Site-directed mutagenesis reveals the interplay between stability, structure, and enzymatic activity in RidA from Capra hircus.

Reactive intermediate deaminase A (RidA) is a highly conserved enzyme that catalyzes the hydrolysis of 2-imino acids to the corresponding 2-keto acids and ammonia. RidA thus prevents the accumulation of such potentially harmful compounds in the cell, as exemplified by its role in the degradation of 2-aminoacrylate, formed during the metabolism of cysteine and serine, catalyzing the conversion of its stable 2-iminopyruvate tautomer into pyruvate. Capra hircus (goat) RidA (ChRidA) was the first mammalian RidA to be isolated and described. It has the typical homotrimeric fold of the Rid superfamily, characterized by remarkably high thermal stability, with three active sites located at the interface between adjacent subunits. ChRidA exhibits a broad substrate specificity with a preference for 2-iminopyruvate and other 2-imino acids derived from amino acids with non-polar non-bulky side chains. Here we report a biophysical and biochemical characterization of eight ChRidA variants obtained by site-directed mutagenesis to gain insight into the role of specific residues in protein stability and catalytic activity. Each mutant was produced in Escherichia coli cells, purified and characterized in terms of quaternary structure, thermal stability and substrate specificity. The results are rationalized in the context of the high-resolution structures obtained by x-ray crystallography.

Fagopyrins from Buckwheat Flowers: Structural and Stereochemical Characterization Through Combined NMR/CD Spectroscopy and Theoretical Calculations.

Fagopyrins are phenantroperylenequinones present in the flowers of Fagopyrum esculentum (buckwheat) endowed with photodynamic activity. It has been reported that fagopyrin extracts actually contain a complex mixture of closely related compounds, differing only on the nature of the perylenequinone substituents. We report our systematic and detailed study on the chemical composition of fagopyrin extracts by a combination of preparative and analytical techniques. The combined use of 1H-NMR and CD spectroscopy was found to be particularly suited to fully characterize all stereochemical aspects of the extracted fagopyrins. For the first time nine isomers have been structurally characterized and their stereochemistry fully elucidated. The presence of two different heterocyclic ring substituents, two stereogenic centers and the inherent axial chirality of the aromatic system provides a complex stereochemical relationships among isomers, thus giving account of the high level of molecular multiplicity found in the extract.

Effect of Combined High-Pressure Homogenization and Biotechnological Processes on Chitin, Protein, and Antioxidant Activity of Cricket Powder-Based Ingredients.

The main objective of this work was to evaluate the combined effect of a biotechnology process, based on selected yeast strains, and a high-pressure homogenization (HPH) treatment on the microbiological quality, structural organization of proteins, chitin content, and antioxidant activity of a mixture of cricket powder (Acheta domesticus) and water. Compared to untreated samples, the cricket matrix treated with HPH four times at 180 MPa promoted the growth of the inoculated Yarrowia lipolytica and Debaryomyces hansenii strains. HPH did not affect the concentration of chitin; however, the combination with microorganisms tended to reduce the content. Although the antioxidant activity increased from 0.52 to 0.68 TAC mM/TE after a 48 h incubation in the control, it was further improved by the combination of HPH and D. hansenii metabolism, reaching a value of 0.77 TAC mM/TE. The combination of the two approaches also promoted a reduction in the intensity of bands with molecular weights between 31 and 21.5 kDa in favor of bands with a lower molecular weight. In addition, HPH treatment reduced the number of accessible thiols, suggesting protein structure changes that may further impact the technological properties of cricket powder.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Effect of Sprouting on Biomolecular and Antioxidant Features of Common Buckwheat (Fagopyrum esculentum).

