Rita Roberti - Academia.edu (original) (raw)

Papers by Rita Roberti

57° Congresso Nazionale Società Italiana di Biochimica e Biologia Molecolare (SIB), 2013

Journal of Proteomics, 2017

Glioblastoma (GBM) are the most common and aggressive brain tumours of adults. The metabolic phen... more Glioblastoma (GBM) are the most common and aggressive brain tumours of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Ourresults show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. Significance Alteration of the glycolytic pathway characterize glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-Bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug.

Lipids, 1975

The transfer of radioactivity from cytidine-5'-diphosphate ethanolamine into 1-alk y 1-2-acyl-sn-... more The transfer of radioactivity from cytidine-5'-diphosphate ethanolamine into 1-alk y 1-2-acyl-sn-glycerophosphorylethanolamine of neuronal and glial cells from adult rabbit brain cortex has been investigated in vitro. The synthesis of 1-alkyl-2a c yl-s n-gly cerophosphorylethanolamine in both cell populations was stimulated 23-25-fold by the addition of 6 mM alkylacylglycerol. The neuronal cell-enriched fraction was found to possess]unit protein a 1.7-1.8-fold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction, when saturating concentrations (6 mM) of alkylacylglycerols were added in the incubation system. The neuronal/glial ratio was 2.6-2.8 in the absence of lipid acceptor or with low concentrations of alkylacylglycerol. Under most favorable conditions, 6.4 and 3.3 nmoles 1-alkyl-2a c y l-sn-gly cerophosphorylethanolamine/rag protein/30 min was obtained for neurons and glia, respectively. Various kinetic properties of the 1-alkyl-2-acylsn-glycerophosphorylethanolamine synthesizing phosphotransferase activity were found to be similar both in neurons and glia.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocatio... more The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacyiglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:.4(n-6)) into ethanolamine phospholipids, known to he catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may he translocated from the outer to the inner leaflet at different rates.

Analytical Biochemistry, 1990

Two alternative procedures are described for the quantitative determination of phosphatidylcholin... more Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.

Antonie van Leeuwenhoek, 2015

A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil... more A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000-to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000-to 64,000fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index-R FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a doseresponse effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.

FEBS Journal, 2008

In cholesterol biosynthesis, lanosterol undergoes removal of the methyl group at C14, leading to ... more In cholesterol biosynthesis, lanosterol undergoes removal of the methyl group at C14, leading to the formation of C14-C15 unsaturated sterol intermediates. The enzymatic activity responsible for the reduction of the introduced double-bond, 3b-hydroxysterol D 14-reductase (EC 1.3.1.70), is carried out by the endoplasmic reticulum (ER) protein delta14-sterol reductase (C14SR) encoded by the TM7SF2 gene

Chemistry and Physics of Lipids, 2008

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2006

3β-hydroxysterol Δ 14-reductase operates during the conversion of lanosterol to cholesterol in ma... more 3β-hydroxysterol Δ 14-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3β-hydroxysterol Δ 14-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3β-hydroxysterol Δ 14-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3β-hydroxysterol Δ 14-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Δ 14-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3β-hydroxysterol Δ 14-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4-and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3β-hydroxysterol Δ 14-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2010

TM7SF2 gene encodes 3β-hydroxysterol Δ 14-reductase, responsible for the reduction of C14-unsatur... more TM7SF2 gene encodes 3β-hydroxysterol Δ 14-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoterluciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in cotransfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Journal of Lipid Research, 1980

Selectivity of CDP-choline: diacylglycerol choline phosphotransferase and CDP-ethanolamine: diacy... more Selectivity of CDP-choline: diacylglycerol choline phosphotransferase and CDP-ethanolamine: diacylglycerol ethanolamine phosphotransferase for molecular species of diglyceride has been studied in rat brain microsomes in vitro. Diglyceride-labeled microsomes were prepared by incubation with labeled sn-glycerol-3-phosphate; the microsomes were then incubated with CDP-choline or CDP-ethanolamine for different time intervals. Experimental data extrapolated to zero-time incubation were taken into account for evaluating species specificity. A small selectivity for diglyceride species has been demonstrated for the choline phosphotransferase, but the ethanolamine phosphotransferase was found to convert hexaenoic diglyceride into phospholipid at the highest rate.-Robed, R., L. Binaglia, and G. Porcellati. Synthesis of molecular species of glycerophospholipids from diglyceride-labeled brain microsomes. J. Lipid Res.

