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Mauro Piacentini

Publications and source records attributed to Mauro Piacentini.

At least 19 recordsLinked to original sources
PubMed2007

Xeno-cannibalism: a survival "escamotage".

Several lines of evidence have demonstrated that self-cannibalism (macroautophagy) is a well regulated process of cell repair as well as of molecule and organelle recycling that allows the cells to survive. However, autophagic activity also represents a cell death pathway characterized by specific features that differentiate autophagy from other cell death processes. We found that cells that are able to exert intense autophagic activity were also able to engulf and digest entire cell siblings. This phenomenon represents a sort of xeno-cannibalism. We wonder whether these two phenomena, self and xeno-cannibalism, could be related the latter being an exacerbation of the first and providing a further survival option to the cells.

Autophagy↗
PubMed2007

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF.

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Calreticulin exposure dictates the immunogenicity of cancer cell death.

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Animals↗
PubMed2006

Transglutaminase 2 ablation leads to defective function of mitochondrial respiratory complex I affecting neuronal vulnerability in experimental models of extrapyramidal disorders.

Transglutaminase 2 (TG2) represents the most ubiquitous isoform belonging to the TG family, and has been implicated in the pathophysiology of basal ganglia disorders, such as Parkinson's disease and Huntington's disease. We show that ablation of TG2 in knockout mice causes a reduced activity of mitochondrial complex I associated with an increased activity of complex II in the whole forebrain and striatum. Interestingly, TG2-/- mice were protected against nigrostriatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is converted in vivo into the mitochondrial complex I inhibitor, 1-methyl-4-phenyl-pyridinium ion. In contrast, TG2-/- mice were more vulnerable to nigrostriatal damage induced by methamphetamine or by the complex II inhibitor, 3-nitropropionic acid. Proteomic analysis showed that proteins involved in the mitochondrial respiratory chain, such as prohibitin and the beta-chain of ATP synthase, are substrates for TG2. These data suggest that TG2 is involved in the regulation of the respiratory chain both in physiology and pathology, contributing to set the threshold for neuronal damage in extrapyramidal disorders.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2006

"Tissue" transglutaminase contributes to the formation of disulphide bridges in proteins of mitochondrial respiratory complexes.

In this study we provide the first in vivo evidences showing that, under physiological conditions, "tissue" transglutaminase (TG2) might acts as a protein disulphide isomerase (PDI) and through this activity contributes to the correct assembly of the respiratory chain complexes. Mice lacking TG2 exhibit mitochondrial energy production impairment, evidenced by decreased ATP levels after physical challenge. This defect is phenotypically reflected in a dramatic decrease of motor behaviour of the animals. We propose that the molecular mechanism, underlying such a phenotype, resides in a defective disulphide bonds formation in ATP synthase (complex V), NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II) and cytochrome c oxidase (complex IV). In addition, TG2-PDI might control the respiratory chain by modulating the formation of the prohibitin complexes. These data elucidate a new pathway that directly links the TG2-PDI enzymatic activity with the regulation of mitochondrial respiratory chain function.

Adenosine Triphosphate↗
PubMed2006

Cloning, expression, and preliminary structural characterization of RTN-1C.

Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.

Circular Dichroism↗
PubMed2006

Genotype-dependent priming to self- and xeno-cannibalism in heterozygous and homozygous lymphoblasts from patients with Huntington's disease.

In the present work, we studied the mitochondrial function and cell death pathway(s) in heterozygous and homozygous immortalized cell lines from patients with Huntington's disease (HD). Heterozygosis was characterized by specific alterations in mitochondrial membrane potential, a constitutive hyperpolarization state of mitochondria, and was correlated with an increased susceptibility to apoptosis. Lymphoblasts from homozygous patients, on the other hand, were characterized by a significant percentage of cells displaying autophagic vacuoles. These cells also demonstrated a striking attitude towards significant cannibalistic activity. Considering the pathogenic role of cell death in HD, our study provides new and useful insights into the role of mitochondrial dysfunction, i.e. hyperpolarization, in hijacking HD heterozygous cells towards apoptosis and HD homozygous cells towards a peculiar phenotype characterized by both self- and xeno-cannibalism. These events can, however, be viewed as an ultimate attempt to survive rather than a way to die. The present work underlines the possibility that HD-associated mitochondrial defects could tentatively be by-passed by the cells by activating cellular 'phagic' activities, including so-called 'mitophagy' and 'cannibalism', that only finally lead to cell death.

Annexin A5↗
PubMed2005

Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism.

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.

Animals↗
PubMed2005

The role of gangliosides in fenretinide-induced apoptosis of neuroblastoma.

Fenretinide is thought to induce apoptosis via increases in ceramide levels but the mechanisms of ceramide generation and the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. In SH-SY5Y neuroblastoma cells, evidence suggests that acid sphingomyelinase activity is essential for the induction of ceramide and apoptosis in response to fenretinide. Downstream of ceramide, apoptosis in response to fenretinide is mediated by increased glucosylceramide synthase activity resulting in increased levels of gangliosides GD3 and GD2 via GD3 synthase. GD3 is a key signalling intermediate leading to apoptosis via the activation of 12-Lipoxygenase, and the parallel induction of GD2 suggests that fenretinide might enhance the response of neuroblastoma to therapy with anti-GD2 antibodies.