Buckwheat is a pseudo-cereal widely grown and consumed throughout the world. Buckwheat is recognized as a good source of nutrients and, in combination with other health-promoting components, is receiving increasing attention as a potential functional food. Despite the high nutritional value of buckwheat, a variety of anti-nutritional features makes it difficult to exploit its full potential. In this framework, sprouting (or germination) may represent a process capable of improving the macromolecular profile, including reducing anti-nutritional factors and/or synthesizing or releasing bioactives. This study addressed changes in the biomolecular profile and composition of buckwheat that was sprouted for 48 and 72 h. Sprouting increased the content of peptides and free-phenolic compounds and the antioxidant activity, caused a marked decline in the concentration of several anti-nutritional components, and affected the metabolomic profile with an overall improvement in the nutritional characteristics. These results further confirm sprouting as a process suitable for improving the compositional traits of cereals and pseudo-cereals, and are further steps towards the exploitation of sprouted buckwheat as a high-quality ingredient in innovative products of industrial interest.

Plasma and cerebrospinal fluid cholesterol esterification is hampered in Alzheimer's disease.

OBJECTIVE: The purpose of this study was to evaluate cholesterol esterification and HDL subclasses in plasma and cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients. METHODS: The study enrolled 70 AD patients and 74 cognitively normal controls comparable for age and sex. Lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF. RESULTS: AD patients have normal plasma lipids but significantly reduced unesterified cholesterol and unesterified/total cholesterol ratio. Lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate (CER), two measures of the efficiency of the esterification process, were reduced by 29% and 16%, respectively, in the plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls but the content of small discoidal preß-HDL particles was significantly reduced. In agreement with the reduced preß-HDL particles, cholesterol efflux capacity mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients' plasma. The CSF unesterified to total cholesterol ratio was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma unesterified cholesterol and unesterified/total cholesterol ratio with Aß1-42 CSF content. CONCLUSION: Taken together our data indicate that cholesterol esterification is hampered in plasma and CSF of AD patients and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF Aß1-42).

Fine Cocoa Fermentation with Selected Lactic Acid Bacteria: Fermentation Performance and Impact on Chocolate Composition and Sensory Properties.

Cocoa fermentation is a central step in chocolate manufacturing. In this research, we performed controlled fermentations of a fine cocoa variety to evaluate the impact of adjunct cultures of selected lactic acid bacteria (LAB) on fermentation parameters, chemical composition, and sensory profile of fine cocoa and chocolate. Improved fermentation processes were carried out at the Centre for the Integral Transformation of Cacao (CETICO) in Dominican Republic. Two strains of LAB, previously isolated from cocoa, and belonging to Lactiplantibacillus fabifermentans and Furfurilactibacillus rossiae species, were employed. Fermentation parameters, protein, peptide and free amino acid profiles of the fermented cocoa and volatile molecules were determined. Sensory analysis of the derived chocolate was also carried out. The obtained results indicated that the addition of the adjunct cultures influences the proteolytic processes and the free amino acid profile. Finally, the adjunct cultures increased the complexity of the flavour profile of the chocolate as they received a higher score for descriptors commonly used for fine chocolate, such as honey and red fruits. The results obtained showed that the selected strains can be an added value to the development of specific flavours that are desirable at industrial level.

Molecular insights into the role of amylose/amylopectin ratio on gluten protein organization.

Waxy (WX) and high-amylose (HA) wheat flours have interesting functional and/or nutritional characteristics, but low technological properties compared to regular wheat. Here a set of three wheat lines, having different amylose content but sharing the same varietal background, were compared to shed light on the role of the amylose/amylopectin ratio on the protein conformational changes that lead to gluten formation. Despite the absence of differences in their protein profile, as also confirmed by thiolomic approaches, both WX and HA lines developed a weaker gluten than the control sample. The altered amylose/amylopectin ratio exerts a matrix effect establishing a competition for water with proteins, leading to a different protein structure and three-dimensional organization of the gluten network. These results add a piece to the understanding of the molecular aspects that oversee matrix effects on gluten formation in wheat, which description can be helpful for a rational optimization of the transformation process.

Pasta from Red Lentils (Lens culinaris): The Effect of Pasta-Making Process on Starch and Protein Features, and Cooking Behavior.

