manner. The ER is considered the major Ca 2 + storage unit inside the cell ([Ca 2 + ] ER ranges from 500 μM to 1 mM), with significant heterogeneity in Ca 2 + levels among its different regions. Upon stimulation with agonists such as histamine or ATP, the ER releases Ca 2 + through the IP 3 R (inositol 1,4,5-trisphosphate receptor), ensuring a large accumulation of Ca 2 + inside the mitochondrial matrix. Mitochondria can rapidly reach high Ca 2 +
    Biochemical Journal 08/2014; 464:5-6. · 4.78 Impact Factor

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