Organic Chimeras Based on Selenosugars, Steroids, and Fullerenes as Potential Inhibitors of the ß-amyloid Peptide Aggregation.

The aggregation of ß-amyloid peptide (Aß) is associated with neurodegenerative diseases such as Alzheimer's disease (AD). Several therapies aimed at reducing the aggregation of this peptide have emerged as potential strategies for the treatment of AD. This paper describes the design and preparation of new hybrid molecules based on steroids, selenosugars, and [60]fullerene as potential inhibitors of Aß oligomerization. These moieties were selected based on their antioxidant properties and possible areas of interaction with the Aß. Cyclopropanations between C60 and malonates bearing different steroid and selenosugar moieties using the Bingel-Hirsch protocol have enabled the synthesis of functionalized molecular hybrids. The obtained derivatives were characterized by physical and spectroscopic techniques. Theoretical calculations for all the selenium compounds were performed using the density functional theory DFT/B3LYP-D3(BJ)/6-311G(2d,p) predicting the most stable conformations of the synthesized derivatives. Relevant geometrical parameters were investigated to relate the stereochemical behavior and the spectroscopic data obtained. The affinity of the compounds for Aß-peptide was estimated by molecular docking simulation, which predicted an increase in affinity and interactions for Aß for the hybrids containing the C60 core. In addition, parameters such as lipophilicity, polar surface area, and dipole moment were calculated to predict their potential interaction with membrane cells.

Low-intensity magnetization pretreatment to enhance biomethane generation and the abundance of key microorganisms for anaerobic digestion of sewage sludge.

This work explores the impact of static magnetic field (SMF) intensity on biomethane production from anaerobic digestion (AD) of sewage sludge. Two different SMF intensities (20 mT and 1.5 T) were applied to magnetize the sludge destined to the AD process. The magnetic pretreatment at 20 mT was particularly effective, as it increased biomethane production by 12.7 % compared to the control test. On the contrary, exposing the sludge to 1.5 T adversely affected biomethane production, resulting in a 15.1 % decrease. The positive correlation observed between low-intensity SMF exposure and enhanced biomethane yield, in contrast to the inhibitory effect of high-intensity SMF, suggests the existence of an optimal intensity threshold within the lower range for maximizing methane production. The impact of magnetic pretreatment on the anaerobic microbial community was investigated through high-throughput sequencing analysis of magnetized sludge samples. This approach enabled the identification of specific shifts in microbial populations associated with SMF exposure, thereby elucidating the role of SMF in modulating key microbial communities for the AD process. The findings of this study provide insights into the potential mechanisms underlying these responses and underscore the potential of SMF application for improving the anaerobic valorization of sewage sludge.

Chitosan/poly-γ-glutamic acid crosslinked hydrogels: Characterization and application as bio-glues.

Ecofriendly hydrogels were prepared using chitosan (CH, 285 kDa) and two fractions of low molecular weight microbial poly-γ-glutamic acid (γ-PGA) (R1 and R2 of 59 kDa and 20 kDa, respectively). The hydrogels were synthesized through sustainable physical blending, employing three CH/γ-PGA mass ratios (1/9, 2/8, and 3/7), resulting in the formation of physically crosslinked materials. The six resulting CH/R1 and CH/R2 hydrogels were physico-chemically characterized and the ones with the highest yields (CH/R1 and CH/R2 ratio of 3/7), analyzed for rheological and morphological properties, showed to act as bio-glues on wood and aluminum compared to commercial vinyl- (V1) and acetovinyl (V2) glues. Lap shear analyses of CH/R1 and CH/R2 blends exhibited adhesive strength on wood, as well as adhesive/cohesive failure like that of V1 and V2. Conversely, CH/R2 had higher adhesive strength and adhesive/cohesive failure on aluminum, while CH/R1 showed an adhesion strength with adhesive failure on the metal similar to that of V1 and V2. Scanning electron microscopy revealed the formation of strong physical bonds between the hydrogels and both substrates. Beyond their use as bio-adhesives, the unique properties of the resulting crosslinked materials make them potentially suitable for various applications in paint, coatings, heritage preservation, and medical sector.

Cold-Azurin, a New Antibiofilm Protein Produced by the Antarctic Marine Bacterium Pseudomonas sp. TAE6080.

Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. Staphylococcus epidermidis, thanks to its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in biofilm-associated infections of medical devices. Therefore, the research of new molecules able to interfere with S. epidermidis biofilm formation has a remarkable interest. In the present work, the attention was focused on Pseudomonas sp. TAE6080, an Antarctic marine bacterium able to produce and secrete an effective antibiofilm compound. The molecule responsible for this activity was purified by an activity-guided approach and identified by LC-MS/MS. Results indicated the active protein was a periplasmic protein similar to the Pseudomonas aeruginosa PAO1 azurin, named cold-azurin. The cold-azurin was recombinantly produced in E. coli and purified. The recombinant protein was able to impair S. epidermidis attachment to the polystyrene surface and effectively prevent biofilm formation.

High density polyethylene (HDPE) biodegradation by the fungus Cladosporium halotolerans.

Polyethylene (PE) is high molecular weight synthetic polymer, very hydrofobic and hardly biodegradable. To increase polyethylene bio-degradability it is very important to find microorganisms that improve the PE hydrophilic level and/or reduce the length of its polymeric chain by oxidation. In this study, we isolated Cladosporium halotolerans, a fungal species, from the gastric system of Galleria mellonella larvae. Here, we show that C. halotolerans grows in the presence of PE polymer, it is able to interact with plastic material through its hyphae and secretes enzymes involved in PE degradation.

CATASAN Is a New Anti-Biofilm Agent Produced by the Marine Antarctic Bacterium Psychrobacter sp. TAE2020.

The development of new approaches to prevent microbial surface adhesion and biofilm formation is an emerging need following the growing understanding of the impact of biofilm-related infections on human health. Staphylococcus epidermidis, with its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in infections of medical devices. In the research of new anti-biofilm agents against S. epidermidis biofilm, Antarctic marine bacteria represent an untapped reservoir of biodiversity. In the present study, the attention was focused on Psychrobacter sp. TAE2020, an Antarctic marine bacterium that produces molecules able to impair the initial attachment of S. epidermidis strains to the polystyrene surface. The setup of suitable purification protocols allowed the identification by NMR spectroscopy and LC-MS/MS analysis of a protein-polysaccharide complex named CATASAN. This complex proved to be a very effective anti-biofilm agent. Indeed, it not only interferes with cell surface attachment, but also prevents biofilm formation and affects the mature biofilm matrix structure of S. epidermidis. Moreover, CATASAN is endowed with a good emulsification activity in a wide range of pH and temperature. Therefore, its use can be easily extended to different biotechnological applications.

Proteomic Characterization of Collagen-Based Animal Glues for Restoration.

Animal glues are widely used in restoration as adhesives, binders, and consolidants for organic and inorganic materials. Their variable performances are intrinsically linked to the adhesive properties of collagen, which determine the chemical, physical, and mechanical properties of the glue. We have molecularly characterized the protein components of a range of homemade and commercial glues using mass spectrometry techniques. A shotgun proteomic analysis provided animal origin, even when blended, and allowed us to distinguish between hide and bone glue on the basis of the presence of collagen type III, which is abundant in connective skin/leather tissues and poorly synthetized in bones. Furthermore, chemical modifications, a consequence of the preparation protocols from the original animal tissue, were thoroughly evaluated. Deamidation, methionine oxidation, and backbone cleavage have been analyzed as major collagen modifications, demonstrating their variability among different glues and showing that, on average, bone glues are less deamidated than hide glues, but more fragmented, and mixed-collagen glues are overall less deamidated than pure glues. We believe that these data may be of general analytical interest in the characterization of collagen-based materials and may help restorers in the selection of the most appropriate materials to be used in conservation treatments.

The effects of two gold-N-heterocyclic carbene (NHC) complexes in ovarian cancer cells: a redox proteomic study.

