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Tatti M.,Oncology and Molecular Medicine | Motta M.,Oncology and Molecular Medicine | Motta M.,University of Rome La Sapienza | Di bartolomeo S.,University of Rome Tor Vergata | And 5 more authors.
Human Molecular Genetics | Year: 2012

Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function. © The Author 2012. Published by Oxford University Press. All rights reserved.

De Zio D.,University of Rome Tor Vergata | Cianfanelli V.,University of Rome Tor Vergata | Cecconi F.,University of Rome Tor Vergata | Cecconi F.,Laboratory of Molecular Neuroembryology
Antioxidants and Redox Signaling | Year: 2013

Significance: When lesions are unrepaired or there are defects in the DNA repair system, DNA damage is often correlated to apoptosis. However, different kinds of lesions and different degrees of lesion severity can trigger numerous signaling responses. Recent Advances: DNA repair proteins involved in specific DNA repair pathways can modulate the function or activity of some apoptotic factors, further emphasizing the crosstalk between DNA damage and cell death. Critical Issues: Here, we discuss the signaling networks that link DNA damage to apoptosis, and we focus on post-translational modifications, leading to crucial changes in protein behavior, following various kinds of DNA damage. Moreover, we analyze the existence of apoptosis-related functions of typical repair proteins, leading to diverse, often-overlapping, DNA damage responses. Future Directions: The better understanding of the regulation and the functionality of key DNA repair proteins, also involved in apoptosis regulation, has the potential of modulating the cell outcomes on DNA damage, particularly in the context of cancer treatment. Antioxid. Redox Signal. 19, 559-571. © 2013, Mary Ann Liebert, Inc.

Cianfanelli V.,Danish Cancer Society | D'Orazio M.,University of Rome Tor Vergata | Cecconi1 F.,Danish Cancer Society | Cecconi1 F.,University of Rome Tor Vergata | Cecconi1 F.,Laboratory of Molecular Neuroembryology
Cell Cycle | Year: 2015

Autophagy-promoting proteins and stimuli are often associated with inhibition of cell proliferation; in this context, we recently described a key role for the pro-autophagic protein AMBRA 1. Indeed, AMBRA1, through its direct interaction with the protein phosphatase PP2A, tightly regulates the stability of the oncoprotein and promitotic factor c-Myc. Moreover, the AMBRA1-mediated regulation of c-Myc affects both cell proliferation rate and tumorigenesis. Interestingly, AMBRA1/ PP2A activity is under the control of the master regulator of autophagy and cell growth, the protein kinase mTOR. Besides the mechanistic details of this regulation pathway which we dissected previously, any possible interplay(s) between AMBRA1 and its interactor BECLIN 1 was not investigated in this scenario. Here we show that both AMBRA1 and BECLIN 1 affect c-Myc regulation, but through two different pathways. Nevertheless, these two proautophagic proteins are, together with PP2A, in the same macromolecular complex, whose functional significance of which will be addressed in future studies © 2015 Taylor & Francis Group, LLC

Pagliarini V.,National Institute For Infectious Diseases Irccs L Spallanzani | Wirawan E.,Molecular Signalling and Cell Death Unit | Wirawan E.,Ghent University | Romagnoli A.,National Institute For Infectious Diseases Irccs L Spallanzani | And 12 more authors.
Cell Death and Differentiation | Year: 2012

Under stress conditions, pro-survival and pro-death processes are concomitantly activated and the final outcome depends on the complex crosstalk between these pathways. In most cases, autophagy functions as an early-induced cytoprotective response, favoring stress adaptation by removing damaged subcellular constituents. Moreover, several lines of evidence suggest that autophagy inactivation by the apoptotic machinery is a crucial event for cell death execution. Here we show that apoptotic stimuli induce a rapid decrease in the level of the autophagic factor Activating Molecule in Beclin1-Regulated Autophagy (Ambra1). Ambra1 degradation is prevented by concomitant inhibition of caspases and calpains. By both in vitro and in vivo approaches, we demonstrate that caspases are responsible for Ambra1 cleavage at the D482 site, whereas calpains are involved in complete Ambra1 degradation. Finally, we show that Ambra1 levels are critical for the rate of apoptosis induction. RNA interference-mediated Ambra1 downregulation further sensitizes cells to apoptotic stimuli, while Ambra1 overexpression and, more efficiently, a caspase non-cleavable mutant counteract cell death by prolonging autophagy induction. We conclude that Ambra1 is an important target of apoptotic proteases resulting in the dismantling of the autophagic machinery and the accomplishment of the cell death program. © 2012 Macmillan Publishers Limited All rights reserved.

Cianfanelli V.,Danish Cancer Society | Cianfanelli V.,Laboratory of Molecular Neuroembryology | Fuoco C.,University of Rome Tor Vergata | Lorente M.,Complutense University of Madrid | And 29 more authors.
Nature Cell Biology | Year: 2015

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene. © 2015 Macmillan Publishers Limited. All rights reserved.

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