Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

A distinctive protein signature induced by lysophosphatidic acid receptor 6 (LPAR6) expression in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed liver malignancy, ranking third in the overall global cancer-related mortality. A complex network of interacting proteins controls HCC growth and progression. Lysophosphatidic acid receptors (LPAR) are commonly overexpressed in HCC. In particular, we have previously reported that the expression of LPAR6 sustains tumorigenesis and growth of HCC and results in a poor prognosis in HCC patients. Here, we applied a comparative proteomic approach to compare protein expression in both LPAR6 expressing (HLE-LPAR6) and nonexpressing HCC cells (HLE-neo). We found changes in the expression levels of 19 proteins, which include carbohydrate metabolism enzymes, redox and detoxification enzymes, and gene-expression regulatory proteins. Our findings support the role of LPAR6 in controlling the expression of a distinctive protein signature in HCC cells, which can offer a valuable resource for the identification of potential theranostic biomarkers.

Decreased amount of vimentin N-terminal truncated proteolytic products in parkin-mutant skin fibroblasts.

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.

Influence of oral administration mode on the efficacy of commercial bovine Lactoferrin against iron and inflammatory homeostasis disorders.

Milk derivative bovine Lactoferrin (bLf), a multifunctional glycoprotein available in large quantities and recognized as safe, possesses high homology and identical functions with human Lactoferrin. There are numerous food supplements containing bLf which, however, can vary in its purity, integrity and, consequently, functionality. Here, we report on a clinical trial where bLf (100 mg two times/day) was orally administered before (Arm A) or during meals (Arm B) to pregnant women with hereditary thrombophilia and suffering from anemia of inflammation. A significant increase of the number of red blood cells (RBCs), hemoglobin (Hb), total serum iron (TSI) and serum ferritin (sFtn) levels, along with a significant decrease of interleukin-6 were detected after 30 days in Arm A, but not in Arm B, thus letting us to hypothesize that bLf inefficacy could be related to its degradation by digestive proteases. To verify this hypothesis, bLf was incubated in gastric juice collected before or after meals. An undigested or a digested profile was observed when bLf was incubated in gastric juice sampled before or after meals, respectively. These results can explain the beneficial effect observed when bLf is administered under fasting conditions, i.e. in the absence of active proteases.

Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome.

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; p<0.05), CD44 antigen (r2 =0.35; p<0.03) and lysosomal alfa glucosidase ( r20.27; p<0.05) abundance with the eGFR. In addition, u-FN (r2 =0.2389; p<0.05) was significantly correlated with ΔeGFR. CONCLUSION: 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.

Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

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.

Rapid peptidomic profiling of peritoneal fluid by MALDI-TOF mass spectrometry for the identification of biomarkers of endometriosis.

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.

Comparative secretome analysis of four isogenic Bacillus clausii probiotic strains.

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.

Surface Layer Protein Pattern of Levilactobacillus brevis Strains Investigated by Proteomics.

The outermost constituent of many bacterial cells is represented by an S-layer, i.e., a semiporous lattice-like layer composed of self-assembling protein subunits called S-layer proteins (Slps). These proteins are involved in several processes, such as protecting against environmental stresses, mediating bacterial adhesion to host cells, and modulating gut immune response. Slps may also act as a scaffold for the external display of additional cell surface proteins also named S-layer associated proteins (SLAPs). Levilactobacillus brevis is an S-layer forming lactic acid bacterium present in many different environments, such as sourdough, milk, cheese, and the intestinal tract of humans and animals. This microorganism exhibits probiotic features including the inhibition of bacterial infection and the improvement of human immune function. The potential role of Slps in its probiotic and biotechnological features was documented. A shotgun proteomic approach was applied to identify in a single experiment both the Slps and the SLAPs pattern of five different L. brevis strains isolated from traditional sourdoughs of the Southern Italian region. This study reveals that these closely related strains expressed a specific pattern of surface proteins, possibly affecting their peculiar properties.

Paraprobiotics: A New Perspective for Functional Foods and Nutraceuticals.

Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as "inactivated microbial cells (non-viable) that confer a health benefit to the consumer," hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Secretome Analysis of Mouse Dendritic Cells Interacting with a Probiotic Strain of Lactobacillus gasseri.

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.

Proteomic Analysis of Mucopolysaccharidosis IIIB Mouse Brain.

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.

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359).

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.

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein.

BACKGROUND: Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. RESULTS: We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. CONCLUSION: Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein.

Proteomics for the Investigation of Surface-Exposed Proteins in Probiotics.

Probiotics are commensal microorganisms that are present in the intestinal tract and in many fermented foods and positively affect human health, promoting digestion and uptake of dietary nutrients, strengthening intestinal barrier function, modulating immune response, and enhancing antagonism toward pathogens. The proteosurfaceome, i.e., the complex set of proteins present on the bacterial surface, is directly involved as leading actor in the dynamic communication between bacteria and host. In the last decade, the biological relevance of surface-exposed proteins prompted research activities exploiting the potentiality of proteomics to define the complex network of proteins that are involved in the molecular mechanisms at the basis of the adaptation to gastrointestinal environment and the probiotic effects. These studies also took advantages of the recent technological improvements in proteomics, mass spectrometry and bioinformatics that triggered the development of ad hoc designed innovative strategies to characterize the bacterial proteosurfaceome. This mini-review is aimed at describing the key role of proteomics in depicting the cell wall protein architecture and the involvement of surface-exposed proteins in the intimate and dynamic molecular dialogue between probiotics and intestinal epithelial and immune cells.

Impact of aerobic and respirative life-style on Lactobacillus casei N87 proteome.

Lactic acid bacteria (LAB) are used as starter, adjunct and/or probiotic cultures in fermented foods. Several species are recognized as oxygen-tolerant anaerobes, and aerobic and respiratory cultivations may provide them with physiological and technological benefits. In this light, mechanisms involved in the adaptation to aerobic and respiratory (supplementation with heme and menaquinone) growth conditions of the O2-tolerant strain Lactobacillus casei N87 were investigated by proteomics. In fact, in this bacterial strain, respiration induced an increase in biomass yield and robustness to oxidative, long-term starvation and freeze-drying stresses, while high concentrations of dissolved O2 (dO2 60%) negatively affected its growth and cell survival. Proteomic results well paralleled with physiological and metabolic features and clearly showed that aerobic life-style led to a higher abundance of several proteins involved in carbohydrate metabolism and stress response mechanisms and, concurrently, impaired the biosynthesis of proteins involved in nucleic acid formation and translation processes, thus providing evidence at molecular level of the significant damage to L.casei N87 fitness. On the contrary, the activation of respiratory pathways due to heme and menaquinone supplementation, led to a decreased amount of chaperones and other stress related proteins. These findings confirmed that respiration reduced oxidative stress condition, allowing to positively modulate the central carbohydrate and energy metabolism and improve growth and stress tolerance features. Results of this study could be potentially functional to develop competitive adjunct and probiotic cultures effectively focused on the improvement of quality of fermented foods and the promotion of human health.

Cytotoxic and apoptogenic activity of a methanolic extract from the marine invertebrate Ciona intestinalis on malignant cell lines.

Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.

Response mechanisms induced by exposure to high temperature in anthers from thermo-tolerant and thermo-sensitive tomato plants: A proteomic perspective.

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.

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I.

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.

Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus.

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 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees 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.