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Vermezovic J.,Instituto Fondazione Italiana Per La Ricerca Sul Cancro Of Oncologia Molecolare | Adamowicz M.,Instituto Fondazione Italiana Per La Ricerca Sul Cancro Of Oncologia Molecolare | Santarpia L.,Instituto Of Ricovero E Cura A Carattere Scientifico | Rustighi A.,Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie | And 10 more authors.
Nature Structural and Molecular Biology | Year: 2015

The DNA-damage response (DDR) ensures genome stability and proper inheritance of genetic information, both of which are essential to survival. It is presently unclear to what extent other signaling pathways modulate DDR function. Here we show that Notch receptor binds and inactivates ATM kinase and that this mechanism is evolutionarily conserved in Caenorhabditis elegans, Xenopus laevis and humans. In C. elegans, the Notch pathway impairs DDR signaling in gonad germ cells. In mammalian cells, activation of human Notch1 leads to reduced ATM signaling in a manner independent of Notch1 transcriptional activity. Notch1 binds directly to the regulatory FATC domain of ATM and inhibits ATM kinase activity. Notch1 and ATM activation are inversely correlated in human breast cancers, and inactivation of ATM by Notch1 contributes to the survival of Notch1-driven leukemia cells upon DNA damage. © 2015 Nature America, Inc. All rights reserved.


Peche L.Y.,Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie | Ladelfa M.F.,University of Buenos Aires | Toledo M.F.,University of Buenos Aires | Mano M.,International Center for Genetic Engineering and Biotechnology | And 4 more authors.
Journal of Biological Chemistry | Year: 2015

MageB2 belongs to the melanoma antigen gene (MAGE-I) family of tumor-specific antigens. Expression of this gene has been detected in human tumors of different origins. However, little is known about the protein function and how its expression affects tumor cell phenotypes. In this work, we found that human MageB2 protein promotes tumor cell proliferation in a p53-independent fashion, as observed both in cultured cells and growing tumors in mice. Gene expression analysis showed that MageB2 enhances the activity of E2F transcription factors. Mechanistically, the activation of E2Fs is related to the ability of MageB2 to interact with the E2F inhibitor HDAC1. Cellular distribution of MageB2 protein includes the nucleoli. Nevertheless, ribotoxic drugs rapidly promote its nucleolar exit.Weshow that MageB2 counter acts E2F inhibition by ribosomal proteins independently of Mdm2 expression. Importantly, MageB2 plays a critical role in impairing cell cycle arrest in response to Actinomycin D. The data presented here support a relevant function for human MageB2 in cancer cells both under cycling and stressed conditions, presenting a distinct functional feature with respect to other characterized MAGE-I proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.


PubMed | Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie, International Center for Genetic Engineering and Biotechnology and University of Trieste
Type: Journal Article | Journal: Nucleic acids research | Year: 2015

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.


Bublik D.R.,Laboratorio Nazionale del Consorzio Interuniversitario Per le Biotecnologie | Scolz M.,Laboratorio Nazionale del Consorzio Interuniversitario Per le Biotecnologie | Triolo G.,International Center for Genetic Engineering and Biotechnology | Monte M.,International Center for Genetic Engineering and Biotechnology | And 3 more authors.
Journal of Biological Chemistry | Year: 2010

p21CIP1/WAF1 belongs to the CIP/KIP family of Cdk inhibitors, and its expression is tightly controlled during the cell cycle, mainly by transcriptional and post-translational mechanisms. Fine regulation of p21 CIP1/WAF1 levels is critical for cell cycle control and for cellular response to stress. In the present work, we describe a novel mechanism to modulate p21CIP1/WAF1 levels mediated by the human GTSE-1 (G 2 and S phase-expressed-1) protein. Our results provide evidence that hGTSE-1 protects p21CIP1/WAF1 from proteasome-dependent degradation as part of a functional complex containing the Hsp90-bindingTPR protein WISp39. We further show that the hGTSE-1 N-terminal portion is sufficient for p21 CIP1/WAF1 binding and stabilization. Finally, we demonstrate that hGTSE-1 mediated-p21CIP1/WAF1 stabilization is clearly involved in the ability of cells to counter-act cytotoxicity induced by the microtubule poison paclitaxel. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Giorgi C.,University of Ferrara | Bonora M.,University of Ferrara | Sorrentino G.,Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie | Missiroli S.,University of Ferrara | And 11 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.


