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P Pucci

Publications and source records attributed to P Pucci.

At least 19 recordsLinked to original sources
PubMed2006

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.

Antigens, Neoplasm↗
PubMed2006

Indole-3-acetic acid improves Escherichia coli's defences to stress.

Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in many living organisms. To elucidate the physiological changes induced by IAA treatment, we used Escherichia coli K-12 as a model system. By microarray analysis we found that 16 genes showed an altered expression level in IAA-treated cells. One-third of these genes encode cell envelope components, or proteins involved in bacterial adaptation to unfavourable environmental conditions. We thus investigated the effect of IAA treatment on some of the structural components of the envelope that may be involved in cellular response to stresses. This showed that IAA-treated cells had increased the production of trehalose, lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated further that IAA triggers an increased tolerance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and different toxic compounds (antibiotics, detergents and dyes) and this correlates with higher levels of the heat shock protein DnaK. We suggest that IAA triggers an increased level of alert and protection against external adverse conditions by coordinately enhancing different cellular defence systems.

Adaptation, Physiological↗
PubMed2006

Separation and measurement of flame-formed high molecular weight polycyclic aromatic hydrocarbons by size-exclusion chromatography and laser desorption/ionization time-of-flight mass spectrometry.

The partial contribution of polycyclic aromatic hydrocarbons (PAH), capable of being detected by gas chromatography (GC-PAH), both to the total mass of the extractable organic fraction of flame-formed carbon particulates and to its UV-visible absorption and fluorescence spectra, has been determined by previous work. This contribution indicates the presence of PAH of molecular weight (MW) greater than 400 Da not accessible to conventional analysis. The detection of species in this higher MW range is important for both their potential toxicology and their possible role in soot formation. In the present work extracts of soots have been analyzed by linear mode laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) to extend the MW range that can be analyzed beyond the GC-PAH. The results have been compared with both analysis by reflector mode LDI-TOF-MS and the MW evaluation obtained by SEC analysis, as the shortcomings and advantages of both techniques appear to be complementary. Matching the results from the two techniques could give interesting insights in the molecular mass range between GC-PAH and the first soot particles (of mass > 2000 Da). Mass spectra in this molecular mass range have been obtained with a main ion sequence spacing of 24 Th and a minor ion sequence also with a spacing of 24 Th but off-set by 12 Th with respect to the main sequence. The two ion progressions have been interpreted by attributing the predominant peaks mainly to PAH with even-carbon numbers and the smaller ones to cyclopenta-fused ring PAH. These distributions indicate the occurrence of two competitive mechanisms in the growth of PAH and soot nucleation, i.e. the addition of acetylene (HACA mechanism) and the incorporation of pentagons by large polycyclic aromatic molecules into their aromatic bonding network.

Carbon↗
PubMed2006

Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli.

The physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1.1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle, the glyoxylate shunt and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme, involved in the production of pyruvate, and pyruvate dehydrogenase, required for the channelling of pyruvate into acetyl-CoA, were also induced in IAA-treated cells. Moreover, it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression, enzyme activity and metabolite level involved in central metabolic pathways in E. coli.

Amino Acids↗
PubMed2005

Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.

Amyloid↗
PubMed2003

The N-terminus of the fragile X mental retardation protein contains a novel domain involved in dimerization and RNA binding.

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). The emerging picture is that FMRP is involved in repression of translation through a complex network of protein-protein and protein-RNA interactions. Very little structural information is, however, available for FMRP that could help to understand its function. In particular, no structural studies are available about the N-terminus of the protein, a highly conserved region which is involved in several molecular interactions. Here, we explore systematically the ability of the FMRP N-terminus to form independently folded units (domains). We produced deletion mutants and tested their fold and functional properties by mutually complementary biophysical and biochemical techniques. On the basis of our data, we conclude that the N-terminus contains a domain, that we named NDF, comprising the first 134 amino acids. Most interestingly, NDF comprises two copies of a newly identified Agenet motif. NDF is thermally stable and has a high content of beta structure. In addition to being able to bind to RNA and to recognize some of the FMRP interacting proteins, NDF forms stable dimers and is able to interact, although weakly, with the full-length protein. Our data provide conclusive evidence that NDF is a novel motif for protein-protein and protein-RNA interactions and contains a previously unidentified dimerization site.

Amino Acid Sequence↗
PubMed2003

A novel zinc finger transcriptional repressor, ZNF224, interacts with the negative regulatory element (AldA-NRE) and inhibits gene expression.

The interaction between the negative cis-element (AldA-NRE) and p97 repressor nuclear protein is a key step in modulating transcription of the human and mouse aldolase A (AldA) gene during the cell cycle and differentiation. In an attempt to clarify the role of transcriptional repression in regulating gene expression, we purified, from HeLa cells, the nuclear protein that specifically binds to the AldA negative regulatory element (NRE). Matrix-assisted laser desorption ionization-time of flight analysis and examination of protein profiles from the SwissProt database revealed that the previously defined p97 repressor is ZNF224, a zinc finger protein. We demonstrate that ZNF224, a Kruppel-like zinc finger transcription factor, is the repressor protein that specifically binds to the negative cis-element AldA-NRE and affects the AldA-NRE-mediated transcription.