Antineoplastic Agents↗
PubMed2005

Fenretinide: a p53-independent way to kill cancer cells.

The synthetic retinoid fenretinide [N-(4 hydroxyphenyl)retinamide] induces apoptosis of cancer cells and acts synergistically with chemotherapeutic drugs, thus providing opportunities for novel approaches to cancer therapy. The upstream signaling events induced by fenretinide include an increase in intracellular levels of ceramide, which is subsequently metabolized to GD3. This ganglioside triggers the activation of 12-Lox (12-lipoxygenase) leading to oxidative stress and apoptosis via the induction of the transcription factor Gadd153 and the Bcl-2-family member protein Bak. Increased evidence suggests that the apoptotic pathway activated by fenretinide is p53-independent and this may represent a novel way to treat tumors resistant to DNA-damaging chemotherapeutic agents. Therefore, fenretinide offers increased clinical benefit as a novel agent for cancer therapy, able to complement the action of existing chemotherapeutic treatment regimes. Furthermore, synergy between fenretinide and chemotherapeutic drugs may facilitate the use of chemotherapeutic drugs at lower concentrations, with possible reduction in treatment-associated morbidity.

Animals↗
PubMed2005

p53-A pro-apoptotic signal transducer involved in AIDS.

P53 is a well-characterized tumor suppressor protein, which can induce apoptosis, either by inducing transcription of pro-apoptotic genes or by direct effects on mitochondrial membranes. Roughly 50% of human cancers are affected by the genetic or epigenetic inactivation of p53. Recently, p53 has been incriminated to play a cardinal role in the destruction of the immune system by human immunodeficiency virus (HIV-1) infection. This suspicion is based on several lines of evidence: (i) p53 exhibits activating phosphorylations in a subset of peripheral blood mononuclear cells and lymph node cells from HIV-1 carriers; (ii) some p53 target genes (e.g., PUMA, a pro-apoptotic member of the Bcl-2 family) are overexpressed in HIV-1 carriers; (iii) in vitro, p53 and/or PUMA are rate-limiting for the induction of cell death by HIV-1 infection or, in particular, by the HIV-1 Envelope (Env), in a variety of model systems, including the apoptosis of syncytia elicited by Env or cell death induced by the Env constituent gp120. Thus, p53 may constitute a novel therapeutic target for the treatment of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Transglutaminase type II is a key element in the regulation of the anti-inflammatory response elicited by apoptotic cell engulfment.

A key feature of the macrophage-dependent clearance of apoptotic cells is the down-regulation of proinflammatory cytokines. Deficiency in the phagocytosis of apoptotic cells is often associated with the development of inflammatory reactions, resulting in chronic inflammatory and autoimmune diseases. The molecular mechanisms that regulate the engulfment process and particularly the immunomodulatory factors involved are still largely unknown in mammals. We have previously reported that the ablation of transglutaminase type II (TG2) in mice results in the defective clearance of apoptotic cells associated with the development of splenomegaly, autoantibodies, and glomerulonephritis. In this study we have investigated the mechanisms at the basis of the development of inflammation/autoimmunity associated with the defective clearance of apoptotic cells characterizing TG2 knockout mice. To this aim we compared the macrophage response to apoptotic cell exposure in wild-type vs TG2-null mice. We demonstrated that the lack of TG2 results in an impaired capacity of macrophages to engulf, but not to bind, apoptotic cells, which is paralleled by an abnormal inflammatory response both in vivo and in vitro. We have identified a differential response in the release of several cytokines in TG2(-/-) vs wild-type mice. Particularly relevant is the finding that both TGF-beta and IL-12 regulations were significantly altered in the absence of TG2. These results help explain the autoimmune phenotype developed by these mice and suggest that TG2 is a key regulatory element of the anti-inflammatory features of apoptosis.

Animals↗
PubMed2005

Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope.

The proapoptotic activity of the transcription factor p53 critically depends on the phosphorylation of serine 46 (p53S46P). Here, we show that syncytia containing p53S46P could be detected in lymph node biopsies from human immunodeficiency virus (HIV)-1 carriers, in the brain of patients with HIV-1-associated dementia and in cocultures of HeLa expressing the HIV-1 envelope glycoprotein complex (Env) with HeLa cells expressing CD4. In this latter model, cell death was the result of a sequential process involving cell fusion, nuclear fusion (karyogamy), phosphorylation of serine 15 (p53S15P), later on serine 46 (p53S46P), and transcription of p53 target genes. Cytoplasmic p38 mitogen-activated protein kinase (MAPK) was found to undergo an activating phosphorylation (p38T180/Y182P [p38 with phosphorylated threonine 180 and tyrosine 182]) before karyogamy and to translocate into karyogamic nuclei. p38T180/Y182P colocalized and coimmunoprecipitated with p53S46P. Recombinant p38 phosphorylated recombinant p53 on serine 46 in vitro. Inhibition of p38 MAPK by pharmacological inhibitors, dominant-negative p38, or small interfering RNA, suppressed p53S46P (but not p53S15P), the expression of p53-inducible genes, the conformational activation of proapoptotic Bax and Bak, the release of cytochrome c from mitochondria, and consequent apoptosis. p38T180/Y182P was also detected in HIV-1-induced syncytia, in vivo, in patients' lymph nodes and brains. Dominant-negative MKK3 or MKK6 inhibited syncytial activation of p38, p53S46P, and apoptosis. Altogether, these findings indicate that p38 MAPK-mediated p53 phosphorylation constitutes a critical step of Env-induced apoptosis.

