Biomedical subjects

Piero Pucci

Publications and source records attributed to Piero Pucci.

36 records · Page 2Linked to original sources

PubMed2004

Tuber borchii fruit body: 2-dimensional profile and protein identification.

The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.

Amino Acid Sequence↗

PubMed2004

Hb Cardarelli [beta86(F2)Ala-->Pro]: a new unstable and hyperaffine variant in association with beta(+)-thalassemia.

Hb Cardarelli [beta86(F2)Ala-->Pro] is a new unstable and high oxygen affinity variant found in several members of a family from Naples, Southern Italy. A detailed structural and functional characterization of the variant was performed on two subjects, at both the protein and DNA level. The first patient exhibited 43% of the variant hemoglobin (Hb) without major hematological problems. The proband showed 82% of the abnormal Hb in association with beta(+)-thalassemia (thal) that caused relevant erythrocytosis requiring frequent phlebotomies. Structural investigation of the Hb variant by mass spectrometric methodologies identified the amino acid replacement as Ala-->Pro at beta86. The corresponding DNA mutation GCC-->CCC at codon 86 of the beta-globin gene was assessed by both DNA sequencing and amplification refractory mutation system (ARMS) techniques. Functional studies carried out on whole blood and diluted hemolysates from both patients demonstrated increased oxygen affinity, decreased Bohr effect, reduced heme-heme interaction and nearly halved 2,3-diphosphoglycerate (2,3-DPG) and chloride effects.

Adult↗

PubMed2004

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

Base Sequence↗

PubMed2003

Thermal stability and aggregation of sulfolobus solfataricus beta-glycosidase are dependent upon the N-epsilon-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of beta-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native beta-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of beta-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of beta-glycosidase from S. solfataricus.

Amino Acids↗

PubMed2003

Structural and biochemical characterization of a new type of lectin isolated from carp eggs.

A previously unidentified glycoprotein present in the eggs of the carp ( Cyprinus carpio ) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N -acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N -acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp ( Carassius auratus gibelio ) and 78% identity with a gene in the cDNA library of the zebrafish ( Danio rerio ).

Amino Acid Sequence↗

PubMed2003

Purification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds.

The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an M(r) of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51-44% identical positions) with other plant LTP2s previously described.

Amino Acid Sequence↗

PubMed2003

The FCP1 phosphatase interacts with RNA polymerase II and with MEP50 a component of the methylosome complex involved in the assembly of snRNP.

RNA polymerase II transcription is associated with cyclic phosphorylation of the C-terminal domain (CTD) of the large subunit of RNA polymerase II. To date, FCP1 is the only specific CTD phosphatase, which is required for general transcription and cell viability. To identify FCP1-associated proteins, we constructed a human cell line expressing epitope-tagged FCP1. In addition to RAP74, a previously identified FCP1 interacting factor, we determined that FCP1-affinity purified extracts contain RNAPII that has either a hyper- or a hypo-phosphorylated CTD. In addition, by mass spectrometry of affinity purified FCP1-associated factors, we identified a novel FCP1-interacting protein, named MEP50, a recently described component of the methylosome complex that binds to the snRNP's Sm proteins. We found that FCP1 specifically interacts with components of the spliceosomal U small nuclear ribonucleoproteins. These results suggest a putative role of FCP1 CTD-phosphatase in linking the transcription elongation with the splicing process.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Hexafluoroisopropanol and acid destabilized forms of apomyoglobin exhibit structural differences.

The conformational properties of partially folded states of apomyoglobin have been investigated using an integrated approach based on fluorescence spectroscopy and hydrogen/deuterium exchange followed by mass spectrometry. The examined states were those obtained: (i) by adding 4% v/v hexafluoroisopropanol to native myoglobin, HFIP-MG(N); (ii) by adding 4% v/v hexafluoroisopropanol to acid unfolded myoglobin, HFIP-MG(U); (iii) at pH 3.8, I-1 state; and (iv) at pH 2.0-0.2 M NaCl, A state. Proteolytic digestion of the hydrogen/deuterium exchanged proteins showed that, in I-1 state, the helices C, D, E, and F incorporate more deuterium, whereas in HFIP-MG(N) the exchange rate is similar for all protein regions. These results suggest that I-1 contains the ABGH domain in a native-like organization, whereas HFIP-MG(N) loses a large number of tertiary interactions, thus acquiring a more flexible structure. The fluorescence data are consistent with the above picture. In fact, the tryptophan/ANS energy transfer is much less efficient for the ANS-HFIP-MG(N) complex than for the other complexes, thus suggesting that the distances between the fluorophores might be increased. Moreover, fluorescence polarization measurements indicated that the rotational motion of HFIP-MG(N) occurs on a longer time scale than the other partially folded states, thus suggesting that the volume of this state could be larger. The overall results indicate that addition of hexafluoroisopropanol to native myoglobin results in the formation of a true molten globule where tertiary interactions are reduced, while the secondary structure and the globular compactness are conserved.

