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Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU−/− mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

Probiotics play a key role in the modulation of the gut immune system in health and disease and their action is mediated by molecules exposed on the microorganism surface or secreted probiotic-derived factors. In particular, Lactobacillus gasseri OLL2809, a probiotic microorganism isolated from human feces, has the potential to modulate various immune responses. The dendritic cells (DCs) are considered the main players in orchestrating the immune response, and their contact with intestinal microbiota is crucial for the development and homeostasis of gut immunity. To gain a perspective on the molecular mechanisms involved in the maturation process of DCs and investigate factors that could modulate these processes, a differential proteomic analysis was performed on the secretome of immature DCs, mature DCs (mDCs, induced by lipopolysaccharide (LPS)), and immature DCs challenged with L. gasseri OLL2809 before treatment with LPS (LGmDCs). The maturation process of DCs was associated to profound changes in the protein secretome and probiotic pre-treatment led to a dramatic modulation of several secreted proteins of mDC, not only classical immune mediators (i.e., cytokines, complement factors, T cell Receptor ligands) but also proteins involved in the contractile and desmosome machineries. The latter data highlight a novel mechanism by which L. gasseri can modulate the maturation process of DCs, reinforcing the concept of a protective anti-inflammatory role ascribed to this probiotic strain.

Vimentin, a member of cytoskeleton intermediate filaments proteins, plays a critical role in cell structure and dynamics. The present proteomic study reveals reduced amount of six different lengths, N-terminal truncated proteolytic products of vimentin, in the primary skin fibroblasts from two unrelated PD patients, as compared to control fibroblasts. The decreased amount of N-terminal truncated forms of vimentin in parkin-mutant fibroblasts, could contribute to impairment of cellular function, potentially contributing to the pathogenesis of Parkinson disease.

Background:/Aims: Renal disease is a common cause of morbidity in patients with Bardet-Biedl syndrome (BBS), however the severity of kidney dysfunction is highly variable. To date, there is little information on the pathogenesis, the risk and predictor factors for poor renal outcome in this setting. The present study aims to analyze the spectrum of urinary proteins in BBS patients, in order to potentially identify 1) disease-specific proteomic profiles that may differentiate the patients from normal subjects; 2) urinary markers of renal dysfunction. Methods: Fourteen individuals (7 males and 7 females) with a clinical diagnosis of BBS have been selected in this study. A pool of 10 aged-matched males and 10 aged-matched females have been used as controls for proteomic analysis. The glomerular filtration rate (eGFR) has been estimated using the CKD-EPI formula. Variability of eGFR has been retrospectively assessed calculating average annual eGFR decline (ΔeGFR) in a mean follow-up period of 4 years (3-7). Results: 42 proteins were significantly over- or under-represented in BBS patients compared with controls; the majority of these proteins are involved in fibrosis, cell adhesion and extracellular matrix organization. Statistic studies revealed a significant correlation between urine fibronectin (u-FN) (r2=0.28; p2 =0.35; p20.27; p2 =0.2389; pConclusion: The present study demonstrates that urine proteome of BBS patients differs from that of normal subjects; in addition, kidney dysfunction correlated with urine abundance of known markers of renal fibrosis.

Assessment of seafood authenticity and origin, mainly in the case of processed products (fillets, sticks, baby food) represents the crucial point to prevent fraudulent deceptions thus guaranteeing market transparency and consumers health. The most dangerous practice that jeopardies fish safety is intentional or unintentional mislabeling, originating from the substitution of valuable fish species with inferior ones. Conventional analytical methods for fish authentication are becoming inadequate to comply with the strict regulations issued by the European Union and with the increase of mislabeling due to the introduction on the market of new fish species and market globalization. This evidence prompts the development of high-throughput approaches suitable to identify unambiguous biomarkers of authenticity and screen a large number of samples with minimal time consumption. Proteomics provides suitable and powerful tools to investigate main aspects of food quality and safety and has given an important contribution in the field of biomarkers discovery applied to food authentication. This report describes the most relevant methods developed to assess fish identity and offers a perspective on their potential in the evaluation of fish quality and safety thus depicting the key role of proteomics in the authentication of fish species and processed products. Biological significance The assessment of fishery products authenticity is a main issue in the control quality process as deceptive practices could imply severe health risks. Proteomics based methods could significantly contribute to detect falsification and frauds, thus becoming a reliable operative first-line testing resource in food authentication.

