Metabolic Effects of the Sweet Protein MNEI as a Sweetener in Drinking Water. A Pilot Study of a High Fat Dietary Regimen in a Rodent Model.

Sweeteners have become integrating components of the typical western diet, in response to the spreading of sugar-related pathologies (diabetes, obesity and metabolic syndrome) that have stemmed from the adoption of unbalanced dietary habits. Sweet proteins are a relatively unstudied class of sweet compounds that could serve as innovative sweeteners, but their introduction on the food market has been delayed by some factors, among which is the lack of thorough metabolic and toxicological studies. We have tried to shed light on the potential of a sweet protein, MNEI, as a fructose substitute in beverages in a typical western diet, by studying the metabolic consequences of its consumption on a Wistar rat model of high fat diet-induced obesity. In particular, we investigated the lipid profile, insulin sensitivity and other indicators of metabolic syndrome. We also evaluated systemic inflammation and potential colon damage. MNEI consumption rescued the metabolic derangement elicited by the intake of fructose, namely insulin resistance, altered plasma lipid profile, colon inflammation and translocation of lipopolysaccharides from the gut lumen into the circulatory system. We concluded that MNEI could represent a valid alternative to fructose, particularly when concomitant metabolic disorders such as diabetes and/or glucose intolerance are present.

Mechanism of 3D domain swapping in bovine seminal ribonuclease.

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 hydrogen­deuterium 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.

The multiple forms of bovine seminal ribonuclease: structure and stability of a C-terminal swapped dimer.

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.

Double domain swapping in bovine seminal RNase: formation of distinct N- and C-swapped tetramers and multimers with increasing biological activities.

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.

Environmental conditions modulate the switch among different states of the hydrophobin Vmh2 from Pleurotus ostreatus.

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, ß-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.

NMR studies on structure and dynamics of the monomeric derivative of BS-RNase: new insights for 3D domain swapping.

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease.

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 ß-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.

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease.

Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.

3D domain swapping provides a minor alternative refolding pathway for ribonuclease A.

The C-terminal ß-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal ß-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C-terminal ß-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit ß-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.

Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants.

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.

Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A oligomers.

RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A "C-dimer", the C-terminal ß-strand is swapped between two subunits. The loop preceding this ß-strand adopts a ß-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal ß-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel ß-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.

Identification of the spiro(oxindole-3,3'-thiazolidine)-based derivatives as potential p53 activity modulators.

Here, we report the design of new analogues of spirooxoindolepyrrolidine nucleus as modulators of p53 activity. Compounds (3R,7aR)-6-(4-chlorobenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (9c) and (3R,7aR)-5'-methyl-6-(3,4,5-trimethoxybenzyl)-1H-spiro[imidazo[1,5-c]thiazole-3,3'-indoline]-2',5,7(6H,7aH)-trione (10d) are the most potent compounds of this series, inhibiting cell growth of different human tumor cells at submicromolar and micromolar concentrations, respectively. Compound 9c induces apoptotic cell death in human melanoma cell line M14 at 24 h, while in the same condition, treatment with 10d showes a clear arrest at G2/M phase inducing delay of cell cycle progression. Possibly, these activities may be due to inhibition of p53-MDM2 interaction and subsequent p53 release and activation.

Comparison of the structural and functional properties of RNase A and BS-RNase: a stepwise mutagenesis approach.

The original structure of bovine seminal ribonuclease (BS-RNase), solved in 1993, represents a milestone in the story of protein structure, because it represented the first X-ray structure showing two polypeptide chains entangled through their terminal regions. It is generally assumed that this structural feature is the basis of several special biological activities, including a potent antitumor activity, but this has not been yet definitely proved. To assess this hypothesis, in this article we have analyzed the effects of the N-terminal hinge region and/or of Arg80 on the swapping propensity and cytotoxicity in newly designed proteins, using a covalent dimeric variant of bovine pancreatic ribonuclease (RNase A) as scaffold. All the proteins have a very poor cytotoxic activity, independently on the swapping propensity, that can even reach the same value of native BS-RNase. Overall our data suggest that the swapping represents still an essential requisite for the cytotoxic activity, because it keeps the dimeric structure stable even in the reducing cytosolic environment, but other features are essential to design dimeric antitumor ribonucleases, including a strong positive potential at the N-terminal face and a quaternary structure able to evade the cytosolic ribonuclease inhibitor, with or without the interchain disulfide bridges.

Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three noncovalent dimeric mutants.

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme.

Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS.

Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis.

Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.

A new mutant of bovine seminal ribonuclease with a reversed swapping propensity.

Bovine seminal ribonuclease (BS-RNase) is made up of two identical subunits bridged through two disulfide bonds. In solution, it exists as a 2:1 equilibrium mixture between two forms, with (MxM) and without swapping (M=M) of the N-terminal arms. The swapping endows BS-RNase with some special biological functions, including antitumor activity, since MxM retains a dimeric structure even under reducing conditions, thus evading the cytosolic ribonuclease inhibitor. To investigate the structural basis of domain swapping in BS-RNase, we have obtained several mutants by replacing selected residues with the corresponding ones of its monomeric counterpart, bovine pancreatic ribonuclease (RNase A). We have already shown that, in contrast with all other cases of swapped proteins, the swapping propensity of BS-RNase does not depend on the specific sequence of the 16-22 hinge loop, which connects the main body to the dislocating arm. In this paper we report the design, the expression, and the structural characterization of two mutants obtained by replacing Arg80 with Ser either in BS-RNase or in the mutant already containing the 16-22 hinge sequence of RNase A. NMR and circular dichroism data indicate that, in the monomeric form of the latter mutant, Ser80 acts as a switch for the conformation of the hinge region. Accordingly, in the dimeric form of the same mutant the MxM:M=M equilibrium ratio is inverted to 1:2. Overall, these data suggest that the presence of Arg80 triggers the swapping of N-terminal ends and plays a relevant role in the stability of the swapped form of BS-RNase.

The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease.

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.

Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.

Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.