The side chain of glutamine 13 is the acyl-donor amino acid modified by type 2 transglutaminase in subunit T of the native rabbit skeletal muscle troponin complex.

Subunit T of the native muscle troponin complex is a recognised substrate of transglutaminase both in vitro and in situ with formation of isopeptide bonds. Using a proteomic approach, we have now determined the precise site of in vitro labelling of the protein. A preparation of troponin purified from ether powder from mixed rabbit skeletal muscles was employed as transglutaminase substrate. The only isoform TnT2F present in our preparation was recognised as acyl-substrate by human type 2 transglutaminase which specifically modified glutamine 13 in the N-terminal region. During the reaction, the troponin protein complex was polymerized. Results are discussed in relation to the structure of the troponin T subunit, in the light of the role of troponins in skeletal and cardiac muscle diseases, and to the rules governing glutamine side chain selection by tissue transglutaminase

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids In vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and re- sistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutami- nase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The aris- ing protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conver- sion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the car- bohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen

Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites

Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure

Flavonoids: a myth or a reality for cancer therapy?

Nutraceuticals are biologically active molecules present in foods; they can have beneficial effects on health, but they are not available in large enough quantities to perform this function. Plant metabolites, such as polyphenols, are widely diffused in the plant kingdom, where they play fundamental roles in plant development and interactions with the environment. Among these, flavonoids are of particular interest as they have significant effects on human health. In vitro and/or in vivo studies described flavonoids as essential nutrients for preventing several diseases. They display broad and promising bioactivities to fight cancer, inflammation, bacterial infections, as well as to reduce the severity of neurodegenerative and cardiovascular diseases or diabetes. Therefore, it is not surprising that interest in flavonoids has sharply increased in recent years. More than 23,000 scientific publications on flavonoids have described the potential anticancer activity of these natural molecules in the last decade. Studies, in vitro and in vivo, show that flavonoids exhibit anticancer properties, and many epidemiological studies confirm that dietary intake of flavonoids leads to a reduced risk of cancer. This review provides a glimpse of the mechanisms of action of flavonoids on cancer cells

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset

Complexation to cationic microspheres of double-stranded peptide nucleic acid-DNA chimeras exhibiting decoy activity

The major aim of this paper was to determine whether cationic microspheres (CM), consisting of the permeable polymer Eudragit\uc2\uae RS 100 plus the cationic surfactant dioctadecyl-dimethyl-ammonium bromide (DDAB18), could bind to double-stranded peptide nucleic acid PNA-DNA-PNA (PDP) chimeras exhibiting decoy activity against NF-\uce\ubaB transcription factors. Microspheres were produced by the 'solvent evaporation method' and centrifugation at 500, 1,000 and 3,000 rpm to obtain different-sized microparticles. Microsphere morphology, size and size distribution were determined by optical and electron microscopy observations. In order to determine their binding activity, double-stranded DNA-based and PDP-based decoy molecules were incubated with different amounts of microparticles in the presence of 100 ng of either32P-labeled DNA-DNA or DNA-PDP hybrid molecules or cold PDP-PDP hybrids. The complexes were analyzed by agarose gel electrophoresis. The resistance of32P-labeled DNA-DNA and DNA-PDP molecules in the presence of serum or cellular extracts was evaluated after binding to CM by gel electrophoresis analysis. DDAB18Eudragit RS 100 microspheres are able to bind to DNA-PDP and PDP-PDP hybrids, to deliver these molecules to target cells and to protect DNA-PDP molecules from enzymatic degradation in simulated biological fluids. In addition, when assayed in ex vivo conditions, DDAB18Eudragit RS 100 microspheres exhibited low toxicity. The results presented in this paper demonstrate that CM can be considered suitable formulations for pharmacogenomic therapy employing double-stranded PDP chimeras

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

<p>Abstract</p> <p>Background</p> <p>Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.</p> <p>Results</p> <p>Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction <it>in vitro</it>. Analysis of the effect of NF90ctv-TAR RNA interaction <it>in vivo </it>showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.</p> <p>Conclusion</p> <p>Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.</p

Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies