on The Journal of biological chemistry
by Viggiani A, Siani L, Notomista E, Birolo L, Pucci P, Di Donato A
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.
on Protein science : a publication of the Protein Society
by Casbarra A, Birolo L, Infusini G, Dal Piaz F, Svensson M, Pucci P, Svanborg C, Marino G
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.
by Pierleoni R, Buffalini M, Vallorani L, Guidi C, Zeppa S, Sacconi C, Pucci P, Amoresano A, Casbarra A, Stocchi V
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.
on Journal of molecular biology
by Monti M, Garolla di Bard BL, Calloni G, Chiti F, Amoresano A, Ramponi G, Pucci P
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.
on The Biochemical journal
by Galliano M, Minchiotti L, Campagnoli M, Sala A, Visai L, Amoresano A, Pucci P, Casbarra A, Cauci M, Perduca M, Monaco HL
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 ).
on Biological chemistry
by Amoresano A, Pucci P, Duro G, Colombo P, Costa MA, Izzo V, Lamba D, Geraci D
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.
on Nucleic acids research
by Licciardo P, Amente S, Ruggiero L, Monti M, Pucci P, Lania L, Majello B
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.
by Sirangelo I, Dal Piaz F, Malmo C, Casillo M, Birolo L, Pucci P, Marino G, Irace G
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.
on FEBS letters
by Medugno L, Costanzo P, Lupo A, Monti M, Florio F, Pucci P, Izzo P
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.
on European journal of biochemistry
by Cafaro V, Scognamiglio R, Viggiani A, Izzo V, Passaro I, Notomista E, Piaz FD, Amoresano A, Casbarra A, Pucci P, Di Donato A
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.