PURPOSE: Ovarian cancer is the fifth leading cause of cancer-related deaths in women. Standard treatment consists of tumor debulking surgery followed by platinum and paclitaxel chemotherapy; yet, despite the initial response, about 70-75% of patients develop resistance to chemotherapy. Gold compounds represent a family of very promising anticancer drugs. Among them, we previously investigated the cytotoxic and pro-apoptotic properties of Au(NHC) and Au(NHC)2PF6, i.e., a monocarbene gold(I) complex and the corresponding bis(carbene) complex. Gold compounds are known to alter the redox state of cells interacting with free cysteine and selenocysteine residues of several proteins. Herein, a redox proteomic study has been carried out to elucidate the mechanisms of cytotoxicity in A2780 human ovarian cancer cells. METHODS: A biotinylated iodoacetamide labeling method coupled with mass spectrometry was used to identify oxidation-sensitive protein cysteines. RESULTS: Gold carbene complexes cause extensive oxidation of several cellular proteins; many affected proteins belong to two major functional classes: carbohydrate metabolism, and cytoskeleton organization/cell adhesion. Among the affected proteins, Glyceraldehyde-3-phosphate dehydrogenase inhibition was proved by enzymatic assays and by ESI-MS studies. We also found that Au(NHC)2PF6 inhibits mitochondrial respiration impairing complex I function. Concerning the oxidized cytoskeletal proteins, gold binding to the free cysteines of actin was demonstrated by ESI-MS analysis. Notably, both gold compounds affected cell migration and invasion. CONCLUSIONS: In this study, we deepened the mode of action of Au(NHC) and Au(NHC)2PF6, identifying common cellular targets but confirming their different influence on the mitochondrial function.

A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Identification and Relative Quantification of hFSH Glycoforms in Women's Sera via MS-PRM-Based Approach.

Follicle-stimulating hormone (FSH) is a glycohormone synthesized by adenohypophysis, and it stimulates ovulation in women and spermatogenesis in men by binding to its receptor (FSHR). FSHR is involved in several mechanisms to transduce intracellular signals in response to the FSH stimulus. Exogenous FSH is currently used in the clinic for ovarian hyperstimulation during in vitro fertilization in women, and for treatment of infertility caused by gonadotropin deficiency in men. The glycosylation of FSH strongly affects the binding affinity to its receptor, hence significantly influencing the biological activity of the hormone. Therefore, the accurate measurement and characterization of serum hFSH glycoforms will contribute to elucidating the complex mechanism of action by which different glycoforms elicit distinct biological activity. Nowadays ELISA is the official method with which to monitor serum hFSH, but the test is unable to distinguish between the different FSH glycovariants and is therefore unsuitable to study the biological activity of this hormone. This study presents a preliminary alternative strategy for identifying and quantifying serum hFSH glycoforms based on immunopurification assay and mass spectrometry (MS), and parallel reaction monitoring (PRM) analysis. In this study, we provide an MS-PRM data acquisition method for hFSH glycopeptides identification with high specificity and their quantification by extracting the chromatographic traces of selected fragments of glycopeptides. Once set up for all its features, the proposed method could be transferred to the clinic to improve fertility treatments and follow-ups in men and women.

Tyrosine Phosphorylation Modulates Peroxiredoxin-2 Activity in Normal and Diseased Red Cells.

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

A versatile and user-friendly approach for the analysis of proteins in ancient and historical objects.

Identification and characterization of ancient proteins still require technical developments towards non-invasiveness, sensitivity, versatility and ease of use of the analyses. We report that the enzyme functionalized films, described in Cicatiello et al. (2018), can be used efficiently on the surface of different objects ranging from fixative-coated paper to canvas to the coating on an albumen photograph, as well as the much harder surfaces of ivory objects and the proteinaceous binders in the decoration of a wooden Egyptian coffin. The mixture of digested peptides that are efficiently captured on the functionalized surface are also amenable to LC-MS/MS analysis, which is necessary to confidently identify chemical modifications induced upon degradation, in order to characterize the conservation state of proteins. Moreover, in a two-step procedure, we have combined the trypsin functionalized film with a PNGaseF functionalized film, which adds a deglycosylation pretreatment allowing improved detection of glycosylated proteins. SIGNIFICANCE: User friendly trypsin functionalized films were implemented to expand their potential as versatile, modular tools that can be widely exploited in the world of diagnosis of cultural heritage objects, ancient proteins, and palaeoproteomics: a procedure that could be carried out by conservators or archaeologists first on-site and later analysed with standard MS techniques.

Protein Glycosylation Investigated by Mass Spectrometry: An Overview.

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms.

Fyn specifically Regulates the activity of red cell glucose-6-phosphate-dehydrogenase.

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we report that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase enzyme catalytic activity (kcat) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn-/-mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress.