Objectives Oral squamous cell carcinoma (OSCC) is the most common epithelial malignant neoplasm affecting the oral cavity. OSCC can mimic oral lesions of inflammatory origin with benign features, often leading to delay in diagnosis and treatment. Early detection is important to greatly increase the chances of a successful treatment. The present study reports a proteomic analysis of a gingival oral squamous cell carcinoma (G-OSCC) and an epulis. Materials and methods Normal mucosae tissue, G-OSCC tissue, and epulis tissue as a comparative sample of benign nature were collected and immediately frozen in liquid nitrogen. Tissue-extracted proteins were separated by two-dimensional gel electrophoresis and subjected to image analysis. Proteins that showed a significant difference in the expression level in the G-OSCC tissue were identified by the nano-ESI-HPLC-MS/MS experiment and database searchi. Results and conclusion The proteomic analysis of G-OSCC tissue enabled the identification of proteins that are potentially related to the disease; these proteins can be considered as signature molecules for diagnostic and prognostic tumor markers.

Constant global warming is one of the most detrimental environmental factors for agriculture causing significant losses in productivity as heat stress (HS) conditions damage plant growth and reproduction. In flowering plants such as tomato, HS has drastic repercussions on development and functionality of male reproductive organs and pollen. Response mechanisms to HS in tomato anthers and pollen have been widely investigated by transcriptomics; on the contrary, exhaustive proteomic evidences are still lacking. In this context, a differential proteomic study was performed on tomato anthers collected from two genotypes (thermo-tolerant and thermo-sensitive) to explore stress response mechanisms and identify proteins possibly associated to thermo-tolerance. Results showed that HS mainly affected energy and amino acid metabolism and nitrogen assimilation and modulated the expression of proteins involved in assuring protein quality and ROS detoxification. Moreover, proteins potentially associated to thermo-tolerant features, such as glutamine synthetase, S-adenosylmethionine synthase and polyphenol oxidase, were identified.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin -mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure–dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

The time course of biosynthesis and accumulation of storage proteins in developing grains of durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) pasta-quality reference cv. Svevo was investigated at the protein level for the first time. Seeds were harvested at key kernel developmental stages, namely, 3 (seed increase 3-fold in size), 5 (kernel development, water-ripe stage), 11 (kernel development, water-ripe stage), 16 (kernel full development, water-ripe stage), 21 (milk-ripe stage), and 30 (dough stage) days postanthesis (dpa). Gliadins and glutenins were fractionated according to their different solubility and individually analyzed after fractionation by reversed-phase high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by liquid chromatography-tandem mass spectrometry of proteolytic peptides. The α- and γ-gliadin were already detected at 3 dpa. The biosynthesis of high molecular mass glutenin Bx7 was slightly delayed (11 dpa). Most of the gluten proteins accumulated rapidly between 11 and 21 dpa, with a minor further increase up to 30 dpa. The expression pattern of gluten proteins in Triticum durum at the early stages of synthesis provides reference data sets for future applications in crop breeding and growth monitoring.

Milk and dairy products are usually submitted to thermal procedures in order to preserve microbiological safety and to prolong their shelf-life; these actions may eventually induce structural modifications of several components. In particular, milk proteins undergo a non-enzymatic glycation, known as the Maillard reaction, in which reducing sugars (such as lactose) mainly react with the ?-amino group of Lys residues, leading to the formation of the Amadori product lactulosyl-lysine. Protein lactosylation leads to a decrease of milk nutritional value as it reduces the bioavailability of the essential amino acid lysine and to a potential increase of the specific allergen activity, due to the formation of new hapten-like antigens. These aspects are of particular importance in products intended for infant diet, such as milk powders and infant milk formulas; accordingly, a detailed structural characterization of the extent and site-specificity of milk protein lactosylation is mandatory. Analytical methodologies based on mass spectrometry and more recently proteomics have been thoroughly applied to the structural analysis of milk proteins and their modified forms, including glycation products. This review highlights the contribution of mass spectrometry to the extensive investigation of lactosylation events in milk proteins, with a particular focus on the most up-to date technological improvements.

Background Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity. Methods SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC–ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour. Results Gliadin and glutenin yields decreased to 7.6 ± 0.5% and 7.5 ± 0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC–ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31–49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31–49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics. Conclusions The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties. General significance Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.

