PubMed Health⌕ Search

Biomedical subjects

F Ascoli

Publications and source records attributed to F Ascoli.

At least 19 recordsLinked to original sources
PubMed2004

Mast cell tryptase, a still enigmatic enzyme.

Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules of all mammalian organisms. These enzymes are released along with other mediators into the extracellular medium upon mast cell activation/degranulation. Among the trypsin-like enzymes, tryptases are unique: they are present as active enzymes in the mast cell granules, but display activity only extracellularly, and have a specificity which is much more restricted than trypsin. Tryptases are mostly tetrameric, and in only few organisms (not in humans) are they inhibited by endogenous inhibitors in vitro. The enzymatic and molecular properties of tryptases are far better characterized that any of their plausible biological functions. On the basis of its structural and functional features it could be predicted that tryptase would not degrade a large number of proteins in vivo due to low accessibility to the tetramer central pore where the active sites face inwards. Although their biological function has not yet been clarified, tryptases seem to be involved in a number of mast cell-mediated allergic and inflammatory diseases. In particular, the involvement of tryptase in asthma, an inflammatory disease of the airways often caused by allergy, has been proposed. Here we review the present knowledge on the structure-function relationship of tryptases from different organisms, with special emphasis on human enzymes, and on their role in a variety of pathophysiological processes.

Animals↗
PubMed2001

Selective inhibition of human mast cell tryptase by gabexate mesylate, an antiproteinase drug.

Gabexate mesylate is a non-antigenic synthetic inhibitor of trypsin-like serine proteinases that is therapeutically used in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. Considering the structural similarity between gabexate mesylate and arginine-based inhibitors of trypsin-like serine proteinases, the effect of gabexate mesylate on human and bovine mast cell tryptase action was investigated. Values of the inhibition constant (K(i)) for gabexate mesylate binding to human and bovine tryptase were 3.4 x 10(-9) M and 1.8 x 10(-7) M (at pH 7.4 and 37.0 degrees ), respectively. Furthermore, gabexate mesylate inhibited the fibrinogenolytic activity of human tryptase. On the basis of the available x-ray crystal structure of human tryptase, the possible binding mode of gabexate mesylate to human and bovine tryptase was analyzed. Human tryptase inhibition by gabexate mesylate may account for the reported prevention of inflammation, erosion, and ulceration of skin and mucosae.

Animals↗
PubMed2001

Structure of the Fe-heme in the hemodimeric hemoglobin from Scapharca inaequivalvis and in the T721 mutant: an X-ray absorption spectroscopic study at low temperature.

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.

Animals↗
PubMed2001

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.

Animals↗
PubMed2000

Scapharca inaequivalvis A and B miniglobin genes: promoter activity of the 5' flanking regions and in vivo transcription.

Globin genes of the bivalve mollusk Scapharca inaequivalvis have the two intron/three exon organization typical of vertebrate and many invertebrate globins, with introns in highly conserved positions. Sequence studies on the A and B globin genes of the mollusk tetrameric hemoglobin gave evidence for the existence of 'minigenes' spanning part of the first and second intron, in-frame with the heme binding domain encoded by the central exon. Putative promoter and regulatory sequences flanking these minigenes were identified in the 3' regions of intron I. Here we report cloning and functional analysis of these regions ( approximately 400bp) and their promoter activity, which was assessed in K562 cells by transient transfection, was established. Moreover, in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) on total cytoplasmatic RNA demonstrated that the A and B minigenes are transcriptionally active in the erythrocytes of S. inaequivalvis. Thus, the present results lead to the conclusion that the present-day organization of the globin genes of S. inaequivalvis tetrameric hemoglobin is still reminiscent of an ancestral globin gene before exon shuffling.

Animals↗
PubMed2000

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system.

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E(o) = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information for a deeper understanding of cytochrome c folding and structural/functional organization.

Animals↗
PubMed2000

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors.

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2000

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

Amino Acid Sequence↗
PubMed1999

Functional modulation of the Thr72-->Ile mutant from Scapharca inaequivalvis homodimeric hemoglobin.

The pH and temperature dependence of both the kinetic and thermodynamic properties of the Thr72-->Ile mutant of Scapharca inaequivalvis homodimeric hemoglobin were investigated between pH 2 and 10 and between 8 degrees C and 36 degrees C, in comparison with the wild-type recombinant protein. Results demonstrate pH-independent O2-binding properties, at least between pH 5 and 10, with the higher affinity of the mutant being related to a less negative entropy change. This observation may relate to a variation in the number of water molecules involved in the intersubunit communication. Furthermore, the kinetic properties of ligand association and dissociation seem to be in keeping with possible structural alterations of water molecules at the subunit interface occurring in the Thr72-->Ile mutant as well as with amino acid residues involved in the modulation of reactivity and cooperativity at the level of (1) the proximal side of the heme pocket and of (2) the heme propionates bridging the two subunits.

Amino Acid Substitution↗
PubMed1998

Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity.

A cluster of interface ordered water molecules has been proposed to act as a key mediator of intersubunit communication in the homodimeric hemoglobin of Scapharca inaequivalvis. Mutations of Thr72 to Val and Ile, which lack the hydroxyl group to hydrogen bond the deoxy interface water molecules, result in sharply altered functional properties. We have determined the high resolution (1.6-1. 8 A) crystal structures of these two mutants in both the deoxygenated and CO-liganded states. These structures show minimal protein structural changes relative to the same native derivatives, despite greater than 40-fold increases in oxygen affinity. In the deoxy state of both mutants two water molecules at the periphery of the water cluster are lost, and the remaining cluster water molecules are destabilized. The CO-liganded structures show key differences between the two mutants including a more optimal interface packing involving Ile72 that acts to stabilize its high affinity (R) state. This additional stabilization allows rationalization of its lowered cooperativity within the context of a two-state model. These studies support a key role of ordered water in cooperative functioning and illustrate how subtle structural alterations can result in significantly altered functional properties in an allosteric molecule.

