A Lactic Acid Bacteria Consortium Impacted the Content of Casein-Derived Biopeptides in Dried Fresh Cheese.

This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides (biopeptides). The designed LAB consortium consisted of three Lactobacillus strains: S-layer carrying Levilactobacillus brevis D6, exopolysaccharides producing Limosilactobacillus fermentum D12 and plantaricin expressing Lactiplantibacillus plantarum D13, and one Enterococcus strain, Enterococcus faecium ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from ß-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

Bimolecular Fluorescence Complementation to Visualize Protein-Protein Interactions in Human Cells Based on Gateway Cloning Technology.

Bimolecular fluorescence complementation (BiFC) is a powerful and sensitive tool to discover new protein-protein interactions (PPIs). It enables visualization and localization of protein-protein interactions (PPIs) in living cells. The idea behind BiFC is to split a fluorescent protein, for example yellow fluorescent protein (YFP), into two parts that are unable to emit fluorescent signal on their own. Therefore, in order to regain fluorescence the split protein fragments must establish close proximity. This is accomplished by fusing the split fragments to proteins that are postulated to interact, and expressing them in living cells. Subsequently, detection of fluorescence indicates interaction of given proteins. Since complementation is practically irreversible it can capture weak and transient interactions. Using suitable vectors for human protein expression, thus avoiding viral cell transfection, we introduced Gateway-based cloning features to the BiFC system, thereby enabling time efficient vector construction in order to maximize the full potential of the BiFC approach to investigate many protein-protein interactions in a high-throughput fashion. This protocol explains steps in a typical protein-protein interaction survey, from the vector selection, cell transfection, and visualization of the fluorescent signal.

Mutations in S-adenosylhomocysteine hydrolase (AHCY) affect its nucleocytoplasmic distribution and capability to interact with S-adenosylhomocysteine hydrolase-like 1 protein.

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.

1-ethyl-3-(6-methylphenanthridine-8-il) urea modulates TLR3/9 activation and induces selective pro-inflammatory cytokine expression in vitro.

We have previously demonstrated the nucleic acid binding capacity of phenanthridine derivatives (PHTs). Because nucleic acids are potent inducers of innate immune response through Toll-like receptors (TLRs), and because PTHs bear a structural resemblance to commonly used synthetic ligands for TLR7/8, we hypothesized that PHTs could modulate/activate immune response. We found that compound M199 induces secretion of IL-6, IL-8 and TNFα in human PBMCs and inhibits TLR3/9 activation in different cellular systems (PBMCs, HEK293 and THP-1 cell lines).

Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein-Protein Interactions.

Protein interaction networks are the basis for human metabolic and signaling systems. Interaction studies often use bimolecular fluorescence complementation (BiFC) to reveal the formation and cellular localization of protein complexes. However, large-scale studies were either far from native conditions in human cells or limited by laborious restriction/ligation cloning techniques. Here, we describe a new tool for protein interaction screening based on Gateway-compatible BiFC vectors. We made a set of four new vectors that permit fusion of candidate proteins to the N or C fragment of Venus in all fusion positions. We have validated the vectors and confirmed self-association of AHCY, AHCYL1, and galectin-3. In a high-throughput BiFC screen, we identified new AHCY interaction partners: galectin-3 and PUS7L. We also describe additional steps in protein interaction analysis, applied for AHCY-galectin-3 interaction. First, we classified the interaction in intracellular vesicles using CellCognition, machine learning free software. Then we identified the vesicles as endosomal pathway compartments, in line with known galectin-3 trafficking route. This offers a platform to rapidly identify and localize new protein interactions inside living cells, a prerequisite to validate in silico interactome data, and ultimately decode complex protein networks.

Low or No Inhibitory Potency of the Canonical Galectin Carbohydrate-binding Site by Pectins and Galactomannans.

Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC50 values >10 mg/ml for a few or in most cases no inhibitory activity at all. The galactomannan Davanat® was more active, albeit not a strong inhibitor (IC50 values ranging from 3 to 20 mg/ml depending on the galectin). Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site.

Ligand induced galectin-3 protein self-association.

Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 µM) as compared with wild type (∼1 µM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>∼50 µM) in agreement with published results and perhaps due to type-N self-association.

Galectin-3 endocytosis by carbohydrate independent and dependent pathways in different macrophage like cell types.

BACKGROUND: Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells. METHODS: Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma. RESULTS: Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the R186S mutation in the galectin-3 carbohydrate recognition domain (CRD). In M1 cells galectin-3 endocytosis could be inhibited only by chlorpromazine and by interference with the non-CRD N-terminal part of galectin-3. In all the cell types galectin-3 entered early endosomes within 5-10 min, to be subsequently targeted mainly to non-degradative vesicles, where it remained even after 24 h. CONCLUSIONS: Galectin-3 endocytosis in M1 cells is receptor mediated and carbohydrate independent, while in M2 cells it is CRD mediated, although the non-CRD galectin-3 domain is also involved. General significance The demonstration that galectin-3 endocytosis in M1 macrophages is carbohydrate independent and different from M2 macrophages and non-macrophage cells, suggests novel, immunologically significant interactions between phagocytic cells, galectin-3 and its ligands.

Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients.

Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7-2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8-3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20-80) in cancer sera and about 30% (range 25-50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.