Biomedical subjects

Piero Pucci

Publications and source records attributed to Piero Pucci.

At least 19 recordsLinked to original sources

PubMed2024

Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

Humans↗

PubMed2006

Recombinant amyloidogenic domain of ApoA-I: analysis of its fibrillogenic potential.

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1-93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

Amyloid↗

PubMed2006

Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation.

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.

Animals↗

PubMed2006

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting.

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.

Archaea↗

PubMed2006

Pancreatic cancer-derived S-100A8 N-terminal peptide: a diabetes cause?

BACKGROUND: Our aim was to identify the pancreatic cancer diabetogenic peptide. METHODS: Pancreatic tumor samples from patients with (n=15) or without (n=7) diabetes were compared with 6 non-neoplastic pancreas samples using SDS-PAGE. RESULTS: A band measuring approximately 1500 Da was detected in tumors from diabetics, but not in neoplastic samples from non-diabetics or samples from non-neoplastic subjects. Sequence analysis revealed a 14 amino acid peptide (1589.88 Da), corresponding to the N-terminal of the S100A8. At 50 nmol/L and 2 mmol/L, this peptide significantly reduced glucose consumption and lactate production by cultured C(2)C(12) myoblasts. The 14 amino acid peptide caused a lack of myotubular differentiation, the presence of polynucleated cells and caspase-3 activation. CONCLUSIONS: The 14 amino acid peptide from S100A8 impairs the catabolism of glucose by myoblasts in vitro and may cause hyperglycemia in vivo. Its identification in biological fluids might be helpful in diagnosing pancreatic cancer in patients with recent onset diabetes mellitus.

Adult↗

PubMed2006

H-prune-nm23-H1 protein complex and correlation to pathways in cancer metastasis.

Cancer is a multi-step process, one of the latest events correspond to metastasis formation and dissemination, to date the major cause of deaths. The h-prune-nm23-H1 protein complex and its activation of PDE-cAMP activity have been shown to correlate with breast cancer progression and metastasis formation. Here, we describe the protein complex formation and its involvement in cell migration. By gene expression studies and protein-protein pull-down analyses coupled to mass spectrometry we have identified new genes and pathways along which the h-prune-nm23-H1 complex exerts its function. We review here h-prune binding to the glycogen synthase kinase (GSK-3beta) and identify a new h-prune protein partner, Gelsolin, an ATP severing protein acting in focal adhesions, in a MDA-435 breast cancer cellular model. The results presented here underline the importance of this protein complex leading to new translational studies involved into the inhibition of cell migration, thus enhancing the potential of using this knowledge to direct inhibition of metastases formation in humans.

Breast Neoplasms↗

PubMed2006

Exploring the mechanism of formation of native-like and precursor amyloid oligomers for the native acylphosphatase from Sulfolobus solfataricus.

Over 40 human diseases are associated with the formation of well-defined proteinaceous fibrillar aggregates. Since the oligomers precursors to the fibrils are increasingly recognized to be the causative agents of such diseases, it is important to elucidate the mechanism of formation of these early species. The acylphosphatase from Sulfolobus solfataricus is an ideal system as it was found to form, under conditions in which it is initially native, two types of prefibrillar aggregates: (1) initial enzymatically active aggregates and (2) oligomers with characteristics reminiscent of amyloid protofibrils, with the latter originating from the structural reorganization of the initial assemblies. By studying a number of protein variants with a variety of biophysical techniques, we have identified the regions of the sequence and the driving forces that promote the first aggregation phase and show that the second phase consists in a cooperative conversion involving the entire globular fold.

Acid Anhydride Hydrolases↗

PubMed2005

Proteomics of beta2-microglobulin amyloid fibrils.

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.

Amino Acid Sequence↗

PubMed2005

Functional proteomics.

BACKGROUND: With the increase in the number of genome sequencing projects, there is a concomitant exponential growth in the number of protein sequences whose function is still unknown. Functional proteomics constitutes an emerging research area in the proteomic field whose approaches are addressed towards two major targets: the elucidation of the biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. METHODS: The identification of interacting proteins in stable complexes in vivo is essentially achieved by affinity-based procedures. The basic idea is to express the protein of interest with a suitable tag to be used as a bait to fish its specific partners out from a cellular extract. Individual components within the multi-protein complex can then be identified by mass spectrometric methodologies. RESULTS AND CONCLUSIONS: The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism is strongly suggestive of the biological function of the protein. Moreover, the identification of protein partners interacting with a given protein will lead to the description of cellular mechanisms at the molecular level. The next goal will be to generate animal models bearing a tagged form of the bait protein.

Humans↗

PubMed2005

Pancreatic cancer-associated diabetes mellitus: an open field for proteomic applications.

BACKGROUND: Diabetes mellitus is associated with pancreatic cancer in more than 80% of the cases. Clinical, epidemiological, and experimental data indicate that pancreatic cancer causes diabetes mellitus by releasing soluble mediators which interfere with both beta-cell function and liver and muscle glucose metabolism. METHODS: We analysed, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), a series of pancreatic cancer cell lines conditioned media, pancreatic cancer patients' peripheral and portal sera, comparing them with controls and chronic pancreatitis patients' sera. RESULTS: MALDI-TOF analysis of pancreatic cancer cells conditioned media and patients' sera indicated a low molecular weight peptide to be the putative pancreatic cancer-associated diabetogenic factor. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of tumor samples from diabetic and non-diabetic patients revealed the presence of a 1500 Da peptide only in diabetic patients. The amino acid sequence of this peptide corresponded to the N-terminal of an S-100 calcium binding protein, which was therefore suggested to be the pancreatic cancer-associated diabetogenic factor. CONCLUSIONS: We identified a tumor-derived peptide of 14 amino acids sharing a 100% homology with an S-100 calcium binding protein, which is probably the pancreatic cancer-associated diabetogenic factor.