Quantification of Polyphenols and Metals in Chinese Tea Infusions by Mass Spectrometry.

Chemical compounds within tea (Camellia sinensis) are characterized by an extensive heterogeneity; some of them are crucial for their protective and defensive role in plants, and are closely connected to the benefits that the consumption of tea can provide. This paper is mainly focused on the characterization of polyphenols (secondary metabolites generally involved in defense against ultraviolet radiation and aggression by pathogens) and metals, extracted from nine Chinese tea samples, by integrating different mass spectrometry methodologies, LC-MS/MS in multiple reaction monitoring (MRM) and inductively coupled plasma mass spectrometry (ICP-MS). Our approach allowed to identify and compare forty polyphenols differently distributed in tea infusions at various fermentation levels. The exploration of polyphenols with nutraceutical potential in tea infusions can widely benefit especially tea-oriented populations. The worldwide consumption of tea requires at the same time a careful monitoring of metals released during the infusion of tea leaves. Metal analysis can provide the identification of many healthy minerals such as potassium, sodium, calcium, magnesium, differently affected by the fermentation of leaves. Our results allowed us: (i) to draw up a polyphenols profile of tea leaves subjected to different fermentation processes; (ii) to identify and quantify metals released from tea leaves during infusion. In this way, we obtained a molecular fingerprint useful for both nutraceutical applications and food control/typization, as well as for frauds detection and counterfeiting.

Gelling behavior of bio-tofu coagulated by microbial transglutaminase combined with lactic acid bacteria.

The aim of this study was to investigate the gelling behavior of proteins in bio-tofu (soymilk-cow milk mixture gel) coagulated by microbial transglutaminase (MTGase) combined with lactic acid bacteria (LAB). It was shown that MTGase (3.0 U/g protein) treatment of soymilk-cow milk mixture (SCMM) could not induce gelation at 43℃ even if the incubation was lasting 4 h. However, the concomitant use of LAB (0.025 UC/L) along with MTGase could induce the formation of denser and finer gel network with smaller pores and higher storage modulus (G') compared to SCMM treated with only LAB. Electrophoresis and mass spectrometry results indicated that LAB improve MTGase-dependent polymerization of proteins. In addition, this study investigates the effect of LAB and MTGase treatment on the rheology behavior of the derived gel products. In general, the use of both bio-coagulants for the manufacture of a mixed protein gel, might open new horizons in the field of novel nutrional and functional foods.

Fiano, Greco and Falanghina grape cultivars differentiation by volatiles fingerprinting, a case study.

The biomolecular characterization of edible products is gaining an increasing importance in food chemistry. The characteristic aroma or bouquet of a wine is the result of complex interactions of volatile molecules and odor receptors. Its characterization is the subject of many different studies, aimed at the development of new methods to be used for the discovery of frauds and for the typization of Protected Designation of Origin (P.D.O.) or Protected Geographic Indication (P.G.I.) wines. We previously outlined the proteomic profile of three cultivars of Vitis vinifera from South Italy (Campania) used for white wine production (Fiano, Greco and Falanghina) during the ripening. In this work, we present a mass spectrometry based study aimed at obtaining the profile of volatiles on the same samples using solid phase micro extraction coupled to gas chromatography. We demonstrated that some of the main constituents of aroma (namely terpenes, alcohols, aldehydes, etc.) were characteristic of certain grapes and absent in others.

Mass spectrometry based proteomics for the molecular fingerprinting of Fiano, Greco and Falanghina cultivars.

The official methodologies used for the identification and comparison of vine cultivars are ampelography and ampelometry. These methodologies are essentially based on qualitative assessments or biometric dependent morphological features of the plant. The heterogeneity of cultivars and consequently the increasing demand for a more detailed product typization, led to the introduction of new methodologies for the varietal characterization. In this scenario, proteomics has already proved to be a very useful discipline for the typization of many kinds of edible products. In this paper, we present a proteomic study carried out on three cultivars of Vitis vinifera peculiar of south Italy (Campania) used for white wine production (Fiano, Greco and Falanghina) by advanced biomolecular mass spectrometry approach. Our data highlight variations in the proteomic profiles during ripening for each cultivar and between analyzed cultivars, thus suggesting a new way to outline the biomolecular signature of vines.

Structural effects of methylglyoxal glycation, a study on the model protein MNEI.

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.