Antonelli R.,International School for Advanced Studies | Pizzarelli R.,International School for Advanced Studies | Pedroni A.,International School for Advanced Studies | Fritschy J.-M.,University of Zürich | And 5 more authors.
Nature Communications | Year: 2014

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2' s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABA A receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1â '/â ') associated with an increase in amplitude of spontaneous GABA A -mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction. © 2014 Macmillan Publishers Limited. All rights reserved.


Peche L.Y.,Laboratorio Nazionale Del Consorzio Interuniversitario per le Biotecnologie | Scolz M.,Laboratorio Nazionale Del Consorzio Interuniversitario per le Biotecnologie | Ladelfa M.F.,University of Buenos Aires | Monte M.,University of Buenos Aires | And 2 more authors.
Cell Death and Differentiation | Year: 2012

MAGE-A genes are a subfamily of the melanoma antigen genes (MAGEs), whose expression is restricted to tumor cells of different origin and normal tissues of the human germline. Although the specific function of individual MAGE-A proteins is being currently explored, compelling evidence suggest their involvement in the regulation of different pathways during tumor progression. We have previously reported that MageA2 binds histone deacetylase (HDAC)3 and represses p53-dependent apoptosis in response to chemotherapeutic drugs. The promyelocytic leukemia (PML) tumor suppressor is a regulator of p53 acetylation and function in cellular senescence. Here, we demonstrate that MageA2 interferes with p53 acetylation at PML-nuclear bodies (NBs) and with PMLIV-dependent activation of p53. Moreover, a fraction of MageA2 colocalizes with PML-NBs through direct association with PML, and decreases PMLIV sumoylation through an HDAC-dependent mechanism. This reduction in PML post-translational modification promotes defects in PML-NBs formation. Remarkably, we show that in human fibroblasts expressing RasV12 oncogene, MageA2 expression decreases cellular senescence and increases proliferation. These results correlate with a reduction in NBs number and an impaired p53 response. All these data suggest that MageA2, in addition to its anti-apoptotic effect, could have a novel role in the early progression to malignancy by interfering with PML/p53 function, thereby blocking the senescence program, a critical barrier against cell transformation. © 2012 Macmillan Publishers Limited. All rights reserved.


PubMed | Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie
Type: Journal Article | Journal: Cell death and differentiation | Year: 2012

MAGE-A genes are a subfamily of the melanoma antigen genes (MAGEs), whose expression is restricted to tumor cells of different origin and normal tissues of the human germline. Although the specific function of individual MAGE-A proteins is being currently explored, compelling evidence suggest their involvement in the regulation of different pathways during tumor progression. We have previously reported that MageA2 binds histone deacetylase (HDAC)3 and represses p53-dependent apoptosis in response to chemotherapeutic drugs. The promyelocytic leukemia (PML) tumor suppressor is a regulator of p53 acetylation and function in cellular senescence. Here, we demonstrate that MageA2 interferes with p53 acetylation at PML-nuclear bodies (NBs) and with PMLIV-dependent activation of p53. Moreover, a fraction of MageA2 colocalizes with PML-NBs through direct association with PML, and decreases PMLIV sumoylation through an HDAC-dependent mechanism. This reduction in PML post-translational modification promotes defects in PML-NBs formation. Remarkably, we show that in human fibroblasts expressing RasV12 oncogene, MageA2 expression decreases cellular senescence and increases proliferation. These results correlate with a reduction in NBs number and an impaired p53 response. All these data suggest that MageA2, in addition to its anti-apoptotic effect, could have a novel role in the early progression to malignancy by interfering with PML/p53 function, thereby blocking the senescence program, a critical barrier against cell transformation.


PubMed | Laboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2010

p21(CIP1/WAF1) belongs to the CIP/KIP family of Cdk inhibitors, and its expression is tightly controlled during the cell cycle, mainly by transcriptional and post-translational mechanisms. Fine regulation of p21(CIP1/WAF1) levels is critical for cell cycle control and for cellular response to stress. In the present work, we describe a novel mechanism to modulate p21(CIP1/WAF1) levels mediated by the human GTSE-1 (G(2) and S phase-expressed-1) protein. Our results provide evidence that hGTSE-1 protects p21(CIP1/WAF1) from proteasome-dependent degradation as part of a functional complex containing the Hsp90-binding TPR protein WISp39. We further show that the hGTSE-1 N-terminal portion is sufficient for p21(CIP1/WAF1) binding and stabilization. Finally, we demonstrate that hGTSE-1 mediated-p21(CIP1/WAF1) stabilization is clearly involved in the ability of cells to counteract cytotoxicity induced by the microtubule poison paclitaxel.

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