Fusarium oxysporum f. sp. radicis-lycopersici (FORL) leading to fusarium crown and root rot is considered one of the most destructive tomato soilborne diseases occurring in greenhouse and field crops. In this study, response to FORL infection in tomato roots was investigated by differential proteomics in susceptible (. Monalbo) and resistant (. Momor) isogenic tomato lines, thus leading to identify 33 proteins whose amount changed depending on the pathogen infection, and/or on the two genotypes. FORL infection induced accumulation of pathogen-related proteins (PR proteins) displaying glucanase and endochitinases activity or involved in redox processes in the Monalbo genotype. Interestingly, the level of the above mentioned PR proteins was not influenced by FORL infection in the resistant tomato line, while other proteins involved in general response mechanisms to biotic and/or abiotic stresses showed significant quantitative differences. In particular, the increased level of proteins participating to arginine metabolism and glutathione S-transferase (GST; EC 2.5.1.18) as well as that of protein LOC544002 and phosphoprotein ECPP44-like, suggested their key role in pathogen defence. © 2013 Elsevier Masson SAS.

Mass spectrometry is a well-known technology used for the analysis of pure compounds as well as mixtures, widely applied in large-scale studies such proteomic studies. The result of mass spectrometric analyses is a mass spectrum, a profile of mass/charge values and corresponding intensity values originated from the analyzed compounds. In the case of large-scale analyses, raw mass spectra comparisons are difficult due to different drawback typologies: data defects, unusual distributions, underlying disturbs and noise, bad data calibration. A bunch of data elaborations is essential, from data processing to feature extraction, in order to obtain a list of peaks from different mass spectra. In this work, a workflow has been developed to process raw mass spectra and compare the new tidy ones with the aim of defining a robust procedure, suitable for real applications and reusable for different kind of studies. A similarity measure has been used for comparison purposes, in order to verify similarity among replicates and differences among analyzed samples, and a clustering method has been performed on fish species, in order to discover how they cluster statistically. A case study is shown with the application of the processing method to data obtained from the analysis of different fish species.

Consumer demand for fish is constantly increasing due to the awareness of beneficial effects of fishery products on human health. Furthermore, the opening of new markets and the use of a growing number of fish species strongly recall the urgent need to guarantee safety, traceability and authenticity of seafood. Recent European Union directives and regulations for quality control of food products have prompted the development of new methods for large-scale tests to ensure consumer protection. MALDI-TOF mass spectrometry has provided a significant contribution to food science, proving to be a key tool in the analysis of several food matrices, including fish, especially in studies aimed to assess food quality, safety and authenticity. The present chapter is focused on an innovative molecular profiling strategy based on MALDI-TOF mass spectrometric analysis of sarcoplasmic protein extracts from fish muscle, successfully applied to fish authentication. This method allows to rapidly discriminate different fish species, to verify commercial product authenticity and to detect fraudulent substitutions

Probiotics are living microorganisms that confer beneficial effects to human health when supplied in adequate amounts, by promoting digestion and uptake of dietary nutrients, strengthening intestinal barrier function, modulating immune response and enhancing antagonism towards pathogens. The purpose of the present article is to focus on microbial proteomics, pointing out its usefulness in the investigation of molecular mechanisms underlying probiotic effects. It deals, in particular, with molecular strategies responsible for adaptation to the harsh physical-chemical environment of the gastro-intestinal tract, bacterial adhesion to host epithelial cells and intestinal mucosa and probiotic immunomodulatory properties, as analyzed by proteomics in the past few years.

This is the first report describing the purification and enzymatic properties of a native invertase (beta-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85 degrees C at pH 6.0. At temperatures of 80-85 degrees C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K(m) and k(cat)/K(m) values (at 85 degrees C and pH 6.0) of about 14 mM and 5.2 x 10(8) M(-1) s(-1), respectively.

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.

Aggregation process affects the ability of Lactobacillus crispatus, a probiotic, to survive into the gastro-intestinal environment and to adhere to the intestinal mucosa. To elucidate mechanisms underlying this process, a comparative proteomic study was carried out on a wild type strain M247 and its spontaneous isogenic mutant Mu5, which had lost the aggregative phenotype. Results highlighted an overall lower amount of enzymes involved in carbohydrate transport and metabolism in strain M247 compared to strain Mu5, suggesting a reduction in the general growth rate, probably caused by nutrient limitation in cell aggregates, coherently with the phenotypic traits of the strains. Moreover, the up-regulation of a putative elongation factor Tu in the wild type M247 strain could suggest a role of this particular protein in the adhesion mechanism of L. crispatus.

