The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.

Characterization of the proteins present in the mucus of the flatworm Artioposthia triangulata (Dendy)

In common with other terrestrial flatworms, the mucus produced by Artioposthia triangulata may have a number of functions, including protection from environmental factors and from predators, and it provides the flatworm with lubrication for movement and adhesion. No previous work has been carried out on the characterization of proteins present in the mucus of A. triangulata and this study was a preliminary investigation of the mucus. Mucus was analysed by SDS-polyacrylamide gel electrophoresis, biotinylated peptide affinity probes and the API ZYM enzyme detection kit. Results have revealed the presence of at least 40 polypeptides in the mucus and further studies with biotinylated probes have characterised one of them as a chymotrypsin-like serine protease. (C) 1998 Elsevier Science Inc.

Molecular dynamics studies of the STAT3 homodimer:DNA complex: relationships between STAT3 mutations and protein-DNA recognition.

Signal Transducers and Activators of Transcription (STAT) proteins are a group of latent cytoplasmic transcription factors involved in cytokine signaling. STAT3 is a member of the STAT family and is expressed at elevated levels in a large number of diverse human cancers and is now a validated target for anticancer drug discovery.. Understanding the dynamics of the STAT3 dimer interface, accounting for both protein-DNA and protein-protein interactions, with respect to the dynamics of the latent unphosphorylated STAT3 monomer, is important for designing potential small-molecule inhibitors of the activated dimer. Molecular dynamics (MD) simulations have been used to study the activated STAT3 homodimer:DNA complex and the latent unphosphorylated STAT3 monomer in an explicit water environment. Analysis of the data obtained from MD simulations over a 50 ns time frame has suggested how the transcription factor interacts with DNA, the nature of the conformational changes, and ways in which function may be affected. Examination of the dimer interface, focusing on the protein-DNA interactions, including involvement of water molecules, has revealed the key residues contributing to the recognition events involved in STAT3 protein-DNA interactions. This has shown that the majority of mutations in the DNA-binding domain are found at the protein-DNA interface. These mutations have been mapped in detail and related to specific protein-DNA contacts. Their structural stability is described, together with an analysis of the model as a starting-point for the discovery of novel small-molecule STAT3 inhibitors.

Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues Conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to (he dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds Conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1.3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo ill a number of human tumor xenograft models.

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1ß secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1ß secretion and pyroptosis. Moreover, IL-1ß secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1ß secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

Mitochondrial proteins Bnip3 and Bnip3L are involved in anthrax lethal toxin-induced macrophage cell death

Anthrax lethal toxin (LeTx) induces rapid cell death of RAW246.7 macrophages. We recently found that a small population of these macrophages is spontaneously and temporally refractory to LeTx-induced cytotoxicity. Analysis of genome-wide transcripts of a resistant clone before and after regaining LeTx sensitivity revealed that a reduction of two closely related mitochondrial proteins, Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) and Bnip3-like (Bnip3L), correlates with LeTx resistance. Down-regulation of Bnip3 and Bnip3L was also found in "toxin-induced resistance" whereby sublethal doses of LeTx induce resistance to subsequent exposure to cytolytic toxin doses. The role of Bnip3 and Bnip3L in LeTx-induced cell death was confirmed by showing that overexpression of either Bnip3 or Bnip3L rendered the resistant cells susceptible to LeTx, whereas down-regulation of Bnip3 and Bnip3L in wild-type macrophages conferred resistance. The down-regulation of Bnip3 and Bnip3L mRNAs by LeTx occurred at both transcriptional and mRNA stability levels. Inhibition of the p38 pathway by lethal factor was responsible for the destabilization of Bnip3/Bnip3L mRNAs as confirmed by showing that p38 inhibitors stabilized Bnip3 and Bnip3L mRNAs and conferred resistance to LeTx cytotoxicity. Therefore, Bnip3/Bnip3L play a crucial role in LeTx-induced cytotoxicity, and down-regulation of Bnip3/Bnip3L is a mechanism of spontaneous or toxin-induced resistance of macrophages.