LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.

The effect of allosteric modulators on the kinetics of agonist-G protein-coupled receptor interactions in single living cells.

Allosteric binding sites on adenosine -A(1) and -A(3) receptors represent attractive therapeutic targets for amplifying, in a spatially and temporally selective manner, the tissue protective actions of endogenous adenosine. This study has directly quantified the kinetics of agonist/G protein-coupled receptor interactions at the single-cell level, reflecting the physiological situation in which intracellular signaling proteins can exert major allosteric effects on agonist-receptor interactions. The association and dissociation rate constants at both A(1) and A(3) receptors, and therefore the affinity of the fluorescent adenosine derivative ABA-X-BY630 (structure appears in J Med Chem 50:782-793, 2007), were concentration-independent. The equilibrium dissociation constants of ABA-X-BY630 at A(1) and A(3) receptors were approximately 50 and 10 nM, respectively, suggesting that, even in live cells, low agonist concentrations predominantly detect high-affinity receptor states. At A(1) receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (k(off) = 1.95 +/- 0.09 min(-1)) compared with the presence of the allosteric enhancer (2-amino-4,5-dimethyl-3-thienyl)(3-(trifluoromethyl)phenyl)-methanone (PD81,723; 10 microM; k(off) = 0.80 +/- 0.03 min(-1)) and allosteric inhibitor 4-methoxy-N-(7-methyl-3-(2-pyridinyl)-1-isoquinolinyl)benzamide (VUF5455; 1 microM; k(off) = 1.48 +/- 0.16 min(-1)). In contrast, ABA-X-BY630 dissociation from A(3) receptors was significantly slower in the absence (k(off) = 0.78 +/- 0.18 min(-1)) than in the presence of the allosteric inhibitors VUF5455 (1 microM; k(off) = 3.15 +/- 0.12 min(-1)) and PD81,723 (10 microM; k(off) = 2.46 +/- 0.18 min(-1)). An allosteric mechanism of action has previously not been identified for PD81,723 at the A(3) receptor or VUF5455 at the A(1) receptor. Furthermore, the marked enhancement in fluorescent agonist dissociation by VUF5455 in living cells contrasts previous observations from broken cell preparations and emphasizes the need to study the allosteric regulation of agonist binding in living cells.

Localisation of a family of complex-forming ß-barrels in the T. vaginalis hydrogenosomal membrane.

Crucial to organellogenesis was the development of membrane translocases responsible for delivering proteins to new cellular compartments. This investigation examines the Trichomonas vaginalis hydrogenosome, a mitochondrially derived organelle. We identify an expanded family of putative ß-barrel proteins (THOM A-I) comprising nine related sequences. Sub-cellular localisation by immunofluorescence and biochemical fractionation is consistent with THOMs being localised to the hydrogenosomal membrane. Native gel electrophoresis and chemical cross-linking support the ability of THOM proteins to be components of membrane-bound oligomeric protein complexes, consistent with a role in protein translocation.

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis.

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.

Dimerization of ABCG2 analysed by bimolecular fluorescence complementation.

ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.