NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

Cardiotonic steroids: potential endogenous sodium pump ligands with diverse function

The highly conserved cardiotonic steroid (CS) binding site present on the ubiquitous membrane sodium pump, sodium, potassium-ATPase, appears to have been conserved by no force other than its capacity to bind CS: a family that includes plant-derived cardiac glycosides and putative endogenous vertebrate counterparts. Binding of ligand is inhibited by increased extracellular potassium. This implies functional coordination because inhibition of the sodium pump would be counterproductive when extracellular potassium is elevated. The interesting biology of the CS binding site continues to stimulate investigations into the identity of endogenous ligands, their role as pump regulators at the cellular level, and as mediators of body fluid balance and blood pressure regulation. In addition to inhibition of sodium and potassium transport, there is considerable recent evidence suggesting that the sodium pump may act as a cell signaling receptor activated by CS binding and responding by coordination of intracellular signaling pathways that can be dependent on and also independent of the reduction in transmembrane ion flux resulting directly from pump inhibition. This signaling may influence cell survival, growth, and differentiation. Recent insight into the biology of pump regulation by CS is reviewed.     

Identification and expression of Ima,a novel Ral-interacting Drosophila protein

We report the identification of Ima, a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays. The gene is expressed in distinct spatiotemporal patterns throughout embryonic development. Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner.     

Lipid radical generation in polar (Laternula elliptica) and temperate (Mya arenaria) bivalves

Lipid peroxidation in Laternula elliptica was assessed by detecting lipid radicals by electronic paramagnetic resonance. The values were compared with data from the temperate mud clam Mya arenaria. Lipid radical content was higher in the Antarctic bivalve than in the temperate mud clam, even within the range of its habitat temperature. The rate of generation of lipid radicals was affected by the iron content in the samples. The iron content in individual samples of digestive glands in L. elliptica ranged from 3 to 6 nmol g⁻¹ fresh weight (fwt) and in M. arenaria from 0.6 to 2.7 nmol g⁻¹ fwt. Arrhenius plots, developed from the rates obtained in the presence of 25 μM iron, showed no significant differences between the activation energy calculated for digestive glands of L. elliptica and M. arenaria. The Fe³⁺ reduction rate in L. elliptica was higher than in M. arenaria (4.7±0.9 vs. 1.8±0.4 nmol mg⁻¹ protein min⁻¹, respectively). L. elliptica had a higher content of α-tocopherol and β-carotene than M. arenaria. Our data suggest that increased lipid radical content in the membranes of cold-adapted organisms could be related to iron content.     

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration

We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca(2+)](i) and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCepsilon to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.     

Crystallization of membrane proteins from media composed of connected-bilayer gels

The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.     

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.