Acids↗

PubMed2003

Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen.

Par j 2.0101, a major allergen of the Parietaria judaica pollen, was expressed in E. coli, purified to homogeneity and fully characterised both at the structural and the functional level. The recombinant rPar j 2.0101 protein showed an allergenic activity in histamine release, skin prick tests and capacity to bind IgE, almost identical to that of the native allergens purified from aqueous pollen extract. The complete pattern of S-S bridges of rPar j 2.0101 was determined by enzymatic digestion with endoproteinase Lys-C followed by mass spectrometric analysis of the resulting peptide mixtures. The eight cysteines occurring in the allergenic protein were found to be paired into the following four disulphides: Cys35-Cys83, Cys45-Cys60, Cys61-Cys106 and Cys81-Cys121. This structural information probes Par j 2.0101 to attain a 3-D fold consistent with that of the non-specific lipid transfer protein (ns-LTP) family and it represents an effective molecular basis to develop modified antigens by selective site-directed mutagenesis for immunotherapy.

Allergens↗

PubMed2002

Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M.

Petrosaspongiolide M (PM) is an anti-inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A(2) (PLA(2)) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA(2) and gamma-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA(2) has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA(2) adduct is consistent with the formation of a Schiff base by reaction of a PLA(2) amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM gamma-hydroxybutenolide ring. Proteolysis of the modified PLA(2) by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA(2) alpha-amino terminal group of the Ile-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA(2) was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the epsilon -amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA(2) binding site were obtained for manoalide, a paradigm for irreversible PLA(2) inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the gamma-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA(2) binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA(2) inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA(2) secretory enzymes.

Amino Acid Sequence↗

PubMed2002

Structural characterization of the M* partly folded intermediate of wild type and P138A aspartate aminotransferase from Escherichia coli.

A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N(2) forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.

Amino Acid Sequence↗

PubMed2002

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin.

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.

Binding Sites↗

PubMed2002

Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

Biochemical Phenomena↗

PubMed2002

Hb Vila Real [beta36(C2)Pro-->His] in Italy: characterization of the amino acid substitution and the DNA mutation.

A rare high oxygen affinity hemoglobin variant was identified in a 22-year-old male patient from Napoli (Naples, Italy) affected by erythrocytosis. A detailed structural characterization of the variant hemoglobin was carried out, both at the protein and DNA levels essentially by mass spectrometric procedures and allele-specific amplification techniques. The amino acid substitution was determined by liquid chromatography tandem mass spectrometric analysis of the tryptic digest as beta36(C2)Pro-->His; the corresponding DNA mutation was identified as C-->A at the second position of codon 36 of the beta chain (CCT-->CAT). These variations identified the presence of Hb Vila Real, described only once before in a Portuguese woman. Haplotype analysis of DNA polymorphisms showed that the beta-globin gene of Hb Vila Real was associated with haplotype I.

Adult↗

PubMed2002

The effect of prime-site occupancy on the hepatitis C virus NS3 protease structure.

We recently reported a new class of inhibitors of the chymotrypsin-like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S' site of the protease, which is not generally used by the natural substrates. The effect of prime-site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis-mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S' site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S' site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N-terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P-P' inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.

Binding Sites↗

PubMed2002

Topological investigation of amyloid fibrils obtained from beta2-microglobulin.

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.

Amyloid↗

PubMed2002

Description of the topographical changes associated to the different stages of the DsbA catalytic cycle.

This paper provides a description of the surface topography of DsbA, the bacterial disulfide-bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA-substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease-accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site.

Amino Acid Sequence↗

PubMedDate not supplied

Interaction proteomics.

The term proteome is traditionally associated with the identification of a large number of proteins within complex mixtures originating from a given organelle, cell or even organism. Current proteome investigations are basically focused on two major areas, expression proteomics and functional proteomics. Both approaches rely on the fractionation of protein mixtures essentially by two-dimensional polyacrylamide gel electrophoresis (2D-gel) and the identification of individual protein bands by mass spectrometric techniques (2D-MS). Functional proteomics approaches are basically addressing two main targets, the elucidation of the biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. In the cell many processes are governed not only by the relative abundance of proteins but also by rapid and transient regulation of activity, association and localization of proteins and protein complexes. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular process would then be strongly suggestive of its biological function. The identification of interacting proteins in stable complexes in a cellular system is essentially achieved by affinity-based procedures. Different strategies relying on this simple concept have been developed and a brief overview of the main approaches presently used in functional proteomics studies is described.

Chromatography, Affinity↗