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We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA–miRNA–Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N&prime, N&prime, tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO4 fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70 degrees C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 μg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways.

The essential micronutrient zinc has long been known to be a functional component of diverse structural proteins and enzymes. More recently, important roles for free or loosely bound intracellular zinc as a signaling factor have been reported. Insufficient zinc intake was shown to exacerbate symptoms in mouse models of inflammation such as experimental colitis, while zinc supplementation was found to improve intestinal barrier function. Herein, we provide evidence that intracellular zinc is essential for maintaining intestinal epithelial integrity when cells are exposed to the inflammatory cytokine Tumor Necrosis Factor (TNF)α. Using the human intestinal Caco-2/TC7 cell line as an in vitro model, we demonstrate that depletion of intracellular zinc affects TNFα-triggered signaling by shifting intestinal cell fate from survival to death. The mechanism underlying this effect was investigated. We show that TNFα promotes a zinc-dependent survival pathway that includes modulation of gene expression of transcription factors and signaling proteins. We have identified multiple regulatory steps regulated by zinc availability which include the induction of cellular Inhibitor of APoptosis (cIAP2) mRNA, possibly through activation of Nuclear Factor-Kappa B (NF-κB), as both nuclear translocation of the p65 subunit of NF-κB and up-regulation of cIAP2 mRNA were impaired following zinc depletion. Moreover, X-linked inhibitor of apoptosis protein level was profoundly reduced by zinc depletion. Our results provide a possible molecular explanation for the clinical observation that zinc supplements ameliorate Crohn's disease symptoms and decrease intestinal permeability in experimental colitis.

Motivation: Analysis of the human genome revealed that the amount of transcribed sequence is an order of magnitude greater than the number of predicted and well-characterized genes. A sizeable fraction of these transcripts is related to alternatively spliced forms of known protein coding genes. Inspection of the alternatively spliced transcripts identified in the pilot phase of the ENCODE project has clearly shown that often their structure might substantially differ from that of other isoforms of the same gene, and therefore that they might perform unrelated functions, or that they might even not correspond to a functional protein. Identifying these cases is obviously relevant for the functional assignment of gene products and for the interpretation of the effect of variations in the corresponding proteins. Results: Here we describe a publicly available tool that, given a gene or a protein, retrieves and analyses all its annotated isoforms, provides users with three-dimensional models of the isoform(s) of his/her interest whenever possible and automatically assesses whether homology derived structural models correspond to plausible structures. This information is clearly relevant. When the homology model of some isoforms of a gene does not seem structurally plausible, the implications are that either they assume a structure unrelated to that of the other isoforms of the same gene with presumably significant functional differences, or do not correspond to functional products. We provide indications that the second hypothesis is likely to be true for a substantial fraction of the cases. Availability: http://maistas.bioinformatica.crs4.it/. Contact: anna.tramontano@uniromal.it

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ERbeta but not for ERalpha. In addition, in ERbeta-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ERbeta translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling gamma- and delta-TT effects.

Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.

Zinc is an essential micronutrient playing fundamental roles in cellular metabolism. It acts mostly through binding a wide range of proteins, thus affecting a broad spectrum of biological processes, which include cell division, growth and differentiation. Full annotation of zinc-binding proteins showed them to represent about 10 % of the human proteome, with over 300 enzymes containing zinc ions within their catalytic domains. Also, hundreds of key regulatory proteins, including transcription factors, require zinc for their activity. In this study, the whole set of zinc-binding proteins together with their direct interactors was listed and defined as the zinc proteome (ZNP). We interrogated pathway analysis tools to identify the cellular processes that are predicted to be affected by zinc availability. Network and functional enrichment analyses highlighted biological processes potentially affected by deregulated zinc homeostasis. This computational approach was also tested on a real case study: The possible involvement of ZNP network proteins in Crohn’s disease pathogenesis was assessed on genes transcriptionally regulated in the intestine of patients affected by this condition. The analysis produced a network of pathways likely to be influenced by zinc and associated with Crohn’s disease. These results highlight a central role for zinc in the tissue remodeling process which occurs upon gut inflammation, pointing at novel disease pathways whose effect could be worsened by zinc dyshomeostasis and impaired zinc fluxes in specific damaged areas. Overall, our computational approach could provide novel insights into pathological conditions and could therefore be used to drive mechanistic research in under-investigated fields of research. An interactive version of the determined ZNP network is available at URL http://93.63.165.11/ZNnetwork/ .

The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and BUSHY AND DWARF2 (BUD2) is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper (HD- ZIP) III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein ARABIDOPSIS THALIANA HOMEOBOX8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as LIKE AUXIN RESISTANT2 (LAX2) and LAX3. We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD-ZIP III genes. © The Author 2014.

Glucosinolates are a class of secondary plant metabolites particularly occurring in Cruciferae with potential health-promoting properties, as their hydrolysis products, isothiocyanates, possess chemopreventive and antioxidant activities. In the present study, we systematically studied the in vitro redox behaviour of 15 glucosinolates, by using a range of analytical methods measuring different activities: (i) radical scavenging activity toward peroxyl and toward ABTS radical (chain-breaking activity); (ii) capacity in modulating the in vitro resistance of human low-density lipoprotein (LDL) catalysed by copper (chelating and chain-breaking activity). Data obtained from different assays were compared and analysed by principal component analysis (PCA). PCA allowed us to identify a big cluster of glucosinolates (10 out 15 tested) that do not possess any antioxidant capacity; while, the other five glucosinolates showed moderate and specific antioxidant capacity. Notably, sinalbin and gluconasturtiin were highly active in scavenging ABTS radical and in protecting LDL from copper-catalysed oxidation, respectively. The overall results of this study indicate that just few glucosinolates can act as antioxidants.