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 °C and is characterized by extreme thermodynamic stability (Tm, 110 °C that increases to 120 °C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 °C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.

Proteomics for Studying Molecular Mechanisms of Probiotic Action: Adaptation Mechanisms to the GIT Environment; Adhesion Mechanisms to the Host Mucosa, Molecular Mechanisms of Probiotic Immunomodulatory Effects, Probiotics and Prebiotics

Dietary polyphenols are bioactive molecules that beneficially affect human health, due to their anti-oxidant, anti-inflammatory, cardio-protective and chemopreventive properties. They are absorbed in a very low percentage in the small intestine and reach intact the colon, where they are metabolized by the gut microbiota. Although it is well documented a key role of microbial metabolism in the absorption of polyphenols and modulation of their biological activity, molecular mechanisms at the basis of the bacteria-polyphenols interplay are still poorly understood. In this context, differential proteomics was applied to reveal adaptive response mechanisms that enabled a potential probiotic Lactobacillus acidophilus strain to survive in the presence of the dietary polyphenol rutin. The response to rutin mainly modulated the expression level of proteins involved in general stress response mechanisms and, in particular, induced the activation of protein quality control systems, and affected carbohydrate and amino acid metabolism, protein synthesis and cell wall integrity. Moreover, rutin triggered the expression of proteins involved in oxidation-reduction processes.This study provides a first general view of the impact of dietary polyphenols on metabolic and biological processes of L. acidophilus.

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.

The mucosal lesion in coeliac disease (CD) is an immune-mediated injury triggered by gliadin and associated with HLA-DQ2 and HLA-DQ8. In view of this, an approach that re-induces tolerance to this antigen should be considered as possible alternative to a strict gluten-free diet in treating CD. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper the efforts to identify single component(s) useful for tolerance induction. To address this issue, a recombinant alpha-gliadin was tested in tolerance experiments in HLA-DQ8 transgenic mice. As tissue transglutaminase (tTG) treatment of gliadin, previously reported to enhance its stimulatory activity in CD, did not increase its immunogenicity when parenterally administered in these mice, untreated gliadin was used as immunogen. A decrease in systemic T cell responses to the recombinant alpha-gliadin was found after nasal administration of antigen, reflected by lymphocytes proliferation assay. Interestingly, while the immunisation protocol induced transcription of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, the tolerisation protocol down-regulated significantly only the IFN-gamma mRNA expression. More important, the recombinant alpha-gliadin induced a similar down-regulatory effect in mice immunised with a commercial preparation of wheat gliadin, that is a mixture of many different gliadin components. As the Th1 phenotype and the HLA-DQ8 molecule play a role in the pathogenesis of CD, our data underlined the potential usefulness of this recombinant protein for the immunomodulation of this disease.

Peptidomic profiling of peritoneal fluid by Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF-MS) may represent a promising, suitable, rapid method for early diagnosis and staging of endometriosis. In a case-control study, peritoneal fluid was collected from 23 patients affected by endometriosis (eight minimal/mild endometriosis and 15 moderate/severe endometriosis) and six “endometriosis free” women undergoing laparoscopy. MALDI-TOF mass spectra of the peptide fraction extracted from peritoneal fluid samples lead to identify biomarkers potentially suitable for discriminating between peritoneal fluid samples from women affected by minimal/mild endometriosis and those from women affected by moderate/severe endometriosis. Peptidomic analysis of peritoneal fluid samples may define putative peptide biomarkers suitable for staging endometriosis and improve our understanding of the pathogenesis of endometriosis.

Hazelnuts exhibit functional properties due to their content in fatty acids and phenolic compounds that could positively affect human health. The food industry requires precise traits for morphological, chemical, and physical kernel features so that some cultivars could be more suitable for specific industrial processing. In this study, agronomical and morphological features of 29 hazelnut cultivars were evaluated and a detailed structural characterization of kernel polyphenols was performed, confirming the presence of protocatechuic acid, flavan-3-ols such as catechin, procyanidin B2, six procyanidin oligomers, flavonols, and one dihydrochalcone in all the analyzed cultivars. In addition, an innovative methodology based on the MALDI-TOF mass spectrometric analysis of peptide/protein components extracted from kernels was developed for the authentication of the most valuable cultivars. The proposed method is rapid, simple, and reliable and holds the potential to be applied in quality control processes. These results could be useful in hazelnut cultivar evaluation and choice for growers, breeders, and food industry. © 2014 American Chemical Society.

