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L Alberghina

Publications and source records attributed to L Alberghina.

At least 19 recordsLinked to original sources
PubMed2006

Ras-dependent carbon metabolism and transformation in mouse fibroblasts.

Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.

Animals↗
PubMed2005

Glucose modulation of cell size in yeast.

Saccharomyces cerevisiae cells grown in glucose have larger average size than cells grown in ethanol. Besides, yeast must reach a carbon source-modulated critical cell size in order to enter S phase at Start. This control is of outmost physiological relevance, since it allows us to coordinate cell growth with cell cycle progression and it is responsible for cell size homeostasis. The cell sizer mechanism requires the overcoming of two sequential thresholds, involving Cln3 and Far1, and Clb5,6 and Sic1, respectively. When both thresholds are non-functional, carbon source modulation of cell size at Start is completely abolished. Since inactivation of extracellular glucose sensing through deletion of either the GPR1 or the GPA2 gene causes a marked, but partial, reduction in the ability to modulate cell size and protein content at Start, it is proposed that both extracellular and intracellular glucose signalling is required for properly setting the cell sizer in glucose media.

Cell Size↗
PubMed2005

Acquired glucose sensitivity of k-ras transformed fibroblasts.

Mutational activation of the ras gene is critical for the onset of different malignant phenotypes. We constructed a dominant negative mutant (GEF-DN) of a Ras activator protein (guanine nucleotide-exchange factor) that upon over-expression in k-ras transformed NIH 3T3 fibroblasts strongly reduces intracellular Ras*GTP, reverting these cells to wild-type phenotype for morphology, anchorage-independent growth and reduction of tumour formation in nude mice. Here we review evidence showing that the enhanced proliferation potential of NIH-ras cells requires high initial glucose concentration in the medium and sustained Ras pathway activation. The exquisite sensitivity of NIH-ras fibroblasts to a shortage in nutrient and energy supply highlights an acquired fragility of cancer cells that may be exploited for therapeutic purposes.

Animals↗
PubMed2001

Towards a blueprint of the cell cycle.

The understanding of the organisation of cell cycle events is of utmost importance to devise effective therapeutic strategies for cancer. In this article we gather evidences from the literature in support of a system model of the cell cycle, in which a growth-sensitive threshold controls entry into S phase and the sequential activation of cyclin-dependent kinases. The cycle is terminated by an End function, that comprises events from the onset of mitosis to cell division and that may also be modulated by the increase of cell size. This blueprint allows quantitative predictions by computer simulations of steady and transitory states. In fact, we show that the proposed control system applies to budding yeast populations during nutritional shift-up and following hyperactivation of the cAMP signalling pathway. Besides the growth-sensitive control system it is shown to apply to mammalian cells both in the exit from quiescence and in active proliferation. The putative molecular determinants that set the threshold controlling S phase entry are consistently altered in cancer cells. Finally, we discuss an input/output analysis based on the simulated behaviour derived from the blueprint as a new tool to investigate the road to cancer.

Animals↗
PubMed2000

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase.

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

Base Sequence↗
PubMed2000

Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.

The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression.

Amino Acid Sequence↗
PubMed2000

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Animals↗
PubMed2000

Relating growth dynamics and glucoamylase excretion of individual Saccharomyces cerevisiae cells.

We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae. The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such as fluorescein isothiocyanate (FITC). The staining conditions did not perturb cell growth after resuspension of stained cells in growth medium. Decrease in fluorescence was found to correlate with excretion of glucoamylase into the growth medium. The analysis of the staining pattern over time provides information on the behaviour of individual cells belonging to different cell-cycle phases and can be used to calculate the specific excretion rate of the overall population.

Culture Media↗
PubMed2000

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae.

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.

Colony Count, Microbial↗
PubMed2000

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

Asparagine↗
PubMed2000

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells.

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

Fungal Proteins↗
PubMed1999

Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm).

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF(W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21(ras), and strongly reduce affinity of the nucleotide-free ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

Animals↗
PubMed1999

Chromosome separation and exit from mitosis in budding yeast: dependence on growth revealed by cAMP-mediated inhibition.

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.

Anaphase↗
PubMed1999

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Aerobiosis↗
PubMed1999

Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts.

Interest in the production of L-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovine L-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into a K. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter(-1), 0.91 g liter(-1) h(-1), and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those usual for bacterial processes.

Animals↗
PubMed1998

Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25Mm originate both loss-of-function and gain-of-function proteins.

The role of two residues within the catalytic domain of CDC25Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25Mm(E1048K) and CDC25Mm(S1122V) were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25Mm(S1122A) showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.

3T3 Cells↗
PubMed1998

Analysis of the secondary structure of the catalytic domain of mouse Ras exchange factor CDC25Mm.

The minimal active domain (GEF domain) of the mouse Ras exchange factor CDC25Mm was purified to homogeneity from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism (CD) spectra in the far UV region indicate that this domain is highly structured with a high content of alpha-helix (42%). Near UV CD spectra evidenced good signal due to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras proteins.

Amino Acid Sequence↗