on International journal of biological macromolecules
by Esposito L, Donnarumma F, Ruggiero A, Leone S, Vitagliano L, Picone D
Structural roles of loop regions are frequently overlooked in proteins. Nevertheless, they may be key players in the definition of protein topology and in the self-assembly processes occurring through domain swapping. We here investigate the effects on structure and stability of replacing the loop connecting the last two β-strands of RNase A with the corresponding region of the more thermostable Onconase. The crystal structure of this chimeric variant (RNaseA-ONC) shows that its terminal loop size better adheres to the topological rules for the design of stabilized proteins, proposed by Baker and coworkers . Indeed, RNaseA-ONC displays a thermal stability close to that of RNase A, despite the lack of Pro at position 114, which, due to its propensity to favor a cis peptide bond, has been identified as an important stabilizing factor of the native protein. Accordingly, RNaseA-ONC is significantly more stable than RNase A variants lacking Pro114; RNaseA-ONC also displays a higher propensity to form oligomers in native conditions when compared to either RNase A or Onconase. This finding demonstrates that modifications of terminal loops should to be carefully controlled in terms of size and sequence to avoid unwanted and/or potentially harmful aggregation processes.
on Frontiers in molecular biosciences
by Emendato A, Guerrini R, Marzola E, Wienk H, Boelens R, Leone S, Picone D
Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.
on Microbial cell factories
by Boumaiza M, Colarusso A, Parrilli E, Garcia-Fruitós E, Casillo A, Arís A, Corsaro MM, Picone D, Leone S, Tutino ML
Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey.
on Molecular and cellular biochemistry
by Leone S, Fonderico J, Melchiorre C, Carpentieri A, Picone D
The reaction of free amino groups in proteins with reactive carbonyl species, known as glycation, leads to the formation of mixtures of products, collectively referred to as advanced glycation endproducts (AGEs). These compounds have been implicated in several important diseases, but their role in pathogenesis and clinical symptoms' development is still debated. Particularly, AGEs are often associated to the formation of amyloid deposits in conformational diseases, such as Alzheimer's and Parkinson's disease, and it has been suggested that they might influence the mechanisms and kinetics of protein aggregation. We here present the characterization of the products of glycation of the model protein MNEI with methylglyoxal and their effect on the protein structure. We demonstrate that, despite being an uncontrolled process, glycation occurs only at specific residues of the protein. Moreover, while not affecting the protein fold, it alters its shape and hydrodynamic properties and increases its tendency to fibrillar aggregation. Our study opens the way to in deep structural investigations to shed light on the complex link between protein post-translational modifications, structure, and stability.
on The Journal of biological chemistry
by Emendato A, Milordini G, Zacco E, Sicorello A, Dal Piaz F, Guerrini R, Thorogate R, Picone D, Pastore A
Increasing evidence shows that Aβ peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible non-enzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the Aβ peptides although little is known about the mechanism. Here, we used the natural metabolic byproduct methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the Aβ40 and Aβ42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent β-conversion to mature fibres. We propose that the previously reported higher toxicity of the glycated Aβ peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species.
by Castiglia D, Leone S, Tamburino R, Sannino L, Fonderico J, Melchiorre C, Carpentieri A, Grillo S, Picone D, Scotti N
Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins. The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25-30 mg of pure protein/plant.
on Biochimica et biophysica acta
by Pica A, Leone S, Di Girolamo R, Donnarumma F, Emendato A, Rega MF, Merlino A, Picone D
MNEI and its variant Y65R-MNEI are sweet proteins with potential applications as sweeteners in food industry. Also, they are often used as model systems for folding and aggregation studies.
on The Biochemical journal
by Fagagnini A, Pica A, Fasoli S, Montioli R, Donadelli M, Cordani M, Butturini E, Acquasaliente L, Picone D, Gotte G
Onconase® (ONC), extracted by the oocytes of the frog Rana Pipiens , is a monomeric member of the secretory "pancreatic-type" RNase super-family. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast to other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases of about four times after lyophilization from acetic acid solutions. Differently from RNase A and the bovine seminal ribonuclease (BS-RNase), which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping (3D-DS) of its N-termini, being the C-termini blocked by a disulfide bond. Also, an homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in the dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.
on Journal of inorganic biochemistry
by Picone D, Donnarumma F, Ferraro G, Gotte G, Fagagnini A, Butera G, Donadelli M, Merlino A
Cisplatin (CDDP) can form interprotein cross-links, leading to the formation of platinated oligomers. A dimer, a trimer and higher oligomers of bovine pancreatic ribonuclease (RNase A) obtained upon reaction with CDDP in 1:10 protein to metal ratio at 37°C have been previously characterized. Here, we verify the ability of carboplatin and oxaliplatin to induce RNase A oligomerization under the same experimental conditions. The amount of formed RNase A oligomers was compared with that obtained in the reaction of the protein with CDDP. Among the three anticancer agents, CDDP is the most reactive and the most effective in inhibiting the ribonucleolytic activity of the protein. Oxaliplatin is the least potent oligomerization agent. Biophysical characterizations of structure and stability of platinated dimers formed in the presence of carboplatin and oxaliplatin suggest that they have a similar thermal stability and are more prone to dissociation than the corresponding dimer obtained with CDDP. Oligomers obtained in the presence of carboplatin are the most active. X-ray structures of the monomeric adducts that RNase A forms with the three drugs provide a rational basis to explain the different effects of the three anticancer agents on enzymatic activity and protein aggregation. Although platinated oligomers of RNase A formed upon reaction with CDDP, carboplatin and oxaliplatin retain a residual ribonuclease activity, they do not show cytotoxic action, suggesting that protein aggregation processes induced by Pt-based drugs can represent a collateral drawback, which affects the functional state of protein targets and reduces the efficacy of Pt-based drug treatment.
on Environmental science and pollution research international
by Rega MF, Siciliano A, Gesuele R, Lofrano G, Carpentieri A, Picone D, Guida M
Low-calorie sweeteners are widespread. They are routinely introduced into commonly consumed food such as diet sodas, cereals, and sugar-free desserts. Recent data revealed the presence in considerable quantities of some of these artificial sweeteners in water samples qualifying them as a class of potential new emerging contaminants. This study aimed at evaluating the ecotoxicity profile of MNEI and Y65R-MNEI, two engineered products derived from the natural protein monellin, employing representative test organism such as Daphnia magna, Ceriodaphnia dubia, and Raphidocelis subcapitata. Potential genotoxicity and mutagenicity effects on Salmonella typhimurium (strain TA97a, TA98, TA100, and TA1535) and Escherichia coli (strain WP2 pkM101) were evaluated. No genotoxicity effects were detected, whereas slight mutagenicity was highlighted by TA98 S. typhimurium. Ecotoxicity results evidenced effects approximately up to 14 and 20% with microalgae at 500 mg/L of MNEI and Y65R-MNEI, in that order. Macrophytes and crustaceans showed no significant effects. No median effective concentrations were determined. Overall, MNEI and Y65R-MNEI can be classified as not acutely toxic for the environment.