The effect of pasta-making processes on starch and protein features, as well as cooking behavior, and nutritional properties (i.e., resistant starch and starch in vitro digestibility) were assessed. Pasta from raw red lentils (R) was prepared by conventional extrusion (C_R) and extrusion-cooking (EC_R), whereas heat-treated red lentils (HT) were processed into pasta by conventional extrusion (C_HT). A "high protein" and "high fiber" pasta was prepared. Using HT was effective in increasing the luminosity (that was about 88, 91, and 96 for EC_R, C_R, and C_HT, respectively), and decreasing the presence of defects on the pasta surface (heterogeneity was 5%, 36%, and 45% for C_HT, EC_R, and C_R, respectively). Heat treatment on grains or flour significantly increased starch susceptibility to α-amylase (6.6, 7.4, and 8.6% for C_R, C_HT, and EC_R, respectively) and decreased the final viscosity (from 335 BU in C_R to 287 and 291 BU in EC_R and C_HT), resulting in a significant increase in starch digestibility (slowly digestible starch was about 41, 27, and 26% in C_R, C_HT, and EC_R, respectively). As regards proteins, the main effect on their structure was observed in C_HT, where the cooking behavior was much improved and cooking losses were lowest (5.7%). On the other hand, protein and starch organization in EC_R might have accounted for pasta resistance in overcooking.

Exploration of Lactiplantibacillus fabifermentans and Furfurilactobacillus rossiae as potential cocoa fermentation starters.

AIMS: To investigate the characteristics of two minority autochthonous LAB species, with particular regard to those properties that could be exploited in an improved cocoa fermentation process from a quality and safety point of view. METHODS AND RESULTS: Bacterial, yeast and mould strains characteristic of spontaneously fermented Dominican cocoa beans were isolated and identified by 16S or 26S rRNA gene sequencing. The potential of two autochthonous strains of LAB belonging to the species Lactiplantibacillus fabifermentans and Furfurilactibacillus rossiae were investigated. The two selected LAB strains were able to utilize glucose and fructose, produced mainly D-L lactic acid and had a good ability to resist to cocoa-related stress conditions such as low pH, high temperature and high osmotic pressure, as well as to grow in sterile cocoa pulp. The strains did not inhibit the growth of yeasts and acetic acid bacteria, that are essential to the cocoa fermentation process, and possessed a complex pool of peptidases especially active on hydrophobic amino acids. The strains also showed antifungal activity against mould species that can be found at the final stages of cocoa fermentation, as Aspergillus tamarii, A. nidulans, Lichtheimia ornata and Rhizomucor pusillus. CONCLUSIONS: The tested strains are good candidates for the design of starter cultures for a controlled cocoa fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: This research showcases the potential of two alternative LAB species to the dominating Lactiplantibacillus plantarum and Limosilactibacillus fermentum as cocoa fermentation starters, with an interesting activity in improving the safety and quality of the process.

Protein interactions in the biological assembly of iron-sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps.

This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli isc-encoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues.

Apis mellifera RidA, a novel member of the canonical YigF/YER057c/UK114 imine deiminase superfamily of enzymes pre-empting metabolic damage.

The Reactive intermediate deiminase (Rid) protein family is a group of enzymes widely distributed in all Kingdoms of Life. RidA is one of the eight known Rid subfamilies, and its members act by preventing the accumulation of 2-aminoacrylate, a highly reactive enamine generated during the metabolism of some amino acids, by hydrolyzing the 2-iminopyruvate tautomer to pyruvate and ammonia. RidA members are homotrimers exhibiting a remarkable thermal stability. Recently, a novel subclass of RidA was identified in teleosts, which differs for stability and substrate specificity from the canonical RidA. In this study we structurally and functionally characterized RidA from Apis mellifera (AmRidA) as the first example of an invertebrate RidA to assess its belonging to the canonical RidA group, and to further correlate structural and functional features of this novel enzyme class. Circular dichroism revealed a spectrum typical of the RidA proteins and the high thermal stability. AmRidA exhibits the 2-imino acid hydrolase activity typical of RidA family members with a substrate specificity similar to that of the canonical RidA. The crystal structure confirmed the homotrimeric assembly and the presence of the typical structural features of RidA proteins, such as the proposed substrate recognition loop, and the ß-sheets ß1-ß9 and ß1-ß2. In conclusion, our data define AmRidA as a canonical member of the well-conserved RidA family and further clarify the diagnostic structural features of this class of enzymes.