The mucosal lesion present in coeliac disease is an immune-mediated injury triggered by gliadin and restricted by a particular assortment of major histocompatibility complex genes. In view of this, an immunomodulatory approach that induces tolerance to this antigen appears to be a possible alternative to a strict gluten-free diet in treating coeliac disease. We have shown that intranasal administration of multiple doses of whole gliadin is required to specifically inhibit T helper 1-like T-cell reactivity in BALB/c mice immunized parenterally with whole gliadin. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper efforts to identify single component(s) useful for tolerance induction. In this study, gliadin fractions were purified and administered intranasally to study their ability to induce tolerance to whole gliadin in our animal model. We found that the alpha fraction was particularly effective in downregulating both the in vitro gliadin-specific T-cell proliferation and interferon-γ production to whole gliadin. In particular, a purified α-gliadin was able to suppress the immune response to the entire gliadin mixture. These results demonstrate how an immune response to a complex antigen may be controlled by treatment with a purified component and specifically indicate α-gliadin to be a good candidate for further identification of short peptides to be used as tolerogens in this model.

International audience; Snake curaremimetic toxins are short all-$\beta$ proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a “disulfide $\beta$-cross”- fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the “disulfide $\beta$-cross”. To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin $\alpha$ ($Naja\ nigricollis$) and erabutoxin b ($Laticauda\ semifasciata$), and two synthetic homologues, the $\alpha$ mutants, $\alpha$60 and $\alpha$62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins

Background: The spore-bearing alkaliphilic Bacillus species constitute a large, heterogeneous group of microorganisms, important for their ability to produce enzymes, antibodies and metabolites of potential medical use. Some Bacillus species are currently being used for manufacturing probiotic products consisting of bacterial spores, exhibiting specific features (colonization, immune-stimulation and antimicrobial activity) that can account for their claimed probiotic properties. In the present work a comparative proteomic study was performed aimed at characterizing the secretome of four closely related isogenic O/C, SIN, N/R and T B. clausii strains, already marketed in a pharmaceutical mixture as probiotics. Results: Proteomic analyses revealed a high degree of concordance among the four secretomes, although some proteins exhibited considerable variations in their expression level in the four strains. Among these, some proteins with documented activity in the interaction with host cells were identified, such as the glycolytic enzyme enolase, with a putative plasminogen-binding activity, GroEL, a molecular chaperone shown to be able to bind to mucin, and flagellin protein, a structural flagella protein and a putative immunomodulation agent. Conclusion: This study shows, for the first time, differences in the secretome of the OC, SIN, NR and T B. clausii strains. These differences indicate that specific secretome features characterize each of the four strains despite their genotypic similarity. This could confer to the B. clausii strains specific probiotic functions associated with the differentially expressed proteins and indicate that they can cooperate as probiotics as the secretome components of each strain could contribute to the overall activity of a mixed probiotic preparation.

The growth of Lactobacillus plantarum WCFS1 and of its ΔccpA ery mutant, WCFS1-2, was compared in batch fermentations in a complex medium at controlled pH (6.5) and temperature (30 °C) with or without aeration, in order to evaluate the effect of ccpA inactivation and aeration on growth, metabolism and stress resistance. Inactivation of ccpA and, to a lesser extent, aeration, significantly affected growth, expression of proteins related to pyruvate metabolism and stress, and tolerance to heat, oxidative and cold/starvation stresses. The specific growth rate of the mutant was ca. 60% of that of the wild type strain. Inactivation of ccpA and aerobic growth significantly affected yield and production of lactic and acetic acid. Stationary phase cells were more stress tolerant than exponential phase cells with little or no effect of inactivation of ccpA or aeration. On the other hand, for exponential phase cells inactivation of ccpA impaired both heat stress and cold/starvation stress, but increased oxidative stress tolerance. For both strains, aerobically grown cells were more tolerant of stresses. Evidence for entry in a viable but non-culturable status upon prolonged exposure to cold and starvation was found. Preliminary results of a differential proteomic study further confirmed the role of ccpA in the regulation of carbohydrate catabolism and class I stress response genes and allow to gain further insight on the role of this pleiotropic regulator in metabolism and stress. This is the first study in which the impact of aerobic growth on stress tolerance of L. plantarum is evaluated. Although aerobic cultivation in batch fermentations does not improve growth it does improve stress tolerance, and may have significant technological relevance for the preservation of starter and probiotic cultures.

