Biomedical subjects

G Raugei

Publications and source records attributed to G Raugei.

At least 55 records · Page 3Linked to original sources

PubMed1996

Characterization of a novel nucleolytic activity of acylphosphatases.

A novel enzymatic activity on nucleic acids was discovered in both muscle type (MT) and erythrocyte or common type (CT) isoforms of acylphosphatase, an enzyme that was previously known as a hydrolase (E.C.3.6.1.7). Both deoxyribonucleic and ribonucleic hydrolitic activity were assayed on a variety of substrates. Our results demonstrate that acylphosphatase possesses both Mg++ dependent deoxyribonuclease and ribonuclease activities, at pH ranging from 5.0 to 6.8. Furthermore, we present evidences, for both isoenzymatic forms, of the coexistence of exonucleolytic and endonucleolytic activities on DNA.

Acid Anhydride Hydrolases↗

PubMed1996

Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate.

Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes.

Acid Anhydride Hydrolases↗

PubMed1995

Expression, purification and kinetic behaviour of fission yeast low M(r) protein-tyrosine phosphatase.

A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast. Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S-transferase in E. coli and its kinetic characterisation. Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M(r) protein phosphatase previously known to exist only in mammalian species. Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M(r) protein tyrosine phosphatases present in mammalian tissues. These differing kinetic characteristics are mainly due to the sequence 45-56 that is spatially close to the active site pocket.

Amino Acid Sequence↗

PubMed1995

Differential modulation of expression of the two acylphosphatase isoenzymes by thyroid hormone.

The modulation of expression of the skeletal muscle and erythrocyte acylphosphatase isoenzymes by thyroid hormone has been investigated. Our results indicate a differential regulation of the two enzymic isoforms by tri-iodothyronine (T3) in K562 cells in culture: an increase in the specific mRNA during T3-stimulation is shown only for the skeletal muscle isoenzyme. A fast and transient T3 induction of the accumulation of the specific mRNA can be observed, reaching a maximum 8 h after hormone treatment and then rapidly decreasing almost to the steady-state level after 24 h. A nuclear run-on assay was performed to explore the mechanisms of this regulation. These studies indicate that T3 induction of skeletal muscle acylphosphatase mRNA is due, at least in part, to a fast and transient increase in the rate of gene transcription, within 4 h after hormone administration. A very rapid decrease is then observed within a further 2 h. T3-dependent accumulation of the mRNA for the skeletal muscle acylphosphatase requires ongoing protein synthesis, as confirmed by inhibition with cycloheximide or puromycin. These findings indicate that the transcriptional regulation of the gene may be indirect.

Acid Anhydride Hydrolases↗

PubMed1995

PDGF receptor as a specific in vivo target for low M(r) phosphotyrosine protein phosphatase.

Low M(r) phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW-PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW-PTP substrate/s we have expressed in NIH-3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW-PTP expressing cells has enabled us to demonstrate an association between LMW-PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW-PTP on the phosphorylated platelet derived growth factor receptor.

3T3 Cells↗

PubMed1995

Cloning and expression of the cDNA coding for the erythrocyte isoenzyme of human acylphosphatase.

Three independent cDNAs coding for the erythrocyte isoform of human acylphosphatase were isolated and characterized. All the clones were incomplete at the 5' end, but Northern blot analysis using the cDNA as a probe showed the presence of an unusually long mRNA 5'-untranslated region. The transcript was present in a variety of human cell lines of different origins, although at different levels. Southern blot analysis on DNA from different individuals revealed a simple hybridization pattern. Large amounts of pure enzyme with kinetic characteristics very similar to those of the native protein were expressed in E. coli.

Acid Anhydride Hydrolases↗

PubMed1995

Properties of N-terminus truncated and C-terminus mutated muscle acylphosphatases.

Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and 1H NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (delta 6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The delta 6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.

Base Sequence↗

PubMed1995

Expression, purification, and characterization of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase.

A genetic construct consisting of the synthetic gene coding for human muscle acylphosphatase linked to the gene for glutathione S-transferase has been prepared. This gene was transformed into and expressed by the Escherichia coli strains DB1035 and TB1, respectively. The fusion protein was purified by affinity chromatography and subsequently cleaved to the fully active acylphosphatase, which was further purified by gel filtration chromatography. Such a purification procedure is very rapid and suitable for obtaining considerable amounts of enzyme at a very high yield. The purified human muscle acylphosphatase was fully active and showed structural features, as well as kinetic and stability parameters, identical to those of the native enzyme.

Acid Anhydride Hydrolases↗

PubMed1994

Arginine-23 is involved in the catalytic site of muscle acylphosphatase.

Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.

Acid Anhydride Hydrolases↗

PubMed1994

Aspartic-129 is an essential residue in the catalytic mechanism of the low M(r) phosphotyrosine protein phosphatase.

The crystal structure of the bovine liver low M(r) phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by 1H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k2) and dephosphorylation (k3) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.

Animals↗

PubMed1994

Inhibition of cellular response to platelet-derived growth factor by low M(r) phosphotyrosine protein phosphatase overexpression.

The role of low M(r) phosphotyrosine protein phosphatase (PTPase) in the control of cell proliferation was studied. A synthetic gene coding for PTPase was transfected and expressed in NIH/3T3 fibroblasts. The effects of the enzyme were particularly evident when cells were stimulated by platelet-derived growth factor (PDGF). The mitogenic response to PDGF was decreased and the inhibition reached 90%. This effect was more pronounced with respect to fetal calf serum stimulation. Hormone-dependent autophosphorylation of the PDGF receptor was significantly reduced. These results demonstrate that low M(r) PTPase, a cytosolic enzyme, not only affects cellular response to PDGF but also reduces the membrane receptor autophosphorylation.