Animals↗

PubMed2005

CysMap and CysJoin: database and tools for protein disulphides localisation.

We have developed a computer program able to make user-customised databases derived from the public PIR non-redundant reference protein database. When the database of interest has been created, the user will generate the map of all the possible linear peptides containing one and two cysteines for each protein and combine them to calculate the mass of all the possible clusters of linear peptides linked by a disulphide bridge with a cysteine pair. It is also possible to create selected maps corresponding to peptides formed by the action of specific proteases. In this way, mass spectrometric data obtained from the hydrolysis of proteins of unknown sequence can be related to that contained in the database for quick disulphide assignment and protein identification. To confirm signal attribution, the program will also furnish the expected mass of cluster peptides after performing a cycle of Edman degradation. The utility of the program is discussed and examples of application are given.

Amino Acid Sequence↗

PubMed2005

Biological properties of a human compact anti-ErbB2 antibody.

ErbB2 is a prognostic factor and target of therapy for many carcinomas. In contrast with the other ErbB receptors, ErbB2 lacks a soluble direct ligand, but it is the preferred co-receptor for the ErbB family members, forming heterodimers with more potent and prolonged signalling activity than that of homodimers. We recently produced a new anti-ErbB2 antibody, Erb-hcAb, by fusion of Erbicin, a human, anti-ErbB2 scFv, selectively cytotoxic to ErbB2-positive cells, and a human Fc domain. This fully human antitumour antibody represents a compact version of an IgG1, with the cytotoxicity of the scFv moiety on target cells, combined with the ability of the Fc moiety to induce both antibody- and complement-dependent cytotoxicity. Here, we describe the main properties of Erb-hcAb, using as a reference Herceptin, an anti-ErbB2 humanized monoclonal currently employed in clinical immunotherapy. We found that both bivalent Erb-hcAb and Herceptin increase receptor phosphorylation and downregulation, whereas monovalent Erbicin does not. These results correlate with the finding that Erb-hcAb is capable of inducing apoptosis and inhibiting cell cycle progression in ErbB2-positive cells. Its powerful in vitro antitumour action matched that observed in vivo in experiments with human ErbB2-positive tumour xenografts established in athymic mice. Finally, Erb-hcAb displays a glycosylation profile virtually superimposable to that of a human IgG. These findings suggest that Erb-hcAb is a very promising new agent for the immunotherapy of carcinomas that overexpress the ErbB2 receptor.

Animals↗

PubMed2005

Identification of proteins interacting with the RNAPII FCP1 phosphatase: FCP1 forms a complex with arginine methyltransferase PRMT5 and it is a substrate for PRMT5-mediated methylation.

FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.

Amino Acid Sequence↗

PubMed2004

Direct interactions among Ret, GDNF and GFRalpha1 molecules reveal new insights into the assembly of a functional three-protein complex.

The glial-cell-line-derived neurotrophic factor (GDNF) ligand activates the Ret receptor through the assembly of a multiprotein complex, including the GDNF family receptor alpha1 (GFRalpha1) molecule. Given the neuroprotective role of GDNF, there is an obvious need to precisely identify the structural regions engaged in direct interactions between the three molecules. Here, we combined a functional approach for Ret activity (in PC12 cells) to cross-linking experiments followed by MS-MALDI to study the interactions among the purified extracellular region of the human Ret, GDNF and GFRalpha1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRalpha1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRalpha1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.

Animals↗

PubMed2004

The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1.

Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.

Binding Sites↗

PubMed2004

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

Amino Acid Sequence↗

PubMed2004

The regions of the sequence most exposed to the solvent within the amyloidogenic state of a protein initiate the aggregation process.

Formation of misfolded aggregates is an essential part of what proteins can do. The process of protein aggregation is central to many human diseases and any aggregating event needs to be prevented within a cell and in protein design. In order to aggregate, a protein needs to unfold its native state, at least partially. The conformational state that is prone to aggregate is difficult to study, due to its aggregating potential and heterogeneous nature. Here, we use a systematic approach of limited proteolysis, in combination with electrospray ionisation mass spectrometry, to investigate the regions that are most flexible and solvent-exposed within the native, ligand-bound and amyloidogenic states of muscle acylphosphatase (AcP), a protein previously shown to form amyloid fibrils in the presence of trifluoroethanol. Seven proteases with different degrees of specificity have been used for this purpose. Following exposure to the aggregating conditions, a number of sites along the sequence of AcP become susceptible to proteolytic digestion. The pattern of proteolytic cleavages obtained under these conditions is considerably different from that of the native and ligand-bound conformations and includes a portion within the N-terminal tail of the protein (residues 6-7), the region of the sequence 18-23 and the position 94 near the C terminus. There is a significant overlap between the regions of the sequence found to be solvent-exposed from the present study and those previously identified to be critical in the rate-determining steps of aggregation from protein engineering approaches. This indicates that a considerable degree of solvent exposure is a feature of the portions of a protein that initiate the process of aggregation.

Acid Anhydride Hydrolases↗

PubMed2004

Hierarchical formation of disulfide bonds in the immunoglobulin Fc fragment is assisted by protein-disulfide isomerase.

Antibodies provide an excellent system to study the folding and assembly of all beta-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the C(H)3 and C(H)2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130-Cys-188) belonging to the C(H)3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the C(H)2 domain requires catalysis by PDI.

Animals↗