Distribution of Charged Residues Affects the Average Size and Shape of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

l- to d-Amino Acid Substitution in the Immunodominant LCMV-Derived Epitope gp33 Highlights the Sensitivity of the TCR Recognition Mechanism for the MHC/Peptide Structure and Dynamics.

Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8+ T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wild-type peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2Db, predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2Db/gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2Db in complex with APLs, in which position 6 was subjected to an l- to d-amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the l- to d-modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.

Beta-Lactoglobulin as a Model Food Protein: How to Promote, Prevent, and Exploit Its Unfolding Processes.

Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.

Impact of Thermal Treatment on the Starch-Protein Interplay in Red Lentils: Connecting Molecular Features and Rheological Properties.

Thermal treatments are widely applied to gluten-free (GF) flours to change their functionality. Despite the interest in using pulses in GF formulations, the effects of thermal treatment at the molecular level and their relationship with dough rheology have not been fully addressed. Raw and heat-treated red lentils were tested for starch and protein features. Interactions with water were assessed by thermogravimetric analysis and water-holding capacity. Finally, mixing properties were investigated. The thermal treatment of red lentils induced a structural modification of both starch and proteins. In the case of starch, such changes consequently affected the kinetics of gelatinization. Flour treatment increased the temperature required for gelatinization, and led to an increased viscosity during both gelatinization and retrogradation. Regarding proteins, heat treatment promoted the formation of aggregates, mainly stabilized by hydrophobic interactions between (partially) unfolded proteins. Overall, the structural modifications of starch and proteins enhanced the hydration properties of the dough, resulting in increased consistency during mixing.

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

A novel hotspot of gelsolin instability triggers an alternative mechanism of amyloid aggregation.

Gelsolin comprises six homologous domains, named G1 to G6. Single point substitutions in this protein are responsible for AGel amyloidosis, a hereditary disease causing progressive corneal lattice dystrophy, cutis laxa, and polyneuropathy. Although several different amyloidogenic variants of gelsolin have been identified, only the most common mutants present in the G2 domain have been thoroughly characterized, leading to clarification of the functional mechanism. The molecular events underlying the pathological aggregation of 3 recently identified mutations, namely A551P, E553K and M517R, all localized at the interface between G4 and G5, are here explored for the first time. Structural studies point to destabilization of the interface between G4 and G5 due to three structural determinants: ß-strand breaking, steric hindrance and/or charge repulsion, all implying impairment of interdomain contacts. Such rearrangements decrease the temperature and pressure stability of gelsolin but do not alter its susceptibility to furin cleavage, the first event in the canonical aggregation pathway. These variants also have a greater tendency to aggregate in the unproteolysed forms and exhibit higher proteotoxicity in a C. elegans-based assay. Our data suggest that aggregation of G4G5 variants follows an alternative, likely proteolysis-independent, pathway.

Circular Dichroism to Probe the Synthesis, Transfer, and Stability of Fe-S Clusters.

All Fe-S proteins are characterized by distinctive circular dichroism (CD) features in the visible region of the spectrum due to chiral interaction between the cluster itself and the protein backbone. Therefore, the presence of a CD signal in the visible region relates to the presence of the cluster, whereas the disappearance of the signal refers to cluster breakdown or redox changes. The position of the CD features in the spectrum and the intensity of individual components of the CD signal show great variations among different Fe-S proteins. This feature can provide information on transfer processes between proteins, as well as on possible changes in cluster nuclearity. This method can also be used to detect changes in the chemical nature or spatial organization of cluster ligands that may be concurrent with cluster transfer and associated events.

Monitoring the carryover of egg proteins in pasta making to support allergen risk management.

Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200-2400 kg h-1, the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000-3000 kg (for long pasta) and 3000-4000 kg (for short pasta).