3T3 Cells↗

PubMed1994

The role of His66 and His72 in the reaction mechanism of bovine liver low-M(r) phosphotyrosine protein phosphatase.

Site-directed mutagenesis of a synthetic gene coding for low-M(r) phosphotyrosine protein phosphatase from bovine liver has been carried out. The two histidine residues in the enzyme have been mutated to glutamine; both single and double mutants were produced. The mutated and non-mutated sequences have been expressed in Escherichia coli as fusion proteins, in which the low-M(r) phosphotyrosine protein phosphatase was linked to the C-terminal end of the maltose-binding protein. The fusion enzymes were easily purified by single-step affinity chromatography. The mutants were studied for their kinetic properties. Both single mutants showed decreased kcat. values (30 and 7% residual activities for His66 and His72 respectively), and alterations of the Ki values relative to four-competitive inhibitors were observed. The kinetic mechanism of p-nitrophenyl phosphate hydrolysis in the presence of both single mutants was determined and compared with that of the non-mutated enzyme. The rate-determining step of the catalytic process of the His66-->Gln mutant was the same as that found for non-mutated enzyme, whereas for the His72-->Gln mutant, both the kinetic constant of the step that causes the formation of a phosphoenzyme covalent intermediate, and the kinetic constant of the step that causes the dephosphorylation of the enzyme covalent intermediate, determined the kcat. value. This observation was confirmed by phosphoenzyme covalent intermediate trapping experiments. The participation of both histidine residues (His66 and His72) at the active site is strongly suggested by the results of diethyl pyrocarbonate inactivation of both single mutants, each containing a single histidine residue. Both mutants are completely inactivated by diethyl pyrocarbonate treatment; the competitive inhibitor Pi protects both mutants from inactivation. The His66/His72 double mutant was completely inactive.

Amino Acid Sequence↗

PubMed1994

Expression of human acylphosphatase in Escherichia coli affects intracellular calcium levels.

In vitro experiments demonstrated the ability of acylphosphatase to hydrolyze the phosphorylated intermediate that is formed during the activity of Na+, K(+)- and Ca(2+)-ATPases of mammalian cells membranes. In order to investigate the effect of this enzyme on intracellular cation levels, a synthetic gene for human muscle acylphosphatase has been expressed in E. coli strains BL21 and JM101. Intracellular total steady-state calcium concentration, as measured by isotopic exchange, was significantly higher in transformed cells as compared to controls and the rising was dependent on the level of acylphosphatase expression. Accordingly also free intracellular calcium concentration, as measured by Fura-2 fluorescence, increased in transformed cells. On the other hand, phosphate levels were not affected by the expression of acylphosphatase, while sodium and rubidium levels increase in transformed cells. Intracellular pH resulted to be slightly affected by the expression of acylphosphatase, cytoplasm of transformed JM101 bacteria being more alkaline (pH 7.45) as compared to control cells (pH 7.40). On the basis of these results, it can be suggested that acylphosphatase acts in vivo by regulating the cation transport in E. coli.

Acid Anhydride Hydrolases↗

PubMed1993

Chemical synthesis and expression of a gene coding for human muscle acylphosphatase.

A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.

Acid Anhydride Hydrolases↗

PubMed1993

Negative growth control by a novel low M(r) phosphotyrosine protein phosphatase in normal and transformed cells.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

3T3 Cells↗

PubMed1993

Loop substitution as a tool to identify active sites of interleukin-1 beta.

By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.

Amino Acid Sequence↗

PubMed1993

The role of Cys12, Cys17 and Arg18 in the catalytic mechanism of low-M(r) cytosolic phosphotyrosine protein phosphatase.

Low-M(r) phosphotyrosine protein phosphatase (PTPase), previously known as low-M(r) acid phosphatase, catalyzes the in-vitro hydrolysis of tyrosine phosphorylated proteins, low-M(r) aryl phosphates and natural and synthetic acyl phosphates. Its activity on Ser/Thr-phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl-phosphate hydrolysis was studied by means of non-mutated and mutated PTPase fusion proteins. The mechanism of benzoyl-phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme-catalyzed mechanisms. Nevertheless, the performed phosphoenzyme-trapping experiments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cys17 in the substrate binding was confirmed, its participation a second time in the step that involves the Cys12 dephosphorylation was suggested by the results of phosphoenzyme-trapping experiments. The participation of Arg18 in the substrate-binding site was demonstrated by site-directed mutagenesis that produced the conservative Lys18 and the non-conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme-trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.

Amino Acid Sequence↗

PubMed1993

The role of Cys-17 in the pyridoxal 5'-phosphate inhibition of the bovine liver low M(r) phosphotyrosine protein phosphatase.

Mammalian tissues contain two low M(r) phosphotyrosine protein phosphatase isoforms (type-1 and type-2) that differ in the 40-73 amino-acid sequence. Only one isoform (type-2) is strongly inhibited by pyridoxal 5'-phosphate, whereas the other is poorly inhibited by this compound. The mechanism of pyridoxal 5'-phosphate inhibition of the bovine liver enzyme (a type-2 isoform) has been studied by kinetic methods using a series of pyridoxal 5'-phosphate analogues. These studies indicate that pyridoxal 5'-phosphate interacts with the enzyme in both the phosphate and aldehyde groups. Active site-directed mutagenesis has been used to investigate the sites of pyridoxal 5'-phosphate binding. Our results indicate that Cys-17, essential for enzyme activity, interacts with the phosphate moiety of pyridoxal 5'-phosphate. On the other hand, Cys-12, which is also involved in the catalytic mechanism, does not participate in pyridoxal 5'-phosphate binding.

Animals↗