Apoptosis↗
PubMed2005

Characterization of cell death pathways in human immunodeficiency virus-associated encephalitis.

Human immunodeficiency virus (HIV)-associated dementia is a neurodegenerative syndrome characterized by cognitive decline, personality change, and motor deficits. HIV-associated encephalitis (HAE), the neuropathology responsible for HIV-associated dementia, involves the formation of multinucleated giant cells or syncytia. In this article we describe the apoptotic pathways activated in the brains of HAE-affected patients. Approximately 50% of multinuclear giant cells exhibited apoptotic DNA fragmentation as detected by the terminal dUTP nick-end labeling technique. In addition, the presence of syncytia in the frontal cortex of approximately 35% of HAE patients correlated with the number of cells expressing the HIV-1 protein p24. Histochemical and immunohistochemical analyses revealed that HAE-associated syncytia underwent apoptosis through a mitochondrial pathway previously delineated for HIV-1 envelope-elicited syncytia in vitro. We observed over-expression of the mammalian target of rapamycin (mTOR), a kinase that mediates activation of the pro-apoptotic transcription factor p53, and p53-dependent up-regulation of two effectors of mitochondrial apoptosis, namely the BH3-only proteins Puma and transglutaminase type 2 (TG2). Interestingly, although mTOR activation and Puma induction were observed in dying syncytia and neurons, IkB phosphorylation and TG2 up-regulation were only found in syncytia. These findings provide substantial new information on the cell death mechanisms that regulate HAE, suggesting an important pathogenetic role of syncytia in the disease.

Acquired Immunodeficiency Syndrome↗
PubMed2004

AIF deficiency compromises oxidative phosphorylation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.

Adenosine Triphosphate↗
PubMed2004

Tissue transglutaminase is a multifunctional BH3-only protein.

Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the domain sequence, were able to induce Bax conformational change and translocation to mitochondria, mitochondrial depolarization, release of cytochrome c, and cell death. Moreover, we found that the TG2-BH3 peptides as well as TG2 itself were able to interact with the pro-apoptotic Bcl-2 family member Bax, but not with anti-apoptotic members Bcl-2 and Bcl-X(L). Mutants in the TG2-BH3 domain failed to sensitize cells toward apoptosis. In TG2-overexpressing cells about half of the protein is localized on the outer mitochondrial membrane where, upon cell death induction, it cross-links many protein substrates including Bax. TG2 is the first member of a new subgroup of multifunctional BH3-only proteins showing a large mass size (80 kDa) and enzymatic activity.

Amino Acid Sequence↗
PubMed2004

Gangliosides link the acidic sphingomyelinase-mediated induction of ceramide to 12-lipoxygenase-dependent apoptosis of neuroblastoma in response to fenretinide.

BACKGROUND: The lipid second messenger ceramide, which is generated by acidic and neutral sphingomyelinases or ceramide synthases, is a common intermediate of many apoptotic pathways. Metabolism of ceramide involves several enzymes, including glucosylceramide synthase and GD3 synthase, and results in the formation of gangliosides (GM3, GD3, and GT3), which in turn promote the generation of reactive oxygen species (ROS) and apoptosis. Fenretinide, a retinoic acid derivative, is thought to induce apoptosis via increases in ceramide levels, but the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. METHODS: SH-SY5Y and HTLA230 neuroblastoma cells were treated with fenretinide in the presence or absence of inhibitors of enzymes important in ceramide metabolism (fumonisin B1, inhibitor of ceramide synthase; desipramine, inhibitor of acidic and neutral sphingomyelinases; and PDMP, inhibitor of glucosylceramide). Small interfering RNAs were used to specifically block acidic sphingomyelinase or GD3 synthase activities. Apoptosis, ROS, and GD3 expression were measured by flow cytometry. RESULTS: In neuroblastoma cells, ROS generation and apoptosis were associated with fenretinide-induced increased levels of ceramide, glucosylceramide synthase activity, GD3 synthase activity, and GD3. Fenretinide also induced increased levels of GD2, a ganglioside derived from GD3. Inhibition of acidic sphingomyelinase but not of neutral sphingomyelinase or ceramide synthase, blocked fenretinide-induced increases in ceramide, ROS, and apoptosis. Exogenous GD3 induced ROS and apoptosis in SH-SY5Y cells but not in SH-SY5Y cells treated with baicalein, a specific 12-lipoxygenase inhibitor. Exogenous GD2 did not induce apoptosis. CONCLUSIONS: A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.

Antineoplastic Agents↗