Amino Acid Sequence↗
PubMed2001

Slow folding of three-fingered toxins is associated with the accumulation of native disulfide-bonded intermediates.

Snake neurotoxins are short all-beta proteins that display a complex organization of the disulfide bonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersect when bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide beta-cross". We investigated the oxidative folding of a neurotoxin variant, named alpha62, to define the chemical nature of the three-disulfide intermediates that accumulate during the process in order to describe in detail its folding pathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bonds were identified using a combination of tryptic hydrolysis, manual Edman degradation, and mass spectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lacking the C43-C54 and C17-C41 pairing and therefore named des-[43-54] and des-[17-41], respectively. Both species were individually allowed to reoxidize under folding conditions, showing that des-[17-41] was a fast-forming nonproductive intermediate that had to interconvert into the des-[43-54] isomer before forming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediate precursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin alpha62 which fits with the observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn 2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.

Amino Acid Sequence↗
PubMed2001

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.

Adenine↗
PubMed2001

Identification of Hb Villejuif [beta123(H1)Thr-->Ile] in Southern Italy.

Hb Villejuif [beta123(H1)Thr-->Ile] is a silent and asymptomatic variant described in 1989 in an 87-year-old woman of French origin suffering from coincidental polycythemia vera. This paper reports the second observation of Hb Villejuif in three related subjects from Montesarchio, Southern Italy. All routine techniques for hemoglobin analysis yielded normal results with the exception of a slight increase in the Hb A2 value. The occurrence of a variant beta-globin was rapidly assessed by liquid chromatography mass spectrometric analysis and the abnormal chain purified by high performance liquid chromatography. The amino acid replacement Thr-->Ile at beta123 was determined by tandem electrospray mass spectrometric analysis of the tryptic digest of the variant beta chain. The corresponding DNA mutation was established as C-->T at the second position of codon 123 (ACC-->ATC) by polymerase chain reaction amplification techniques.

Adult↗
PubMed2001

Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1.

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Amino Acid Sequence↗
PubMed2001

Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain.

The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.

Amino Acid Sequence↗
PubMed2000

Binding of alpha-actinin to titin: implications for Z-disk assembly.

Titin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z-disk to the M-line. In the Z-disk, it interacts with alpha-actinin homodimers that are a principal component of the Z-filaments linking actin filaments. The interaction between titin and alpha-actinin involves repeating approximately 45 amino acid sequences (Z-repeats) near the N-terminus of titin and the C-lobe of the C-terminal calmodulin-like domain of alpha-actinin. The conformation of Z-repeat 7 (ZR7) of titin when complexed with the 73-amino acid C-terminal portion of alpha-actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N-labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14-Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin-like domains to target peptides.

Actinin↗
PubMed2000

Trypsin sheds light on the singular case of seminal RNase, a dimer with two quaternary conformations.

Dimeric seminal RNase presents the singular case of a dimer with access at equilibrium to two conformations: one in which the subunits exchange, or swap, their NH(2)-terminal arms; the other with no exchange. Thus a continuous unfolding/refolding of structural elements into two alternative conformations takes place in the native protein at equilibrium. The phenomenon was investigated by kinetic and mass spectrometric analyses of the effects of trypsin on the native protein, on its isolated quaternary forms, as well as on a monomeric derivative of the protein and on homologous dimeric RNase A. The kinetics of tryptic action on the protein forms and on the protein derivatives, as well as the location of the tryptic cleavage sites, and their chronological sequence, led to the identification of relevant interconversion intermediates, to the description of a model for the interconversion process, and to a hypothesis for the unique phenomenon of the dual quaternary conformation of seminal RNase.

Animals↗
PubMed2000

Modern mass spectrometric methodologies in monitoring milk quality.

Structural modifications induced by industrial treatments on milk proteins have been investigated using a new analytical protocol based on mass spectrometric procedures (electrospray and matrix assisted laser desorption ionization mass spectrometry) providing a direct correlation between the severity of the treatment and the damages observed. The application of this procedure to the analysis of whey proteins from milk samples submitted to different thermal processes confirmed that under these conditions protein modification is essentially due to the nonenzymatic glycation of amino groups by lactose (Maillard reaction). A detailed structural investigation of the modification sites, carried out by the mass mapping strategy, revealed the occurrence of preferentially lactosylated sites in both alpha-lactalbumin and beta-lactoglobulin.

Animals↗
PubMed2000

Structural characterization of the oligosaccharide chains of human alpha1-microglobulin from urine and amniotic fluid.

Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed.

Alpha-Globulins↗
PubMed2000

Mass spectrometry study of ecto-5'-nucleotidase from bull seminal plasma.

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.

5'-Nucleotidase↗