Encyclopedia of Cancer, 2015

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987

Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosph... more Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) was solubilized by treating rat brain microsomes with buffered solutions containing octyl glucoside or Triton X-100. The solubilized enzyme was stable both at 4°C and at-WC. A partial purification was obtained using an ion-exchange chromatographic procedure. The partially purified enzyme showed four major bands in SDS-polyacrylamide gel electrophoresis; its specific activity was increased by a factor of 37 compared to that of the membrae-bound enzyme. Glycerol and diacylglycerol were effective as stabilizers. P~~tidyIcholine, ly~ph~phati~lcholine and phosphatidyiserine increased both the specific activity and the s~bili~ of the partially ~fi~ enzyme.

Advances in Experimental Medicine and Biology, 1978

Phosphatidic acid can be synthesized de novo by two different pathways, usually named “glycerol-3... more Phosphatidic acid can be synthesized de novo by two different pathways, usually named “glycerol-3-phosphate pathway” (1,2) and “acyl-dihydroxyacetone-phosphate pathway” (3). A study of the contribution of each one of these pathways to the synthesis of phosphatidic acid in vivo presents many difficulties, due to the high activity of brain diglyceride kinase (4,5,6). In addition, an in vivo study does not easily distinguish the mitocondrial and microsomal synthesis as well as the transfer of the newly formed molecules from one type of membrane to the other.

Journal of Bioenergetics and Biomembranes, 2014

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apopto... more Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate-and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate-or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H 2 O 2 release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.

European Journal of Biochemistry, 2001

Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from ... more Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.

The Italian Journal of Neurological Sciences, 1984

The intraventricular injection of pyridoxal phosphate (PLP; 1 t, mole / brain) to rats causes con... more The intraventricular injection of pyridoxal phosphate (PLP; 1 t, mole / brain) to rats causes convulsive seizures beginning 3 min after injection and lasting for about 20 rain. The incorporation of [2-3H] glycerol into rat brain glycerides has been studied to ascertain whether treatment with PLP affects the incorporation of label into various lipid classes. The labelingpattern of glycerides is changed by the administration of PLP. The observed alterations begin a few min after injection, together with the convulsive seizures. 1 h after the injection the pattern of labeling of brain glycerides returns to normal. Different glycerides are differently affected by PLP. This work demonstrates that the labeling of diglyceride increases whereas that of phosphatidylethanolamine decreases following PLP administration.

Neurochemical Research, 1985

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridox... more Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-3H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3-60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciably vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This 'lipid effect' was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.

Molecular Membrane Biology, 1993

Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) are the enzymes catalyzi... more Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) are the enzymes catalyzing the last step of the de novo pathway for phosphatidylcholine and phosphatidylethanolamine synthesis, respectively. A major limitation for the complete characterization of the reactions catalyzed by the two enzymes derives from their poor stability in detergent-containing buffers. CPT is heavily inactivated, when native membranes are solubilized using a series of detergents, whereas EPT activity is better preserved during solubilization. An investigation of the factors which could play a role in preserving both enzymes from inactivation was carried out. The dramatic loss of enzymatic activities occurring upon dilution of solubilized membranes with detergent-containing buffers can be reduced by supplementing the dilution medium with phospholipids. The addition of Mn2+ ions to the dispersion buffer increases the stability of both enzymes. The procedure previously described for solubilizing EPT from rat brain microsomes has been modified on the basis of this evidence. Microsomes were solubilized in buffered detergent solutions containing Mn2+ ions and both CPT and EPT were partially purified in their active form by anion-exchange chromatography.

Molecular and Cellular Biochemistry, 1996

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the ... more A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.