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA).In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition. © 2012 Elsevier B.V.

Listeria monocytogenes, one of the major food-related pathogens, is the aetiological agent of listeriosis, a potentially life-threatening illness. It is able to survive in hostile environments and stress conditions such as those encountered in food-processing technologies (high salt concentration, wide range of pH and temperature, low water availability) and it also thrives at temperatures ranging from -0.4 to 45 °C. In this study, expression proteomics was applied to gain insight into key cellular events that allow L. monocytogenes to survive and multiply even at refrigeration temperatures. Interestingly, we observed that the adaptation processes mainly affect biochemical pathways related to protein synthesis and folding, nutrient uptake and oxidative stress. Furthermore, proteins implicated in metabolic pathways for energy production, such as glycolysis and Pta-AckA pathway, were present to a higher level in the cells grown at 4 °C. This suggests that, on the whole, cells exhibit an enhanced demand for energy to sustain cold growth. Proteomics may represent a key tool in deciphering specific mechanisms underlying cold adaptation response and, more widely, cell machinery.

Recent EU directives and regulations for quality control and authentication of food products have prompted the development of new methods for large-scale tests to ensure the protection of consumers. In view of this, an innovative method based on MALDI-TOF mass spectrometry has been developed and successfully applied to fish authentication. Highly specific mass spectrometric profiles from 25 different fish species were obtained. Signals generated from proteins with molecular weights of about 11 kDa have been selected as specific biomarkers for unambiguous discrimination. This method is also suitable for verifying commercial product authenticity and to rapidly discriminate species subjected to fraudulent substitutions, such as those belonging to Gadidae and Pleuronectiformes. For example, biomarkers for fillets of sole (m/z 11975.21), European plaice (m/z 11351.73, 11763.63) and Greenland halibut (m/z 11432.38) were defined. Structural characterization by mass spectrometry of several proteins generating biomarker signals allowed us to identify them as parvalbumins, known to be among the major fish allergens.

BACKGROUND & AIMS: Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. METHODS: Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. RESULTS: Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. CONCLUSIONS: Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.

Copper and iron are cofactors of many metallo-proteins that accomplish vital functions, such as oxygen and electron transport. Specific metabolic pathways have been selected through evolution, although still not fully elucidated, to confine the dangerous reactivity of their free ionic forms. Inadequate supply of both metals can severly affect basic physiological funtions. A differential analysis of the rat intestinal proteome evidenced the following dietary copper- and iron-deficiencies, i.e., significant changes in the levels of proteins belonging to different functional classes (glucose and fatty acid metabolism, molecular chaperones, cytoskeleton plasticity, vitamin transporters). The presented results bring new perspectives to understand the role of copper and iron in the metabolic pathways and provide novel diagnostic tools to characterize the effects of subclinical deficiencies of both metals in unbalanced nutritional disorders. Keywords: copper • iron; intestine • proteomics • mass spectrometry • F...

An immunochemical approach has been developed to detect the use of formaldehyde as a bacteriostatic agent in dairy products. A synthetic peptide, reproducing the first five amino acid residues of the γ2-casein sequence, was formylated to generate the novel haptenic structure, already well-recognized in formaldehyde-treated milk and arising out of molecular rearrangement after the addition of formaldehyde to the alpha-amino group of the histidine residue at the N terminus of γ2-casein. A polyclonal antibodies preparation produced against the formylated peptide adduct proved to be a highly specific analytical tool for detecting the formylated adduct of γ2-casein in formaldehyde-treated milk. Polyclonal antibodies obtained against the unmodified peptide were able to detect selectively residual native γ2-casein in ripened cheese. Keywords: Formaldehyde; food additives; antipeptide antibodies; γ2-casein; cheese ripening

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant α-gliadin (r-α-gliadin) showing the most conserved sequence among the fraction of α-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-α-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-α-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120–139) and p23 (aa 220–239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-α-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-γ secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.