Allosteric Regulation↗
PubMed1998

The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes.

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called 'central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (approximately 80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.

Animals↗
PubMed1998

Scapharca inaequivalvis tetrameric hemoglobin A and B genes: evidence for a minigene.

Vertebrate and many invertebrate globin genes have a three-exon/two-intron organization, with introns in highly conserved positions. According to the "intron early" hypothesis, introns are the vestigial segments which flank previously independent coding sequences, thus providing evidence for the assembly of the ancient proteins by "exon shuffling." In this paper, we report the analysis of the genes of the bivalve mollusk Scapharca inaequivalvis tetrameric hemoglobin (HbII), which support this hypothesis, at least for the hemoglobin genes. We show the existence of "minigenes" in the IIA and IIB globin genes, spanning part of the first and second introns, "in frame" with the heme-binding domain coded by the second exon. Further support for the exon shuffling hypothesis can be found in the degree of identity of the "new" translated sequences with those flanking the central protein domain of some invertebrate hemoglobins.

Amino Acid Sequence↗
PubMed1998

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Animals↗
PubMed1998

Bovine mast cell tryptase inactivation: effect of temperature.

The thermal stability of bovine tryptase, a serine proteinase present in the bovine mast cell secretory granules, has been studied by circular dichroism and catalytic activity measurements. Bovine tryptase shows a peculiar dichroic negative band centered at 230 nm. The decrease of this band in a temperature dependent fashion represents a good marker to monitor the native conformation of the enzyme. Bovine tryptase inactivation has been followed in the temperature range between 10 degrees C and 80 degrees C, and reversibility of the process has been also studied. The results obtained show that the temperature dependent loss of activity and the conformational change, as monitored by circular dichroism, are both fully reversible between 10 degrees C and 40 degrees C, while only the CD change displays reversibility in going from 60 degrees C to 10 degrees C. Moreover, a functional analysis of the temperature-dependent enzymatic activity of bovine tryptase toward peptide substrates in the 10 degrees C - 40 degrees C range is reported and compared with the temperature dependence of the enzymatic activity of trypsin.

Animals↗
PubMed1998

Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role.

Some structural features of bovine tryptase were discussed based on spectroscopic analysis. The far UV-CD spectrum of the enzymatically active bovine tryptase is consistent with a structure containing very little, if any alpha-helix, as found for other serine proteases. The analysis of near UV-CD and UV absorption spectra reveals the presence of a high number of Trp residues arranged probably in strong structural motifs. At variance with other tryptases, the bovine enzyme shows an electrophoretic behaviour in native and denaturating conditions compatible with an association state larger than a tetramer (probably a dodecamer). From a biochemical point of view, the bovine tryptase shares with the human counterpart, the preference for cleaving substrates bearing dibasic cleavage sites. Thus, it is hypothesized that tryptase may be involved in some proprotein processing mechanism(s).

Amino Acid Sequence↗
PubMed1997

Histone-tryptase interaction: H2A N-terminal tail removal and inhibitory activity.

The involvement of tryptase, the trypsin-like serine proteinase of mast cell granules, in many (patho)physiological conditions is now recognized. In vitro this enzyme is known to act as a potent growth factor for fibroblasts and epithelial cells. Moreover, a role in inflammatory diseases and in dermatological disorders characterized by increased cell turnover has been suggested for this protease. In an attempt to understand the molecular basis of tryptase activity, we have investigated the interaction in vitro between bovine tryptase and histones. Here we show that tryptase cleaves histone H2A at a specific site (Arg20-Ala21), resulting in the removal of the N-terminal flexible fragment of the molecule. Furthermore, we demonstrate that the H2A major fragment (H2A*, 109 residues) generated by hydrolysis and lacking the N-terminal domain, is a noncompetitive, reversible and highly specific inhibitor (Ki = 29 nM) of tryptase enzymatic activity. H2A* is able to inhibit the hydrolysis of a small substrate as well as the cleavage of fibronectin, a high-molecular-weight substrate of tryptase.

Animals↗
PubMed1997

The Soret circular dichroism spectrum as a probe for the heme Fe(III)-Met(80) axial bond in horse cytochrome c.

A spectroscopic signal sensitive to the strength of the heme iron(III)-Met(80) bond in cytochrome c represents a useful tool that will provide valuable information on the heme pocket region and redox properties of the protein. At present, the 695-nm absorption band is perhaps the simplest diagnostic signal for the axial bond; this band disappears when the Fe(III)-Met(80) bond is disrupted. From the analysis of the Soret region circular dichroism spectrum of cytochrome c under conditions that gradually induce disruption of the Fe(III)-Met(80) bond, we present evidence that the 416-nm spectral dichroic band provides independent information addressing the strength of the axial bond between Fe(III) and Met(80) in cytochrome c. Further, this study demonstrates extension of the diagnostic application to very dilute protein samples based on the useful sample concentration of 5-10 microM vs 200-300 microM required for 695-nm absorbance measurements.

Animals↗