on Biochimica et biophysica acta
by Fiorini C, Cordani M, Gotte G, Picone D, Donadelli M
Onconase® (ONC) is a member of the RNase super-family that is secreted in oocytes and early embryos of Rana pipiens. Over the last years, research interest about this small and basic frog RNase, also called ranpirnase, constantly increased because of its high cytotoxicity and anticancer properties. Onconase is currently used in clinical trials for cancer therapy; however, the precise mechanisms determining cytotoxicity in cancer cells have not yet been fully investigated. In the present manuscript, we evaluate the antitumoral property of onconase in pancreatic adenocarcinoma cells and in non-tumorigenic cells as a control. We demonstrate that ONC stimulates a strong antiproliferative and proapoptotic effect in cancer cells by reporting for the first time that ONC triggers Beclin1-mediated autophagic cancer cell death. In addition, ONC inhibits the expression of mitochondrial uncoupling protein 2 (UCP2) and of manganese-dependent superoxide dismutase (MnSOD) triggering mitochondrial superoxide ion production. ONC-induced reactive oxygen species (ROS) are responsible for Akt/mTOR pathway stimulation determining the sensitivity of cancer cells to mTOR inhibitors and lessening autophagic stimulation. This indicates ROS/Akt/mTOR axis as a strategy adopted by cancer cells to reduce ONC-mediated cytotoxic autophagy stimulation. In addition, we demonstrate that ONC can sensitize pancreatic cancer cells to the standard chemotherapeutic agent gemcitabine allowing a reduction of drug concentration when used in combination settings, thus suggesting a lowering of chemotherapy-related side effects. Altogether, our results shed more light on the mechanisms lying at the basis of ONC antiproliferative effect in cancer cells and support its potential use to develop new anticancer strategies.
by Tuppo L, Spadaccini R, Alessandri C, Wienk H, Boelens R, Giangrieco I, Tamburrini M, Mari A, Picone D, Ciardiello MA
Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein.
on Biochimica et biophysica acta
by Fiorini C, Gotte G, Donnarumma F, Picone D, Donadelli M
Among the large number of variants belonging to the pancreatic-type secretory ribonuclease (RNase) superfamily, bovine pancreatic ribonuclease (RNase A) is the proto-type and bovine seminal RNase (BS-RNase) represents the unique natively dimeric member. In the present manuscript, we evaluate the anti-tumoral property of these RNases in pancreatic adenocarcinoma cell lines and in nontumorigenic cells as normal control. We demonstrate that BS-RNase stimulates a strong anti-proliferative and pro-apoptotic effect in cancer cells, while RNase A is largely ineffective. Notably, we reveal for the first time that BS-RNase triggers Beclin1-mediated autophagic cancer cell death, providing evidences that high proliferation rate of cancer cells may render them more susceptible to autophagy by BS-RNase treatment. Notably, to improve the autophagic response of cancer cells to BS-RNase we used two different strategies: the more basic (as compared to WT enzyme) G38K mutant of BS-RNase, known to interact more strongly than wt with the acidic membrane of cancer cells, or BS-RNase oligomerization (tetramerization or formation of larger oligomers). Both mutant BS-RNase and BS-RNase oligomers potentiated autophagic cell death as compared to WT native dimer of BS-RNase, while the various RNase A oligomers remained completely ineffective. Altogether, our results shed more light on the mechanisms lying at the basis of BS-RNase antiproliferative effect in cancer cells, and support its potential use to develop new anti-cancer strategies.
on The FEBS journal
by Spadaccini R, Ercole C, Graziano G, Wechselberger R, Boelens R, Picone D
3D domain swapping (3D-DS) is a complex protein aggregation process for which no unique mechanism exists. We report an analysis of 3D-DS in bovine seminal ribonuclease, a homodimeric protein whose subunits are linked by two disulfide bridges, based on NMR and biochemical studies. The presence of the covalent bonds between the subunits stabilizes the unswapped dimer, and allows distinct evaluation of the structural and dynamic effects of the swapping with respect to the dimerization process. In comparison with the monomeric subunit, which, in solution has a compact structure without any propensity for local unfolding, both swapped and unswapped dimers show increased flexibility. NMR analysis, together with urea denaturation and hydrogen–deuterium exchange data, indicates that the two dimers have increased conformational fluctuations. Furthermore, we found that the rate-limiting step of both the swapping and unswapping pathways is the detachment of the N-terminal helices from the monomers. These results suggest a new general mechanism in which a dimeric intermediate could facilitate 3D-DS in globular proteins.
on FEBS letters
by Sica F, Pica A, Merlino A, Russo Krauss I, Ercole C, Picone D
Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111-116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.
on FEBS letters
by Gotte G, Laurents DV, Merlino A, Picone D, Spadaccini R
Protein aggregation via 3D domain swapping is a complex mechanism which can lead to the acquisition of new biological, benign or also malignant functions, such as amyloid deposits. In this context, RNase A represents a fascinating model system, since by dislocating different polypeptide chain regions, it forms many diverse oligomers. No other protein displays such a large number of different quaternary structures. Here we report a comparative structural analysis between natural and artificial RNase A dimers and bovine seminal ribonuclease, a natively dimeric RNase with antitumor activity, with the aim to design RNase A derivatives with improved pharmacological potential.
on Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology
by Tuppo L, Alessandri C, Pomponi D, Picone D, Tamburrini M, Ferrara R, Petriccione M, Mangone I, Palazzo P, Liso M, Giangrieco I, Crescenzo R, Bernardi ML, Zennaro D, Helmer-Citterich M, Mari A, Ciardiello MA
Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions.
on Plant science : an international journal of experimental plant biology
by Picone D, Temussi PA
The fruits of a few tropical plants contain intensely sweet proteins. Their common property points to a protein family. Generally, proteins belonging to the same family share similar folds, similar sequences and, at least in part, similar function but sweet proteins constitute an exception to this rule. Apart from sharing the rather unusual taste function, they show no obvious similarities either in their sequences or in three-dimensional structures. In this review we describe the nature, structure and mechanism of action of the best known sweet tasting proteins, including two taste modifying proteins. Sweet proteins stand out among sweet molecules because their volume is not compatible with an interaction with orthosteric active sites of the sweet taste receptor. The best explanation of their mechanism of action is the interaction with the external surface of the sweet taste receptor, according to a model that has been named "wedge model". It is hypothesized that this mode of action may be related to the ability of other members of their protein families to inhibit different enzymes.
by Merlino A, Picone D, Ercole C, Balsamo A, Sica F
3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.
by Longobardi S, Picone D, Ercole C, Spadaccini R, De Stefano L, Rea I, Giardina P
Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca(2+) ions, an assembled state, β-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications.